ChemInform Abstract: The Unique Properties of Dipeptidyl-Peptidase IV (DPP IV / CD26) and the Therapeutic Potential of DPP IV Inhibitors

Current Medicinal Chemistry 2005 12 971-998 971

The Therapeutic Potential of Inhibitors of Dipeptidyl Peptidase IV (DPP IV)and Related Proline-Specific Dipeptidyl Aminopeptidases

K Augustyns P Van der Veken K Senten and A Haemers

Department of Medicinal Chemistry University of Antwerp Universiteitsplein 1 B-2610 Antwerpen Belgium

Abstract In this review the structural and functional aspects of dipeptidyl peptidase IV (DPP IV) will bedescribed and the therapeutic potential of DPP IV inhibitors will be highlighted DPP IV will be situated inclan SC a group of serine proteases that contains several proline specific peptidases Structural aspects of DPPIV and its interaction with different types of inhibitors are recently revealed by the publication of severalcrystal structures Especially the design and development of new DPP IV inhibitors based on the three-dimensional structure substrate specificity and catalytic mechanism will be discussed In the last years therewas an important development of new pyrrolidine-2-nitriles with very promising therapeutic properties for thetreatment of type 2 diabetes The role of DPP IV in peptide metabolism of members of the PACAPglucagonpeptide family neuropeptides and chemokines has been thoroughly investigated during recent years This isdirectly related to the promising therapeutic potential of DPP IV inhibitors in the treatment of type 2 diabetesand in the treatment of immunological disorders Several inhibitors are currently under investigation inclinical trials for the treatment of type 2 diabetes and represent a new class of drugs for the treatment of thisdisease

Keywords Dipeptidyl peptidase IV DPP IV CD26 type 2 diabetes DPP IV inhibitor

1 INTRODUCTION Asp His and which are members of the αβ hydrolase foldfamily [4] This arrangement of the catalytic triad is notcommon for lsquoclassicalrsquo serine-type peptidases of clan SA(His Asp Ser trypsin-like enzymes) and of clan SB (AspHis Ser subtilisin-like enzymes) The position of thescissile Pro-Xaa bond within the substrate can vary sinceclan SC contains several dipeptidyl aminopeptidases insubfamilies S9B and S28 as well as the carboxypeptidaselysosomal Pro-X carboxypeptidase in subfamily S28 and theendopeptidase prolyl oligopeptidase in subfamily S9A Alsonon-human enzymes with Pro-Xaa specificity are found inclan SC (Table 2) The dipeptidyl peptidases are sometimesreferred to as lsquoDPP IV activity- andor structure-homologuesrsquo(DASH) proteins [5] to recognise the status of DPP IV asthe most widely studied member of this family and toindicate the structural or functional similarity of its memberswith DPP IV

This review is a continuation of a review published in1999 in this journal [1] For a review of the early literatureon the subject of dipeptidyl peptidase IV (DPP IV) and thetherapeutic potential of its inhibitors the reader is referred tothat paper Essential elements from publications before1999 necessary to understand more recent data will berepeated in this review Most attention will be given toinhibitors of the enzymatic activity of DPP IV and to theirtherapeutic potential especially in the treatment of type 2diabetes

2 CLASSIFICATION OF DPP IV

Due to the unique structure of proline relatively fewpeptidases are able to cleave peptide bonds containingproline (Fig 1) [2] Many biologically active peptidescontain an evolutionary conserved proline residue as aproteolytic-processing regulatory element and thereforeproline-specific peptidases are expected to be importantldquocheck-pointrdquo controls with great potential as targets fordrug discovery [3] Remarkably in humans these proline-specific enzymes are uniquely found in families of thepeptidase clan SC or in the subfamily M24B of clan MG(Tables 1 and 2)

Enzymes specific for cleavage of the Xaa-Pro bond (P1rsquo =Pro) are members of the subfamily M24B of themetallopeptidase clan MG (Table 2)

The properties of DPP IV and its inhibitors will beextensively discussed in the next chapters Here we willshortly describe the most essential properties of the otherproline specific dipeptidyl aminopeptidases of clan SC (forrecent reviews the reader is refereed to [3456789]) Acomparison with DPP IV will be made in order to get abetter understanding on the importance of the developmentof specific DPP IV inhibitors

All human enzymes specific for cleavage of the Pro-Xaabond (P1 = Pro) are members of clan SC Clan SC containspeptidase families with serine nucleophiles in which thecatalytic triad has been identified as being in the order Ser

Like DPP IV fibroblast activation protein alpha subunit(FAPα seprase) is a type II integral membrane protein ableto cleave peptides with proline as the penultimate aminoacid [1011] FAPα has 54 amino acid sequence identitywith DPP IV In contrast to DPP IV FAPα has gelatinaseand collagenase activity with a highly restricted expressionin vivo FAPα is found at remodelling sites in the liver and

Address correspondence to this author at the Department of MedicinalChemistry University of Antwerp Universiteitsplein 1 B-2610Antwerpen Belgium Tel +32-(0)3-820 27 03 Fax +32-(0)3-820 27 39E-mail KoenAugustynsuaacbe

0929-867305 $5000+00 copy 2005 Bentham Science Publishers Ltd

972 Current Medicinal Chemistry 2005 Vol 12 No 8 Augustyns et al

N

O

NH2

O

R1

HO

N

O

N

O

N

O

NH2

O

R1

HN

NH

R3

O

R

O

NH

HN

O

RO

R

HN

OH

Rn

O

PCP

POP

DPP IVFAP αDPP8DPP9DPP II

Aminopept idase P2X-Pro dipeptidase

DPP IV = dipeptidyl peptidase IV FAP α = fibroblast activation protein α subunit DPP8 = dipept idyl peptidase8 DPP9 = dipept idyl peptidase 9 DPP II = dipept idyl peptidase II POP = prolyl oligopeptidase PCP = lysosomal Pro-X carboxypept idase

Fig (1) Human proline-specific peptidases

in tumours but not in normal tissues [12-20] Thisrestricted expression and the gelatinasecollagenase activitypoint to the possibility that FAPα might be a key cellsurface protease involved in promoting extracellular matrixdegradation in cancer invasion and wound healing [21] Arecent study showed the promotion of tumour growth byFAPα in an animal model [22] Moreover antibodies toFAPα were shown to inhibit tumour growth in this modelHowever a humanised anti-FAPα antibody showed noefficacy in a Phase II clinical trial for metastatic colorectalcancer [2324] Still the highly restricted expression andlack of overt developmental defects in FAPα-deficient mice[25] make FAPα a valuable target for the development oftherapeutics for cancer

nucleophilic serine is replaced with Asp and so it lackspeptidase activity [29] Despite the absence of this catalyticactivity DPP6 exerts an important developmental functionThe mouse white rump mutation which lacks expression ofthe DPP6 gene is embryonic lethal in homozygotes andcauses a pigmentation defect in heterozygotes [30] It isrecently established that DPP6 is a critical component ofneuronal A-type potassium channels [31] This biologicalrole of DPP6 possibly indicates that the other members ofthis subfamily have additional functions apart from theirpeptidase activity This is already shown for DPP IV (videinfra)

Dipeptidylpeptidase homologue DPP10 (DPP10) has asignificant sequence identity with DPP6 (51) DPP IV(32) and FAPα (29) Like DPP6 it has no peptidaseactivity due to a mutation of the catalytic serine to glycine[28] DPP10 is mainly expressed in the brain and pancreasIt has a predicted transmembrane region If this is confirmedDPP10 is a type II membrane protein like DPP IV FAPαand DPP6

Dipeptidyl peptidase 8 (DPP8) shares 27 amino acididentity and 51 amino acid similarity with the proteinsequences of DPP IV and FAPα and this increases to 35amino acid identity and 57 amino acid similarity in thehydrolase domain [9] Like DPP IV DPP8 hydrolyses thep-nitroanilide substrates Ala-Pro Arg-Pro and Gly-Pro witha neutral pH optimum and its tissue expression isubiquitous Unlike DPP IV and FAPα DPP8 is a solublemonomeric protein localised in the cytoplasm [26]

So the subfamily S9B contains next to the 4 peptidases(DPP IV FAPα DPP8 and DPP9) also two enzymes withno peptidase activity (the dipeptidylpeptidase homologuesDPP6 and DPP10) Phylogenetic analysis suggests thatDPP IV is clustered with FAPα DPP8 with DPP9 andDPP6 with DPP10 It is assumed that DPP8 and DPP9 arediverged from the others earlier in evolution DPP8 andDPP9 seem to be more closely related to ancient DPP IVenzymes found in bacteria nematodes and arthropods [28]After bioinformatic search of the human genome it isbelieved that all members of this subfamily have now beenidentified [927]

Dipeptidyl peptidase 9 (DPP9) shares 60 amino acididentity and 77 amino acid similarity with DPP8 DPP9is also ubiquitously expressed and was found in a solubleputative cytosolic form DPP9 is predicted to have DPP IV-like enzymatic activity because of the presence of theGWSYG serine protease motif and a Ser Asp His catalytictriad [27] This was later confirmed by the hydrolysis of afluorogenic Ala-Pro substrate [28]

Dipeptidylpeptidase homologue DPP6 (DPP6 DPP X)contains two of the catalytic residues (Asp His) but the

The Therapeutic Potential of Inhibitors of Dipeptidyl Peptidase IV Current Medicinal Chemistry 2005 Vol 12 No 8 973

Table 1 Peptidases of Clan SC Found in Humansa

Family Subfamily Members Specificity

S9 S9A Prolyl oligopeptidase (POP EC 342126) -Xaa-ProYaa-

Mername AA0072

S9B Dipeptidyl peptidase IV (DPP IV EC 34145) H-Xaa-ProYaa-H-Xaa-AlaYaa-

Fibroblast activation protein α subunit (FAP α) H-Xaa-ProYaa-

Dipeptidyl peptidase 8 (DPP8)


Dipeptidylpeptidase homologue DPP6 (DPP6) No peptidase activity

Dipeptidylpeptidase homologue DPP10 (DPP10)

S9C Acylaminoacyl peptidase (AAP EC 34191) Ac-XaaYaa-

S9X Several putative peptidases

S10 Serine carboxypeptidase A -XaaYaa-OHwith Yaa = hydrophobic or positively charged

Vitellogenic carboxypeptidase-like protein

Serine carboxypeptidase 1

S28 Dipeptidyl peptidase II (DPP II EC 34142)Quiescent cell proline dipeptidase (QPP)

H-Xaa-ProYaa-H-Xaa-AlaYaa-

Lysosomal Pro-X carboxypeptidase(PCP EC 34162)

-Xaa-ProYaa-OHwith Yaa = hydrophobic

Thymus-specific serine peptidase

S33 Epoxide hydrolase No peptidase activity

MEST gp No peptidase activity

CGI-58 putative peptidaseaMerops-The Protease Database httpmeropssangeracuk (searched on October 23 2003)

Although belonging to another family (family S28)dipeptidyl peptidase II (DPP II EC 34142) shows DPP

IV-like peptidase activity [32] In contrast to DPP IV DPP8and DPP9 DPP II has an optimum at acidic pH DPP II

Table 2 Other Proline Specific Peptidasesa

Clan Family Subfamily Members Specificity Homo sapiens

MG M24 M24B X-Pro dipeptidase H-XaaPro-OH Yes

Aminopeptidase P2(EC 34119)

H-XaaPro-Yaa- Yes

Mername AA0019 peptidase H-XaaPro-Pro- No

SC S9 S9B Prolyl tripeptidylpeptidase No

Dipeptidyl aminopeptidase A H-Xaa-ProYaa- No

Dipeptidyl aminopeptidase B

S15 X-Pro dipeptidylpeptidase(EC 341411)

H-Xaa-ProYaa- No

S33 Prolyl aminopeptidase(EC 34115)

H-ProXaa- No

None Membrane Pro-X carboxypeptidase (EC 341716) -Xaa-ProYaa-OH NoaMerops-The Protease Database httpmeropssangeracuk (searched on October 23 2003)


was first identified by McDonald et al [33] More recentlyit is suggested to be identical to human quiescent cellproline dipeptidase (QPP) based on the significant sequencehomology (794) found between human QPP and rat DPPII [34] Human QPP is a 58-kDa glycoprotein functionallyactive as a homodimer formed with a leucine zipper motif Itis targeted to intracellular vesicles that are distinct fromlysozymes and is widely found in the human body It hasbeen shown that QPP inhibitors cause apoptosis in quiescentlymphocytes but not in activated or transformedlymphocytes This process is believed to be independent ofDPP IV because both DPP IV(+) and DPP IV(-) T cellsundergo apoptosis [35-39] No sequence homology has beenfound between DPP IIQPP and DPP IV

a short N-terminal cytoplasmic tail of six amino acids Asoluble form of DPP IV lacking the intracellular part and thetransmembrane region is found in human serum (starting atamino acid 39) and in human seminal plasma (startingaround amino acid 30) [44] Each subunit consists of twodomains an αβ-hydrolase domain and an eight-bladed β-propeller domain The catalytic site is located in a largecavity formed between both domains with each domainparticipating in substrate and inhibitor binding (Fig 2B)The catalytic site is accessible via two openings a large sideopening and a funnel-shaped tunnel through the β-propeller(Fig 2B and 2C) It has been proposed that substrates enterthrough the β-propeller tunnel and products leave the activesite through the side opening [45] but this is still a matterof debate [43]Another enzyme with DPP IV-like activity is dipeptidyl

peptidase IV-β (DPP IV-β) [40] To date this enzyme hasnot been assigned to a family It hydrolyses the samesubstrates as DPP IV but with different kinetics anddifferent susceptibility to inhibitors

Another crystal structure of DPP IV purified fromporcine kidney reveals a tetrameric assembly (Fig 2D) [45]This structure is reported both with a pyrrolidine-2-nitrileinhibitor and without inhibitor Yet other crystal structuresof human DPP IV are reported without inhibitor and incomplex with Ile-Pro-Ile (13) [46] Generally all crystalsreveal an identical overall structure

It is clear that the development of potent and specificinhibitors for each of these enzymes is of utmost importanceto unravel their physiological function and for the validationof their potential as therapeutic target Where possible theselectivity of DPP IV inhibitors with respect to thementioned enzymes will be indicated

In contrast to DPP IV POP is a monomerSuperposition of the POP monomer on the DPP IV dimershows that the monomeric POP in itself fills part of thedimeric structure of DPP IV [43] There is significantstructural homology between the αβ-hydrolase fold of POPand DPP IV [46] Like DPP IV POP has a β-propeller with7 blades instead of 8 POP has a β-propeller tunnel but noside opening which indicates that POP substrates have toenter through the tunnel [41] This probably explains whyPOP is reported to hydrolyse substrates with a maximumsize of about 30 residues only whereas DPP IV canhydrolyse substrates up to about 80 residues long [46]

3 STRUCTURE OF DPP IV

Since the end of 2002 several three-dimensionalstructures of DPP IV have been described and deposited inthe Protein Data Bank (Table 3) Knowledge of thesestructures contributes largely to the understanding of boththe enzymatic and non-enzymatic functions of DPP IV TheDPP IV structures can be compared with prolyloligopeptidase (POP) the first reported crystal structureamong the members of the S9 family [4142] 32 The Active Site

31 The Overall Structure The active site is formed by residues of both the αβ-hydrolase domain and the β-propeller domain Its structurereveals how substrate specificity and inhibitor binding isachieved A crystal structure of human DPP IV in complexwith Ile-Pro-Ile (13 diprotin A) nicely illustrates theinteractions in the active site (Fig 3A) [46] Ile-Pro-Ile is asubstrate with a low turnover rate leading to an apparentcompetitive inhibition [47] This tripeptide is covalentlybound to Ser-630 and trapped as a tetrahedral intermediate ofthe hydrolysis reaction

The first report by Rasmussen et al describes a 25 Aringstructure of the extracellular region (amino acids 39-766) ofhuman DPP IV in complex with the inhibitor Val-pyrrolidide (3) [43] This structure is a homodimer in thecrystal in agreement with previous biochemical datareporting that the active enzyme is a dimer (Fig 2A) TheN-terminus of each subunit is located at the same site of thedimer and is extended in the membrane-bound form with ahydrophobic transmembrane segment (amino acids 7-28) and

Table 3 Structures of DPP IV in the Protein Data Bank

PDB code Source Inhibitor Resolution (Aring) Reference

1N1M Homo sapiens Val-pyrrolidide (3) 250 [43]

1NU6 Homo sapiens None 210 [46]

1NU8 Homo sapiens Ile-Pro-Ile (13) 250 [46]

1ORV Sus scrofa None 180 [45]

10RW Sus scrofa p-Iodo-Phe-Pyrr-CN 284 [45]

1PFQ Homo sapiens None 190 [276]


Fig (2) Three-dimensional representations of dipeptidyl peptidase IV

Figure 2A Human dipeptidyl peptidase IV (DPP IV) in complex with Val-pyrrolidide (PDB code 1N1M) The schematicrepresentations of the N-terminus (residues 1-38) and the cell membrane have been added manually DPP IV forms a homodimer witheach subunit consisting of two domains an αβ-hydrolase domain and a β-propeller domain Subunit B of the homodimer is shownin green The αβ-hydrolase domain consists of amino acids 39-51 and 506-766 and is shown in red in subunit A The β-propellerdomain consists of amino acids 55-497 and is shown in blue in subunit A The connecting amino acids (52-54 and 498-505) areshown in grey in subunit A The complexed inhibitor Val-pyrrolidide is shown in yellow in a space filling model

Figure 2B This figure shows a Gauss-Connolly molecular surface of the homodimer of DPP IV (PDB code 1N1M) The orientation andcolors are the same as in figure 2A The sugar residues are shown in pink The large side opening formed between the αβ-hydrolasedomain and the β-propeller domain is clearly visible This cavity contains the catalytic site in which the Val-pyrrolidide inhibitor(yellow) is bound

Figure 2C Is the same representation as in figure 2A but viewed from the bottom One can see the tunnel in the β-propeller betweenthe bottom of the monomer and the active site containing the inhibitor (shown in yellow)

Figure 2D This figure shows the tetramer formed with two homodimers (PDB code 1ORV) The orientation of the upper dimer (shownin red and green) is the same as in Figure 2A

Figure 2E Molecular surface around the inhibitor Val-pyrrolidide (PDB code 1N1M) The molecular surface is shown in grey theinhibitor in stick representation with carbons green nitrogens blue and oxygen red The S1 pocket optimally suited to fit a 5-membered pyrrolidine is clearly visible The hydrophobic S1 pocket is formed by the side chains of Tyr-666 Tyr-662 Val-711 Val-656 Trp-659 and Tyr-631 The P2 nitrogen and the P2 carbonyl oxygen are in contact with the enzyme The P2 side chain is pointingtowards the solvent explaining the large diversity of P2 amino acids that are allowed


O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

O

N

O O

N O

O

O

O

Tyr54 7

N

NHis7 40

Asp708

3127

28

27

28

2526

26

28

32

33

31

27

31

Glu206

Glu2 05

Tyr631

Ser6 30

O O

O

N

N O

O

O

N

NHis7 40

Asp708

O

Tyr54 7

N O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

27

26

28

2830

27

30

37

28

Glu206

Glu2 05

Tyr631

Ser6 30

O

Tyr54 7

O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

N O

O

O

N

NHis7 40

Asp708

N

I3225

26

30

33

2732

25

30

31

28

Glu206

Glu2 05

Tyr631

Ser6 30

Arg643

N

N

N

O O

N

ON

O

N

N

O

R

N O

O

O

Tyr473

N

NHis680

Asp64 1

ON

3027

2927

38

28

Asn555

Ala55 4

A B

C

D

Fig (3) Schematic representation of the active site of DPP IV and POP

Amino acids forming the S1ndashpocket that fits the pyrrolidine of proline are not shown to improve clarity For the same reasonhydrogen atoms are omitted Dotted lines indicate possible hydrogen bonds or salt bridges Numbers indicate the distances in Aring Fig3A shows Ile-Pro-Ile bound in the active site of DPP IV with serine covalently bound to the inhibitor forming a hemi-acetal (PDBcode 1NU8) Fig 3B shows Val-pyrrolidide bound in the active site of DPP IV (PDB code 1N1M) Fig 3C shows p-iodo-Phe-Pyrr-CNbound in the active site of DPP IV with serine covalently bound to the nitrile forming an imidate adduct (PDB code 1ORW) Fig 3Dshows the P2-P1rsquo part of an octapeptide substrate in the active site of a mutant POP (S554A) (PDB code 1E8N)


The catalytic triad (Ser-630 Asp-708 and His-740) islocated in the large cavity at the interface of the twodomains Ser-630 is found at the tip of a very sharp turncalled the nucleophile elbow which is characteristic ofhydrolases of the αβ type In a crystal without inhibitor theserine hydroxyl is well exposed to the solvent and hydrogenbonded to the catalytic imidazole of His-740 on one side andaccessible to the substrate on the other side The othernitrogen atom of the imidazole of His-740 is hydrogenbonded to one of the oxygens of the side chain of Asp-708

As much of the catalytic power of serine proteasesderives from its preferential binding of the transition statethe tetrahedral intermediate is a well-defined state butnormally with a short lifetime Inspection of the active sitereveals several structural features that could explain theaccumulation of this intermediate of Ile-Pro-Ile during thehydrolysis reaction First the two hydrophobic side chainsof isoleucine are in proximity and point in the samedirection This hydrophobic interaction may stabilise thetripeptide in an unsuitable conformation for the progress ofthe reaction Second a large network of salt bridges andhydrogen bonds stabilise the complex (Fig 3A) It involvesthe carboxylates of Glu-205 and Glu-206 that interact withNH3

+ of the tripeptide Glu-205 makes another salt bridge toArg-125 and this in turn interacts with the C-terminalcarboxylate of the tripeptide It is obvious that this lastinteraction is only present in tripeptidic substrates and thatthis may stabilise the tetrahedral intermediate by protectionof the leaving group

The S1-site is a hydrophobic pocket in the large cavity Itis formed by the side chains of Tyr-666 Tyr-662 Val-711Val-656 Trp-659 and Tyr-631 It is optimally suited toaccommodate the pyrrolidine ring of proline (Fig 2E) andexplains the proline-specificity of DPP IV A majorcontribution to binding of the pyrrolidine is achieved byring stacking to Tyr-662 and Tyr-666 Stacking to Tyr-662is in a parallel fashion to Tyr-666 in an orthogonal fashionOther small uncharged amino acids such as glycine alanineand serine can also fit in the S1-pocket although lessoptimally

Figure 3B shows Val-pyrrolidide (3) bound in the activesite of human DPP IV [43] It shows essentially the sameinteractions as observed for the P2 and P1 residues of Ile-Pro-Ile The complex with a pyrrolidine-2-nitrile inhibitorshows some new features (Fig 3C) [46] The hydroxyl ofSer-630 forms a covalent bond with the nitrile resulting inan imidate The imino nitrogen interacts with the oxyanionhole These interactions explain the high potency of thepyrrolidine-2-nitrile inhibitors

Furthermore the P1 proline is bound in the trans-conformation as was predicted earlier [48] The P1 carbonylis in Ile-Pro-Ile covalently bound to Ser-630 The negativelycharged oxygen of the tetrahedral intermediate is stabilisedin the oxyanion hole This is formed by the main chain NHof Tyr-631 and by the hydroxyl of Tyr-547

The P2 side chain points into the solvent and nointeraction with the protein occurs This explains the largediversity of P2 amino acids that are allowed in DPP IVsubstrates Essential for substrate binding and catalysis isthe N-terminus of the substrates which has to beunprotected and protonated [49] The diprotin A complexshows a strong interaction between the terminal NH3

+ groupand the carboxylates of Glu-205 and Glu-206 Theimportance of both glutamate residues was predicted bysingle point mutations that abolish DPP IV activity [50]This interaction explains why DPP IV is a dipeptidylaminopeptidase This Glu-Glu motif is conserved among themembers of subfamily S9B (DPP IV FAPα DPP8 DPP9DPP6 DPP10) [51] The P2 carbonyl oxygen atom isstabilised by the side chain of Arg-125 and during thehydrolysis reaction probably also by the nitrogen atom ofthe side chain of Asn-710

We superposed the active sites of the complexes of DPPIV with Ile-Pro-Ile (Fig 3A) PDB code 1NU8 [46]) and amutant POP (Ser-554-Ala) with an octapeptide substrate(Fig 3D) PDB code 1E8N [42]) Superposition of the Cαatoms of Ser-630 Asp-708 His-740 Tyr-547 and Tyr-631of DPP IV with respectively the Cα atoms of Ala-554 Asp-641 His-680 Tyr-473 and Asn-555 of POP gives a RMSdeviation of only 024 Aring2 (own unpublished results) Theatoms involved in the oxyanion hole of POP (OH of Tyr-473 and main chain NH of Asn-555) give a perfect fit withthe oxyanion hole of DPP IV The residues of the catalytictriad differ slightly probably because of the absence of thehydroxyl of the active serine in the mutated POP Thisindicates a well conserved catalytic triad and oxyanion holeSimilar to DPP IV a hydrogen bond is formed within thesubstrate between the NH group of P1rsquo and the P2 carbonyloxygen Orientation of the P2 and P1 residues in the activesites of DPP IV and POP is very similar Orientation of theP1rsquo residue reflects the difference between the trigonalground state and the tetrahedral intermediate A majorcontribution of binding the pyrrolidine ring of Pro isachieved by ring stacking to Trp-595 which is equivalent toTyr-662 in DPP IV

Like the P2 side chain the P1rsquo side chain points into thesolvent The NH group of the P1rsquo Ile is in hydrogen bondingdistance to the catalytic imidazole of His-740 This indicatesthat His-740 is protonated by accepting the proton of Ser-630 with the formation of a tetrahedral intermediatefollowed by transfer of the proton from the protonated His-740 to the nitrogen of the P1

rsquo leaving group resulting in theformation of an acylated enzyme The C-terminal carboxylateof the tripeptide makes a salt bridge to Arg-125

Clear differences could be seen in the other interactingresidues The residue of POP equivalent to Asn-710 in DPPIV is Arg-643 The guanidine group of this arginine issituated close to the guanidine of Arg-125 in DPP IV LikeArg-125 and Asn-710 Arg-643 is hydrogen bonded to theP2 carbonyl oxygen The P3 and P4 residues of theoctapeptide substrate in POP coincide with the Glu-205-Glu-206 motif in DPP IV indicating the difference between anendopeptidase and an exopeptidase

Although not reported by Thoma et al [46] we observeda hydrogen bond formed within the substrate between theNH group of P1rsquo and the P2 carbonyl oxygen Thisintramolecular hydrogen bond was also observed in POP(Fig 3D) and might be of catalytic importance [42] Thiswill be discussed further in chapter 5


33 Other Interaction Sites constant for X-Pro-4-nitroanilides and X-Ala-4-nitroanilidesdiffer by a factor 10 to 100 However this discriminationbetween Pro and Ala at P1 is generally much greater fordipeptide chromogenic and fluorogenic substrates than fornatural substrates (see chapter 82) The rate-limiting step forthe hydrolysis of substrates with Pro in P1 is thedeacylation and for Ala in P1 is the acylation reaction [49]

A crystal structure of DPP IV purified from porcinekidney shows the formation of a homotetramer (Fig 2D)[45] This tetramer consists of a dimer of dimers Thecontact area of the dimer is hydrophobic and large (2270 Aring2)and involves residues of the αβ-hydrolase fold Thetetramer interface is more hydrophilic and smaller andinvolves residues of the β-propeller blades 4 and 5 All theglycosylation sites but one are located on the β-propellerdomain (Figs 2B and 2C) One of these glycosylation sites(Asn-281) is involved in tetramerization and glycosylationof this residue might be a regulatory element

In an extended investigation of the substrate specificityusing Ala-X-4-nitroanilides it was concluded that theenzyme can hydrolyse some substrates in which the ringstructure of proline in position P1 has been modified into acyclic imino acid as azetidine-2-carboxylic acid andpiperidine-2-carboxylic acid or an oxa- or thia analogue ofproline The replacement of the proline residue by (S)-azetidine-2-carboxylic acid gives the best substrate accordingto its kcatKm value This substrate has a low affinity that iscounteracted by a high catalytic turnover On the contrarythe substrate with piperidine-2-carboxylic acid has higheraffinity but the lowest kcat value resulting in poor substrateproperties Substituting the methylene group by sulphur oroxygen at position 4 of the pyrrolidine ring slightly affectsthe kcatKm value The affinity however is slightlydecreased for the oxa analogue and increased for the thiaanalogue [58]

It is suggested that tetramerization is a key mechanism toregulate the interaction of DPP IV with other components[45] Tetramerization on the cell surface involves either amembrane-bound dimer and a soluble dimer or dimersbound to membranes of two different cells In the latter caseDPP IV functions as a cell-cell communication moleculeThis is indeed observed when applying soluble DPP IV in acell adhesion model [5253] Alternatively soluble dimerscan assemble to form a homotetramer Furthermore it isreported that DPP IV can form a heterodimer with FAPα(seprase) [54]

DPP IV binds adenosine deaminase (ADA) anassociation that occurs on the cell surface and that preservesthe enzymatic activities of both molecules [55] By usingsite-directed mutagenesis Leu-294 and Val-341 wereidentified as two ADA binding sites [56] These residues arelocated on the β-propeller domain 15 Aring apart from anotherand are involved in tetramerization Therefore ADA bindingwill interfere with tetramerization A recent publicationconfirms that ADA binds at the outer edges of the β-propeller with the binding site stretched across the β-propeller blades 4 and 5 (amino acids 282-295 and 322-350)[57] Also the glycosylation of Asn-281 might influenceADA binding This suggests that tetramerization and properglycosylation of Asn-281 serve as major control mechanismsfor ADA binding [4557]

Studies with short synthetic peptides show that the Prsquo1position accepts all amino acids except secondary aminessuch as N-methylated amino acids proline andhydroxyproline [59]

Any L-amino acid can be placed at the P2 positionprovided that the N-terminal amino function is free andprotonated In general the nature of the side chain of the P2amino acid has only limited influence on binding buthydrophobic aliphatic residues are favoured Substitution ofthe hydroxyl group of Ser or Thr at P2 with phosphateprevents hydrolysis of the substrate [60] On the other handeven bulky structural modifications of the P2 side chain areallowed [60]

With proline as the P1 amino acid the P2 amino acidneeds an L-configuration with alanine at P1 it also acceptsD-amino acids at the N-terminal position [49] Theconformation around the peptide bond between P2 and P1has to be trans for catalytic activity [48] DPP IV can utilisesubstrates with a thioamide peptide bond between P2 andP1 but with a 100-1000 fold reduced selectivity constant[61]

Knowledge of the three-dimensional structure of DPP IVis important for designing new inhibitors for understandingthe structure-activity relationship of known inhibitors andthe structural basis of substrate specificity The substratespecificity and the properties of inhibitors will be discussedin the next chapters

4 SUBSTRATE SPECIFICITYWith the identification of several proline-specific

dipeptidyl aminopeptidases the investigation of thesubstrate specificity was extended to these other enzymesUsing a positional scanning synthetic combinatorialdipeptide substrate library it was shown that both DPP IVand DPPII strongly prefer proline at P1 but that the nextmost preferred residue for DPP II is norleucine (Nle)whereas for DPP IV this is alanine For the P2 subsite DPPII has a preference for Lys Nle Met Ala Ser and Argwhereas DPP IV can tolerate a variety of residues at thisposition [32] Using 4-nitroanilides of Gly-Pro Ala-Pro andArg-Pro there was only a very small difference in hydrolysisbetween DPP IV and DPP8 [26] However comparably littleis known on the substrate specificity of these DPP IV-relatedenzymes

The P2 and P1 substrate specificity of DPP IV is usuallydetermined with dipeptide chromogenic andor fluorogenicsubstrates The specificity of the Prsquo sites can be obtainedfrom synthetic peptides and natural substrates One has totake into account that the information obtained can largelydepend on the substrate Steady-state kinetic analysis ofsubstrates classically produces three parameters the Km (M)or the Michaelis-Menten constant kcat (s-1) or the catalyticrate constant kcatKm (M-1s-1) or the selectivity constant

Besides proline at the penultimate position (P1 position)DPP IV can accommodate although less efficiently alaninedehydroproline and hydroxyproline [49] The selectivity


O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C

Fig (4) Catalytic mechanism of DPP IV

This figure shows the potential catalytic mechanism of DPP IV Fig 4A shows the substrate bound in the active site in the groundstate Fig 4B shows the tetrahedral intermediate formed after the attack of the hydroxyl of Ser-630 on the amide bond Fig 4C showsthe acylated enzyme formed after the decomposition of the tetrahedral intermediate The acylated enzyme is subsequently hydrolysedby water via a second tetrahedral intermediate to the carboxylic acid and the active enzyme


5 CATALYTIC MECHANISM OF DPP IV amino acids such as proline and N-methyl amino acids at theP1rsquo position are not hydrolysed However oligopeptideswith a X-Pro-Pro motif are inhibitors [6566] This indicatesthat these peptides are bound in the ground state but do notproceed to the acylated enzyme This might be explained bythe absence of a NH at P1rsquo with the impossibility of theformation of a hydrogen bond with the P2 carbonyl oxygenFurthermore replacement of the P2 carbonyl oxygen withsulphur strongly reduces catalytic efficiency [61]

The catalytic mechanism of serine peptidases and morespecifically of POP has been thoroughly investigated[4162426364] In view of the similar binding ofsubstrates to a comparable active site around the cleavableamide bond we propose a similar mechanism for DPP IV(Fig 4) The published crystal structures of severalsubstrates and inhibitors are good tools to study the catalyticmechanism Binding of Val-pyrrolidide (3) to DPP IV (Fig3B) and of an octapeptide to a catalytically inactive POP(Fig 3D) represent the ground state of the reaction Bindingof Ile-Pro-Ile to DPP IV (Fig 3A) shows the first tetrahedralintermediate of the reaction The imidate complex of DPPIV with a nitrile inhibitor (Fig 3C) probably represents theacylated enzyme

It has been proposed that the catalytic mechanism forDPP IV involves a cis-trans isomerization of the substrate[6745] This is however not supported by the structure ofthe tetrahedral intermediate of Ile-Pro-Ile bound to DPP IVthat has a trans P2-P1 amide bond

6 DISTRIBUTIONIn serine peptidases the catalytic triad (Ser His Asp)serves as a charge stabilising system in which the aspartatestabilises the protonated histidine that accepted a protonfrom serine In the ground state of the reaction (Fig 4A) thesubstrate is held in the correct conformation by interaction ofthe terminal NH3

+ with the double Glu motif by interactionof the P2 carbonyl oxygen with Arg-125 and Asn-710 byinteraction of the pyrrolidine ring with the S1 pocket and byinteraction of the P1 carbonyl with the oxyanion holeBecause of the very similar structure of the active site ofDPP IV in three different states (Fig 3A-C) and because ofthe well conserved structure of the catalytic triad and theoxyanion hole compared to POP (Fig 3D) we conclude thatthe active site is relatively rigid during the reaction but thatthe substrate moves while hydrolysed The P2 and P1residues are bound very similar in the ground state and thetetrahedral intermediate This indicates that binding of theground state to the enzyme favours the formation of thetetrahedral intermediate [42]

The tissue distribution of DPP IV and its expressionunder normal or pathological conditions has been recentlyreviewed [68-71]

DPP IV has a widespread organ distribution and itsexpression level differs greatly between tissues The finelocalisation of endothelial en epithelial DPP IV was foundto be on the apical membrane [7273] It is found in thekidney liver intestine pancreas spleen and placenta It isexpressed in high density in membrane regions organised asmicrovilli such as the brush-border regions of the smallintestine and the proximal tubuli in the kidney Comparisonof DPP IV activity in human intestine revealed that theactivity was highest in ileum and jejunum low induodenum but not detectable in colon [74] Howeversometimes it is aberrantly expressed in colon tumours [75]

DPP IV has been reported in body fluids such as humanserumplasma and seminal fluid The activity in seminalfluid is high This activity originates from the prostate glandand is accounted for by soluble DPP IV (sDPP IV 20 to30) and prostasomal membrane-bound DPP IV [76-78]Moderate amounts of sDPP IV are detected in serumplasma[794480]

In the ground state of the reaction the Nε2 of His-740accepts a proton from Ser-630 (general base catalysis) with asimultaneous attack of the serine on the amide bond and theformation of a negatively charged tetrahedral intermediate(Fig 4B) The negative charge is stabilised in the oxyanionhole Decomposition of the tetrahedral intermediate requiresthe transfer of a proton from the protonated catalytichistidine to the NH of the leaving group (P1rsquo) We believethat like in POP this general acid catalysis is promoted in asubstrate assisted manner by the formation of a stronghydrogen bond between the P1rsquo NH and the P2 carbonyloxygen [42] This hydrogen bond is observed in thecomplex of Ile-Pro-Ile with DPP IV In this complex thedistance between the P2 carbonyl oxygen and the side chainnitrogen of Asn-710 is at the upper limit for a hydrogenbond So probably during the formation of the tetrahedralintermediate this hydrogen bond is replaced by the one withthe P1rsquo nitrogen

In the hematopoietic system DPP IV is identical to theleukocyte antigen CD26 and is often referred to as DPPIVCD26 Originally described as a T cell activationmolecule surface expressed DPP IVCD26 is now regardedas a general marker of cellular activation in the immunesystem (reviewed in [81]) After T cell stimulation theexpression levels of DPP IVCD26 as well as the percentageof CD26+ T cells increase DPP IVCD26 is absent onresting B and natural killer cells but is induced on their cellsurface after stimulation [82-84]

In resting peripheral blood mononuclear cells a smallsubpopulation of T cells expresses CD26 at high density onthe surface (CD26bright T cells) [85] This CD26bright

population belongs to the CD45R0+ T cells and is necessaryfor the proliferative response to recall antigens andalloantigen induction of cytotoxic T lymphocyte activityagainst alloantigen and induction of B cells to synthesiseIgG Furthermore the CD26bright cells have the capacity oftransendothelial migration After antigenic stimulationCD4+ cells can differentiate in two types of effector cells Thelper 1 (Th1) and T helper 2 (Th2) cells Th1 cells display

Decomposition of the tetrahedral intermediate results inthe formation of an acylated enzyme (Fig 4C) The acylatedenzyme is subsequently hydrolysed by water via a secondtetrahedral intermediate to the carboxylic acid and the activeenzyme

Further arguments for the catalytic importance of theintramolecular hydrogen bond between P2-C=O and P1rsquo-NHcome from substrate specificity Substrates with cyclic


a higher expression of DPP IVCD26 than Th2 cellsAlthough probably not all features of CD26bright cells arefunctionally related to the expression of CD26 a number ofimmune functions can be influenced by targeting CD26 [81]

Thiazolidides were reported as the first potent and stableDPP IV inhibitors [899091] The most potent compoundof this series was Ile-thiazolidide (2) Also Val-pyrrolidide(3) and the analogous N-(ε-(4-nitrobenzyloxycarbonyl)lysyl)compounds 4a and 4b are of similar potency The Ile-thiazolidide (2) is currently being developed by Probiodrugunder the name P3298 Unfortunately these pyrrolididesand thiazolidides at P1 combined with natural amino acids atP2 have a very low selectivity with respect to DPP II Ile-thiazolidide (2) and Val-pyrrolidide (3) are the most selectiveinhibitors in this series but their selectivity is only 20 to 50fold [9293]

Aberrant (increased or decreased) DPP IV levels havebeen reported as clinically relevant for various diseases thatcan be subdivided into four distinct categories (1) solidtumours (2) haematological malignancies (3) (auto)immunediseases inflammation and psychoneuroendocrine disordersand (4) infectious diseases such as AIDS and Hepatitis C(reviewed in[6970])[71]

7 DPP IV INHIBITORSThe structure-activity relationship of the P2 and P1

residue in this class of inhibitors has been thoroughlyinvestigated In a search for an optimal P1 residue it wasshown that changing the 5-membered ring of pyrrolidine orthiazolidine to their 4- 6- or 7-membered analogues or totheir unsaturated analogues decreases potency [94] The sameis true for the acyclic analogues Introduction of asubstituent at the 3-position of the pyrrolidine ring generallydecreased the inhibitory activity for DPP IV Only a smallsubstituent such as fluorine is allowed (5) [94]Nevertheless this investigation yielded a selective andpotent DPP II inhibitor with piperidine at P2 combined with(S)-24-diaminobutyric acid at P1 (6a IC50 DPP II = 013microM IC50 DPP IV gt 1000 microM) [9293] Also somesubstituents on the P1 ring were allowed for DPP IIinhibitors Considering these results it is assumed that theS1 site of DPP II is somewhat larger than the one of DPP IV[93]

DPP IV is inhibited by the typical serine protease markerdiisopropyl fluorophosphate [86] A wide variety ofinhibitors for serine proteases have been reported and mostof them are peptide analogues that have an electrophilicgroup at the P1 amino acid that replaces the normallycleaved amide bond However in case of DPP IV thiselectrophile is responsible for stability problems of theinhibitor After conversion to the cis amide bond acyclization reaction can occur between the free amino groupof the P2 amino acid and the electrophile attached to theproline mimic in P1 as illustrated for a prolyl boronic acidpeptide (Fig 5) This is not surprising since it is wellknown that peptides with proline at the penultimate positionform relatively easy diketopiperazines arising from thenucleophilic attack of the N-terminal nitrogen on the amidecarbonyl between the second and the third amino acidTherefore only a few classes of the classical serine proteaseinhibitors are effective for DPP IV inhibition The differentclasses of inhibitors will be discussed in this chapter For amore complete description of inhibitors reported until 1998the reader is referred to previously published reviews [187]Where available selectivity to other proline-specificpeptidases will be described

In a few examples the P2 carbonyl oxygen was replacedwith sulphur resulting in thioamide analogues of the abovementioned aminoacyl pyrrolidides and thiazolidides [95]However thioxylation led to a slight decrease in inhibitorypotency DPP IV is only inhibited by the Z isomer of thethioamide bond Ile-ψ[CS-N]-Thia (7) has a K i = 0203 microMon DPP IV from pig kidney compared to Ki = 0126 microMfor Ile-Thia (2) In the same paper the authors describe thatthioxylation increases the inhibitory potency towards DPPII thereby reducing the selectivity of these compounds asDPP IV inhibitors However this conclusion may not begeneralised [93]

NHN

OR

O

NH2N

B

OR

HOOH

A BTo establish an optimal P2 side chain a series of

aminoacyl pyrrolidides were prepared showing thatlipophilic amino acids gave more potent compounds Inparticular β-branched α-amino acid derivatives were themost potent compounds with the non-proteinogenic aminoacid cyclohexylglycine providing the most activepyrrolidide (8) [96] Compound 8 is only 60 fold selectivewith respect to DPP II (IC50 DPP IV = 032 microM IC50 DPPII = 19 microM) [97] However very recently this scaffold waselaborated further at Merck and led to the development ofsome very potent and selective DPP IV inhibitors [97]Selectivity profiles of this series of inhibitors were assessedagainst POP aminopeptidase P2 X-Pro dipeptidase DPP IIand FAPα Selectivity problems were only noticed withDPP II Unfortunately DPP8 and DPP9 were not evaluatedIntroduction of an amino moiety at the 4-position of thecyclohexyl ring as a trans isomer was not well tolerated (9)This agrees with our conclusion that substitution or removalof an amino in the side chain of P2 increases DPP IV

Fig (5) Instability of DPP IV inhibitors having an elecrophile

Fig 5A shows a diketopiperazine formed by cyclisation of apeptide having a penultimate proline Fig 5B is a cyclised andhence inactivated boronic acid DPP IV inhibitor

71 Peptide-Derived Reversible Inhibitors

711 Competitive Inhibitors (Fig 6)

These inhibitors are usually substrate or productanalogues Dipeptides (1) resulting from hydrolysis by theenzyme are possible endogenous DPP IV inhibitorsInhibition constants of these competitive product inhibitorsare in the range of Ki = 10-2000 microM [8889]

Product-like compounds lacking the carbonyl function ofthe proline residue such as Aminoacyl Pyrrolidides and


N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)

R = C(O)OCH2C6H4(4-NO 2)4a X = CH24b X = S

5 6a R = NH26b R = NHC(O)OCH 2C6H56c R = H

7

9

10a X = S R1 = NHCONMe2 R2 = H10b X = CH2 R1 = NHCONMe 2 R2 = H10c X = CH2 R1 = NHSO2CH2CF3 R2 = H10d X = CH2 R1 = F R2 = F

11

12 (Gly-P ro-Ile-Thia)

13

Fig (6) Peptide-derived competitive inhibitors

inhibition and decreases DPP II inhibition compared to theunsubstituted amino (compounds 6b and 6c) [93] Indeedalso in this series attenuation of the basicity of the 4-aminoby acylation provided potent and selective DPP IVinhibitors [97] In the thiazolidide series severalsulfonamide amide carbamate and urea substituents onposition 4 were synthesised with differences in selectivityand potency Compound 10a was the most potentthiazolidide (IC50 DPP IV = 75 nM) with a 267 foldselectivity (IC50 DPP II = 2 microM) The epimeric cisanalogues at position 4 were significantly less potent withno selectivity The pyrrolidide analogues were at least 3-foldless potent than their thiazolidide congeners but exhibitedimproved selectivity over DPP II (compound 10b IC50DPP IV = 26 nM IC50 DPP II = 12 microM) Compound 10crepresents the most potent DPP IV inhibitor reported to date

lacking an electrophile on the pyrrolidine ring with a morethan 5000-fold selectivity (IC50 DPP IV = 26 nM IC50DPP II = 15 microM) Representative analogues were selectedfor evaluation of pharmacokinetic properties in the rat andpossible ion channel activity as a measure of general off-target activity On the basis of these results compound 10d(IC50 DPP IV = 88 nM IC50 DPP II = 88 microM) wasselected for further evaluation It has an excellentpharmacokinetic profile in dog with a moderate clearance 6h half-life and oral bioavailability equal to 100 It was alsoorally efficacious at 3 mgkg in an oral glucose tolerance testin lean mice

Novartis synthesised N-(N-substituted glycyl)thiazolidides [98] These are peptoid like molecules in which theside chain has moved from the α-carbon to the terminalnitrogen The most potent compound from this series is


compound 11 with an IC50 = 57 microM on human plasmaDPP IV However no comparison with reference compoundswas made The same peptoid principle was also applied tothe pyrrolidine-2-nitriles (vide infra)

complexed with a nitrile shows the formation of a covalentbond between the oxygen of the catalytic serine and thenitrile resulting in an imidate (Fig 3c)

The structure-activity relationship for the N-terminalresidue developed in the pyrrolidide series correlated well forthe dipeptide nitriles with the cyclohexylglycine derivative14 as a very potent compound This compound has a Ki =14 nM (compared to Ki = 64 nM for 8) and an excellentchemical stability at pH = 74 (t12 gt 48h) [96] Compound14 has also an excellent selectivity profile (post-prolinecleaving enzyme (PPCE an early name for prolyloligopeptidase (POP)) IC50 = 41 microM DPP II IC50 = 102microM) [106] In this series Ferring is currently testing nitrile15 (FE 999011 Ki = 38 nM) as DPP IV inhibitor for thetreatment of type II diabetes [107108] Enhancement oflipophilicity was achieved by introduction of long alkylchains in the side chain of the P2 amino acid (16 Ki = 05nM) [107] In analogy with the pyrrolidides replacement ofthe pyrrolidine-2-nitrile with thiazolidine-4-nitrile enhancedpotency [109]

Probiodrug is developing prodrugs of their DPP IVinhibitor isoleucylthiazolidide (2 Ile-Thia P3298) Oneexample is Gly-Pro-Ile-Thia (12) [99] This peptidederivative is a substrate for DPP IV that cleaves afterproline and thereby releases the inhibitor Ile-Thia It isdescribed that this prodrug has a 30 increased effect onglucose tolerance in Wistar rats compared to the parentcompound This is not due to an increase in bioavailabilitysince the parent compound is 100 available The authorsclaim that through cleavage of the prodrug by DPP IV DPPIV is inhibited gradually what will ultimately result intermination of the activation of the prodrug The uncleavedprodrug will then act as a depot Due to this effect it wouldbe possible to release the inhibitor in an amount adjusted tothe level of DPP IV in individual patients and to the level ofDPP IV in different tissues and at different time points

Apart from the dipeptides and dipeptide analogues someoligopeptides are also described as inhibitors Diprotin A(Ile-Pro-Ile 13) is a competitive inhibitor with a Ki = 22microM [100] This result is rather surprising since thecompound has the overall substrate-like structure needed forDPP IV hydrolysis A more recent report showed that thosetripeptides are indeed substrates with a low turnover rate(kcat) [47] As shown in the crystal structure (Fig 3a) theformation of a stable tetrahedral intermediate explains thisunexpected result

Novartis applied the above mentioned peptoid principlealso to a large series of pyrrolidine-2-nitriles [110] andthiazolidine-4-nitriles [111] As described the thiazolidine-4-nitriles are more potent A very active pyrrolidine-2-nitrile inthis series is NVP-DPP728 (17) with a Ki = 11 nM onhuman plasma DPP IV [112] The potency of NVP-DPP728is strongly dependent upon the presence and chirality of theP1 nitrile By alteration of the orientation (L to D) of thenitrile-pyrrolidine bond approximately 500-fold loss ofpotency was observed By removal of the nitrile substituent(hydrogen replacement) a 1000-fold loss of potencyresulted Similarly placement of a more bulky amidesubstituent in place of the nitrile resulted in a 30000-foldloss of potency Kinetic experiments have established thatNVP-DPP728 derives its potency through a slow-bindinginhibition mechanism Under the assay conditions (pH =74) the P2 site amine can nucleophilically attack the carbonof the pyrrolidine-nitrile to form an inactive cyclic amidineThis intramolecular cyclisation was slow with a half-life ofapproximately 72 h Also 17 is highly selective for DPP IV(IC50 for human plasma DPP IV = 7 nM) over closelyrelated peptidases post-proline cleaving enzyme (PPCE(POP) IC50 = 190000 nM) and dipeptidyl peptidase II(DPP II IC50 = 110000 nM) In addition the in vitrospecificity was profiled in over 100 receptor and enzymeassays and no significant binding was observed (10 microM)[113] A solid phase procedure for the synthesis of analoguesof 17 has been reported [113114]

The HIV-1 Tat (1-86) protein has been reported as aDPP IV inhibitor suggesting that the immunosuppressiveeffects of Tat on non-HIV-1-infected T cells could bemediated by DPP IV [101102] It was shown that the N-terminal Met-Xaa-Pro sequence of this protein wasimportant and that other proteins with this sequence and atleast 6 amino acids in length are inhibitors [6566] This isrelated to the fact that DPP IV can not hydrolyse peptideswith proline as the third amino acid (P1rsquo position) Morerecently the tromboxane A2 receptor peptide TXA2-R(1-9)having an N-terminal Met-Trp-Pro sequence wascharacterised as an even more potent DPP IV inhibitor (Ki =5 microM) So the thromboxane A2 receptor could be anendogenous DPP IV inhibitor possibly playing a functionalrole during antigen presentation by inhibiting T-cellexpressed DPP IV [103] Some of these peptides arecompetitive inhibitors but other inhibit DPP IV accordingto a parabolic mixed-type mechanism indicating binding oftwo inhibitor molecules to two different binding sites at thesame enzyme molecule [104]

A further study in this direction by Novartis afforded the3-hydroxy-adamant-1-yl derivative 18 (NVP-LAF237)[115106] 18 is slightly more potent than 17 (IC50 forhuman plasma DPP IV = 27 nM and 7 nM respectively) Itis even more specific against related peptidases (PPCE(POP) IC50 = 210 microM IC50 DPP II gt 500 microM) It wasalso profiled in over 100 receptor and enzyme assays and nosignificant binding was observed (10 microM) Because of thesterics imposed by the adamantyl group the ability of 18 tocyclize to an inactive amidine was more than 30-fold reducedcompared to that of 14 and 17 [106]

712 Slow Tight-Binding Inhibitors (Fig 7)

Introduction of a nitrile on the place of the cleavableamide bond leads to a series of pyrrolidine-2-nitriles withon average a 100- to 1000-fold increase in potency comparedto the pyrrolidides [96105] However as explained abovethe electrophilic nitrile can cause stability problems due tocyclisation to an inactive amidine Sometimes the nitrilesare more selective DPP IV inhibitors with respect to DPP IIthan the unsubstituted pyrrolidides because the nitrileenhances potency towards DPP II much less significantlythan towards DPP IV [93] The crystal structure of DPP IV

Ferring synthesised prodrugs of their pyrrolidine-2-nitriles in order to prevent cyclisation and hence increase


N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23

Fig (7) Peptide-derived slow tight-binding inhibitors

chemical stability [116] An example of this approach is 19an acetoxyethoxycarbonyl prodrug of 15 (FE 999011) Theseprodrugs show no significant inhibitory activity on DPP IVup to 10 microM indicating that the prodrugs are at least 1000times less potent than the parent compounds Hence it canbe assumed that any in vivo activity seen is due tobioconversion to the parent inhibitors

Unfortunately the authors did not compare the fluoro-olefin21 with the corresponding Ala-Pyrr-2-CN under their assayconditions A Ki of 02 microM has been reported for the lattercompound [105] what seems to indicate that replacement ofamide with fluoro-olefin decreases potency but to a smallerextent than the olefin Likewise it is stated that the fluoro-olefin 21 is very stable but no comparison is made with theparent amide Fluoro-olefin analogues of N-(substitutedglycyl)-pyrrolidine-2-nitriles were also synthesised [118]

In patent literature several analogues of the pyrrolidine-2-nitrile series are described by different companies buteither no biological data are described or the added value isunclear Hence these compounds are not discussed in thisreview

Xaa-boroPro dipeptides were reported among the mostpotent inhibitors with Ki values in the nanomolar rangeThese compounds are reversible transition state analogueswith slow tight-binding kinetics [119] The empty P-orbitalat boron is thought to interact with the catalytic serine toform a stable ldquoaterdquo complex which mimics the transitionstate of amide hydrolysis Separation of L-Pro-DL-boroProinto its diastereoisomers showed that the L-L isomer (22)has a Ki value of 16 pM [120] Unfortunately they have ashort half-life at neutral pH caused by cyclisation of theterminal aminofunction with the boronic acid forming acyclic inactive species containing a B-N bond (Fig 5b)[121-123] The linear chain (active inhibitor) is favoured atlow pH whereas the inactive cyclic compound is favoured athigh pH However cyclic analogues such as 23 (cyclic Val-boroPro) can also be administered orally as prodrug Theacidic conditions in the stomach are sufficient to regeneratethe linear chain and regain its inhibitory activity [124]

In order to increase the chemical stability of aminoacylpyrrolidine-2-nitriles the amide bond has been replaced withan olefin and a (Z)-fluoro-olefin These olefins mimic thepartial double bond character of the trans amide bond Intheory the fluoro-olefin should be a superiorpeptidomimetic since the electronegative fluorine mimics thecarbonyl oxygen of the amide bond The olefin and (Z)-fluoro-olefin should prevent isomerization to a cis bondnecessary for the cyclisation reaction responsible forinstability

Replacement of the amide bond with an olefin in theisoleucyl analogue (20) decreased potency 1000-fold (Ki =17 microM) [107] The (Z)-fluoro-olefin 21 was isolated as twopairs of diastereomers [117] Unexpectedly both pairs hadsimilar inhibitory potency on DPP IV (Ki = 769 and 603microM) This is in contrast with the absolute requirement of aLL-stereochemistry for dipeptide inhibitors with Pro at P1

In a structure-activity relationship of boronic acidinhibitors it was shown that a wide variety of L-amino acids


N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32

Fig (8) Non-peptidic reversible inhibitors

are accepted at the P2 position but D-amino acids αα-disubstituted amino acids and glycine are not tolerated[125] The specificity of these compounds for DPP IVinhibition was investigated and it was shown that mostcompounds that were active on DPP IV were also potentinhibitors of DPP II On the contrary the IC50 values forPOP inhibition were 30 to 1000 fold higher

pharmacokinetic properties in rats and is active in an oralglucose tolerance test in the Zucker obese rat model AlsoBoehringer Ingelheim Pharma KG developed a series ofxanthines with compound 25 as a representative example(IC50 = 3 nM) [128]

Novel DPP IV inhibitors were discovered from thefermentation broth of Aspergillus orizae A374 TMC-2A(26) inhibited DPP IV in a non-competitive manner (Ki =53 microM) with high selectivity An approach usingcombinatorial synthesis identified TSL-225 (27) as thecritical core structure responsible for the inhibitory activity(Ki = 36 microM) In vivo evaluation revealed that bothcompounds had antiarthritic effects although theirmechanism of action remains to be clarified [129130]

72 Non-Peptidic Reversible Inhibitors (Fig 8)

So far the mentioned inhibitors are structurally derivedfrom the dipeptide obtained after DPP IV cleavage At NovoNordisk a completely novel class of inhibitors wasdiscovered structurally unrelated to any DPP IV inhibitorknown so far This class is exemplified by 7-benzyl-13-dimethyl-8-(1-piperazinyl)xanthine (24) [126] It is claimedthat these compounds are furthermore potent competitiveand stable and thus offering a solution to the instabilityproblems often associated with the previously known DPPIV inhibitors [127] Compound 24 has favourable

Compound 28 was identified at Novartis as most potentDPP IV inhibitor (IC50 = 032 microM) in a series of 1-aminomethylisoquinoline-4-carboxylates [131] Within thisseries a primary amine is required for inhibitory activity andthe 68-dimethoxy substitution is optimal


Research at Takeda revealed an analogous seriesconsisting of aminomethylisoquinolones [132] One of themost potent compounds (29 IC50 = 025 microM) hasfavourable pharmacokinetic properties and administration of29 to Wistar fatty rats leads to reduced glucose and increasedinsulin levels after an oral glucose tolerance test

microM and 30 microM respectively) The other pair ofdiastereomers containing S S and R R was less potent (Ki =144 microM) than the first pair of diastereomers but still morepotent than the parent amide Chemical stability in buffer(pH = 76) of the first pair of diastereomers compared to 33was also enhanced (half-life of 103 h versus 88 h)

Compound 30 is reported as a DPP IV inhibitor (IC50 =578 microM) with selectivity towards aminopeptidase N (IC50gt 309 microM) but with cytotoxic properties (IC50 = 187 microM)The authors use an intact-cell assay and report that undertheir conditions 30 is more potent than Pro-boroPro (22IC50 = 882 microM) [133]

Dipeptide-Derived Diphenyl Phosphonate Esters arepotent irreversible inhibitors of DPP IV [140] probablybecause they lead to a phosphorylated serine at the activesite With these inhibitors only a fraction (10 ) of theenzyme activity was regained after 4 weeks in vitroindicating the strong irreversible inhibition [141] In a studyon the role of the P2 amino acid in dipeptide diphenylphosphonates [141142] it was shown that proline in thisposition gives one of the most potent inhibitors (35) Amajor advantage of 35 was its improved stability in humancitrated plasma (t12 = 5 h) compared to the other dipeptidederivatives Intravenous injection of a single dose of 35 (1 5or 10 mg) in rabbits reduces the plasma DPP IV activitywith more than 80 and it takes more than 20 days forcomplete recovery Not only plasma DPP IV was inhibitedbut also DPP IV in circulating lymphocytes and peripheraltissues [143] The role of the P1 amino acid was alsoinvestigated showing that a 6-membered analogue(homoproline) increased potency [140] whereas alaninedecreased potency [141144] With 35 as lead compound wesynthesised a series of diaryl 1-(S)-prolylpyrrolidine-2-(RS)-phosphonates with different substituents on the aryl rings(hydroxyl methoxy acylamino sulfonylamino ureylmethoxycarbonyl and alkylaminocarbonyl) [145] A goodcorrelation was found between the electronic properties of thesubstituent and the inhibitory activity and stability Thismeans that electron-withdrawing substituents increasepotency but also decrease stability The most strikingdivergence of this correlation was the high potencycombined with a high stability of the 4-acetylaminosubstituted derivative (36a IC50 = 04 microM t12 = 320 min)The most potent compound in this series (36b IC50 = 23nM) has a rather short half-life in plasma (t12 = 35 min)This is however sufficient to completely inhibit DPP IVactivity Compounds 36a and 36b are specific with respect

A new DPP IV inhibitor was isolated from the culturebroth of Streptomyces sp MK251-43F3 Sulphostin (31)inhibited DPP IV in a dose dependent manner with an IC50= 22 nM a 100-fold stronger inhibition than that of diprotinA (13) [134]

Researchers at Eisai found carbamoyltriazoles to bepotent DPP IV inhibitors Compound 32 has an IC50 = 035nM and shows activity in a diabetes mouse model [135]

73 Irreversible Inhibitors (Fig 9)

N-peptidyl-O-(4-nitrobenzoyl)hydroxylamines areenzyme-activated irreversible inhibitors for serine peptidases[136] After the attack of the active site serine the inhibitorforms a latent chemically reactive intermediate which formsa stable covalent bond with a functional group at or near theactive site thus leading to an irreversibly modified enzymeAla-Pro-NHO-(4-NO2)-benzoyl (33) is such an irreversibleinhibitor of DPP IV [137138]

Similar to the (Z)-fluoro-olefin nitrile 21 a (Z)-fluoro-olefin N-peptidyl-O-(4-nitrobenzoyl)hydroxylamine 34 wasisolated as two pairs of diastereomers [139] In this exampleit was proven that introduction of a (Z)-fluoro-olefin as apeptidomimetic of a trans amide bond enhances bothpotency and chemical stability The pair of diastereomerscontaining S R (mimic of an L L dipeptide) and R Sexhibited an inhibitory activity superior to 33 (Ki = 019

NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334

3536a R = NHCOCH336b R = CONHCH2 COOEt

Fig (9) Irreversible inhibitors


to POP DPP II membrane alanyl aminopeptidase andelastase and show low cytotoxicity in human peripheralblood mononuclear cells

821 The Pancreatic Polypeptide Family

DPP IV cleaves neuropeptide Y (NPY) and peptide YY(PYY) but not pancreatic polypeptide all members of thepancreatic polypeptide family [153154155] NPY and PYYare both 36 amino acids long and have a N-terminal Tyr-ProThe peptides belonging to the pancreatic polypeptide familyactivate a heterogeneous population of at least five G-proteincoupled receptors (Y1-Y5)

Parallel synthesis of dipeptide diphenyl phosphonateesters led to some potent DPP II inhibitors with goodselectivity with respect to DPP IV Replacement of theprolyl phosphonate at P1 with Ala or other α-aminoalkylphosphonates completely destroyed inhibitory activitytowards DPP IV [144146] NPY is a good substrate for DPP IV in vitro NPY(3-36)

represents 35 of total NPY in porcine brain and exogenousNPY administration was found to be more potent in DPPIV-deficient rats suggesting that in vivo DPP IV isresponsible for cleavage of NPY [156] NPY is the mostabundant neuropeptide in the central and peripheral nervoussystems In the brain NPY is a powerful inhibitoryneurotransmitter leading to anxiolysis stimulation of foodintake and several other effects In the periphery NPY isfound in the sympathetic nervous system co-stored and co-released with norepinephirine where it leads tovasoconstriction It also stimulates vascular smooth musclecell proliferation and angiogenesis Truncation of NPY byDPP IV changes the receptor specificity because NPY(3-36)is inactive on the Y1 receptor and is more specific for Y2Y3 and Y5 receptors [157] Among other effects the Y1receptor mediates anxiolytic effects of NPY In F344 ratsubstrains with extremely reduced DPP IV activity a reducedbehavioural stress response was found probably related to adifferential degradation of NPY [158] This was tested bythe administration of NPY intracerebroventricularly to theseDPP IV-deficient rats These animals exhibited an increasedresponsiveness to the sedative- and anxiolytic-like effects ofNPY which is very likely mediated via a Y1 receptor-dependent mechanism These authors suggest that DPP IVinhibitors specifically targeted to the central nervous systemcould be useful modifiers of the centrally mediated effects ofNPY [156]

8 PHYSIOLOGICAL FUNCTIONS OF DPP IV

DPP IVCD 26 is a multifunctional protein and itsspecific function will be defined by the site of expressionSome of these functions are related to the exopeptidaseactivity like the processing of bioactive peptides and theinvolvement in the resorption of proline-containing peptidesApart from its catalytic activity in the active site DPP IVcontains several other protein binding sites It binds forinstance adenosine deaminase the HIV gp120 proteinfibronectin collagen the chemokine receptor CXCR4 andthe tyrosine phosphatase CD45 The roles of DPP IV in theimmune system and tumour invasion appear to involve bothenzymatic and nonenzymatic actions In this chapter we willbriefly describe all these functions and the reader will bereferred to excellent recent reviews for more in depthdiscussions

81 Role of DPP IV in the Intestinal and RenalHandling of Proline Containing Peptides

The absorption or recycling of proline containingpeptides is a vital process DPP IVrsquos contribution to thisprocess is twofold due to its brush border localisation in thesmall intestine and the kidney (vide supra) It plays anobligatory role in the breakdown of peptides in the intestinaland renal tubular lumen generating the substrates for thepeptide specific transport systems [147148149]

The L cells of the gastrointestinal tract are the majorsource of PYY which exists in two endogenous forms PYY(1-36) and PYY(3-36) The latter is produced by theaction of DPP IV [155] PYY(1-36) activates Y1 Y2 andY5 receptors whereas PYY(3-36) is more selective for Y2In humans peripheral infusion of PYY(3-36) significantlydecreased appetite and reduced food intake It is suggestedthat PYY(3-36) released in response to a meal acts via theY2 receptor in the hypothalamic arcuate nucleus tophysiologically regulate food intake [159]

82 Function of DPP IV in Proteolysis of BioactivePeptides

The in vitro kinetic study of the truncation of bioactivepeptides by DPP IV resulted in the identification of severalexcellent substrates with high selectivity constants(kcatKm) These peptides have proline at the penultimateposition but also alanine serine and glycine have beenobserved in good substrates This indicates that not only thesequence surrounding the scissile bond determines theselectivity but also for instance the length of the peptideand a free and flexible N-terminus are important It has to bestressed that not all in vitro substrates for DPP IV are indeedprocessed by DPP IV in vivo Also truncation by DPP IVmay have different effects on the bioactive peptide inactivation alteration of receptor specificity or no effectThis topic has recently been reviewed [68150-152] We willdescribe the truncation of some relevant bioactive substratesThose substrates for which inhibition of the catalytic activityof DPP IV has therapeutic potential will be discussed inmore detail In the next chapter we will describe data basedon the use of DPP IV inhibitors

822 Other Neuropeptides

Substance P is a widespread neuropeptide that originatesfrom sensory neurons and signals mainly through the G-protein-coupled receptor NK1 Together with the othertachykinins neurokinin A and B substance P is responsiblefor nociceptive transmission from the peripheral to thecentral nervous system It also has a well-established role inimmunity Substance P induces the release of inflammatorymediators from mast cells and causes an increase in vascularpermeability It was shown that the activity of DPP IV ismarkedly reduced in the nasal mucosa of patients sufferingfrom chronic rhinosinusitis and that it increases again aftertreatment and symptom improvement [160] Substance P isinactivated in blood by DPP IV and angiotensin-converting


enzyme [161162] DPP IV sequentially removes Arg-Proand Lys-Pro from the undecapeptide [163] The resultingsubstance P(5-11) is still active as a transmitter of thesensory nerves but has lost its capacity to stimulatehistamine release from mast cells [164] The truncation ofexogenous substance P is decreased in DPP IV-deficient rats[161]

form the hormonal component of the entero insular axisThis term describes the connection between the gut and thepancreatic islets and comprises all stimuli coming from thesmall intestine and influencing the release of different islethormones including hormonal nervous and direct substratestimulation It has been shown that the incretin function isgreatly impaired in type 2 diabetes [175]

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) andendomorphin-2 (Tyr-Pro-Phe-Phe-NH2) are endogenousopioid peptides with high affinity at micro-opioid receptors thatproduce potent analgesia DPP IV liberates the N-terminalTyr-Pro thereby abolishing the analgetic activity[165166167] An endomorphin-2 analogue with D-Pro atthe penultimate position was resistant to degradation andwas more potent and longer lasting to induce analgesia invivo Moreover intracerebroventricularly administered Ala-pyrrolidine-2-nitrile potentiated endomorphin-2 inducedanalgesia [165] Also diprotin A (13) had a similar effect[168169] In investigations on transbuccal peptide deliveryendomorphin-1 proved to be highly unstable The additionof diprotin A (13) provided significant inhibition of thedegradation [166]

Today there is general agreement that the two mostimportant incretin hormones are glucose-dependentinsulinotropic polypeptide (GIP formerly known as gastricinhibitory polypeptide) and glucagon-like peptide-1 (GLP-1) GLP-1 refers to the biologically active form of glucagon-like peptide-1 either amidated (GLP-1 [7-36]) amide or witha C-terminal glycine (GLP-1 [7-37]) Both GIP and GLP-1are potent insulinotropic hormones and both are released byoral glucose as well as ingestion of mixed meals GIP is apeptide of 42 amino acids belonging to the glucagon-secretinfamily It is secreted from specific endocrine cells the so-called K cells which exhibit the highest density in the uppersmall intestine (duodenum and jejunum) Secretion isstimulated by absorbable carbohydrates and lipids GLP-1 isa peptide of 30 amino acids belonging to the same family ofGIP It is secreted from L cells which exhibit the highestdensity in the lower small intestine (ileum) and the colonGLP-1 is one of the most potent insulin-releasing substancesknown its potency actually exceeding that of GIP[173174]

823 The PACAPGlucagon Family of Peptides

The PACAPglucagon family includes glucagonglucagon-like peptide-1 (GLP-1) and ndash2 (GLP-2) secretinvasoactive intestinal peptide (VIP) pituitary adenylatecyclase-activating peptide (PACAP) glucose-dependentinsulinotropic peptide (GIP) growth-hormone-releasingfactor (GRF) and peptide histidine methionine (PHM) Inthis family the intact N-terminus (Tyr-Ala His-Ala or His-Ser) is necessary for biological activity and truncation byDPP IV causes inactivation

As mentioned above interference with the incretin actionof GLP-1 and GIP results in impaired insulin secretion intype 2 diabetic patients It seems that the major componentsof incretin defect in type 2 diabetes are a defective secretionof GLP-1 and a defective insulinotropic activity of GIP Onthe other hand the insulinotropic activity of GLP-1 and thesecretion of GIP are more or less preserved Indeed it wasshown that supraphysiological (pharmacological)concentrations of GLP-1 can normalise elevated bloodglucose levels in type 2 diabetic patients whereassupraphysiological concentrations of GIP have no effect[176] This suggests a therapeutic use of GLP-1 for thetreatment of type 2 diabetes but not of GIP Howeverrecent studies show that modified GIP analogues withincreased stability evoke a substantially larger and moreprotracted insulin response to oral glucose than the nativeGIP in type 2 diabetic patients [177]

Tissue-specific post-translational processing of the N-terminal sequence of proglucagon leads to production ofglucagon in the pancreatic α cells In the intestinal L-cellsand central nervous system GLP-1 and GLP-2 are produced[170] The biological activities of glucagon are directedmostly toward opposing insulin action in the liver in thecontrol of glucose metabolism primarily via stimulation ofglycogenolysis and gluconeogenesis The truncation ofglucagon with a penultimate serine by DPP IV wasunexpected [171172]

The two most important incretin hormones are GLP-1and GIP Since inhibition of their truncation by DPP IV isa new target for the treatment of type 2 diabetes GLP-1 andGIP will be discussed in more detail

GLP-1 has furthermore a number of effects that arehighly desirable in the context of treatment of type 2diabetes (reviewed in [173178179])

The discovery and effects of incretin hormones (incretins)are excellently described in recent reviews [173174] In thisreview we summarise the aspects necessary to understand thedevelopment of DPP IV inhibitors Incretins areinsulinotropic hormones of the gut that are released bynutrients and that stimulate insulin secretion inphysiological concentrations in the presence of elevatedblood glucose levels It was observed that glucoseadministered orally gave rise to much higher insulin levelsthan intravenously administered glucose in spite of similaror even higher plasma glucose levels obtained with thelatter This incretin effect is defined as the ratio between theintegrated insulin response to an oral glucose load and anisoglycaemic intravenous glucose infusion The incretins

bull GLP-1 is released upon absorption of nutrients fromthe lower gut followed by a glucose-dependentincrease in the secretion of insulin The glucosedependency of secretion and insulinotropic effect ofGLP-1 has been interpreted as safeguard againsthypoglycaemia

bull GLP-1 stimulates all steps of insulin biosynthesisand insulin gene transcription

bull GLP-1 upregulates the genes involved in insulinsecretion

bull GLP-1 stimulates β-cell proliferation and enhancesthe differentiation of new β-cells in rodents


bull GLP-1 strongly inhibits glucagon secretion in aglucose-dependent manner

reduced energy intake and concomitant increase in energyexpenditure They furthermore show improved metaboliccontrol resulting in improved insulin sensitivity reducedpancreatic islet hypertrophy and protection againststreptozotocin-induced loss of β-cell mass andhyperglycaemia Despite reports proposing a role for DPPIVCD26 in T-cell activation the knockout mice developnormally with no obvious defects in the immune system Ahistological study on more than 40 organs in these animalsreveal no remarkable differences compared to the wild typesuggesting that long-term DPP IV inhibition is tolerated

bull GLP-1 inhibits gastrointestinal secretion motilityand gastric emptying

bull GLP-1 inhibits appetite and food intake

Near normalisation of diurnal plasma glucoseconcentrations were obtained during continuous intravenousinfusion of GLP-1 in type 2 diabetic patients [180]However it turned out that simple subcutaneous injectionsof GLP-1 are ineffective [181] The reason for this is thatGLP-1 is metabolised extremely rapidly by the ubiquitousenzyme dipeptidyl peptidase IV (DPP IV EC 34145)DPP IV cleaves a dipeptide from GLP-1 which is therebyinactivated In fact the metabolite (GLP-1 [9-36]) may act asa GLP-1 receptor antagonist [182]

As for glucagon the cleavage of the N-terminal His-Serfrom VIP and PACAP by DPP IV was unexpected [154]VIP is a 28 amino acid neuropeptide and PACAP38 is a 38amino acid peptide with a C-terminally truncated 27-residuesplicing variant PACAP27 DPP IV sequentially cleaves offdipeptides from VIP PACAP27 and PACAP38 In vitrothey are rather poor substrates [154] but in vivo DPP IVplays a major role in the degradation of circulatingPACAP38 but not of PACAP27 [195] The resultingPACAP(3-38) is inactive Recent studies with PACAPreceptor-deficient mice and PACAP-deficient mice suggest arole for this neuropeptide in glucose control lipidmetabolism and adaptive thermogenesis PACAP signals viathree receptor subtypes (PAC1 VPAC1 and VPAC2) ThePAC1 receptor is specific for PACAP VPAC1 and VPAC2are shared with VIP PAC1-deficient mice display impairedglucose tolerance and fed hyperinsulinemia [196] PACAP-deficient mice have an impaired response to insulin-inducedhypoglycemia and a 2-3 fold increase in serum triglyceridesand cholesterol [197] Loss of PACAP in these animals alsoresults in inadequate heat production due to reducednorepinephrine stimulation of brown adipose tissue [198]This suggests that DPP IV inhibitors may have therapeuticutility extending beyond improvements in glucose controlspecifically in the regulation of lipid metabolism andoradaptive thermogenesis [195] Like PACAP VIP is releasedafter stimulation of the parasympathetic nerves in thepancreas and increases insulin secretion in a glucose-dependent manner

The inactivation of GLP-1 and GIP by DPP IV both invitro and in vivo has clearly been established [183184] Ithas been shown that endogenous GLP-1 is metabolisedalready before it leaves the gut Whereas all of the peptidestored in the L-cells is intact 23 of what leaves the gut isdegraded by DPP IV present in the endothelium of thecapillaries surrounding the gut [185] Also the majority ofexogenous GLP-1 whether administered intravenously orsubcutaneously is present in the circulation as the truncatedinactive metabolite [186] Thus it is clear that GLP-1 itselfcannot be used for the treatment of type 2 diabetes

To solve this problem a number of different strategieshave been explored

bull Development of small molecule agonists for theGLP-1 receptor Until now this approach has notbeen successful

bull Development of DPP IV-resistant analogues (egreplacement of Ala at the penultimate position)[187188189]

bull Alternative routes of application such as continuoussubcutaneous infusion of GLP-1 [187]

bull Inhibition of DPP IV [190]DPP IV also releases His-Ala from PHM [183] VIP and

PHM generally exert similar biological effects but PHM isless potent

The use of DPP IV inhibitors for the treatment of type 2diabetes has been investigated extensively As discussedbelow DPP IV inhibitors improve glucose tolerance duringshort-term studies in normal and diabetic rodents pigsmonkeys healthy volunteers and type 2 diabetic patientsFurther evidence for the importance of DPP IV inhibitioncomes from two different animal models Targetedinactivation of the CD26 gene in mice (CD26(--) mice)yielded apparently healthy mice that have normal bloodglucose levels in the fasted state but reduced glycaemicexcursion after a glucose challenge Levels of glucose-stimulated circulating insulin and intact GLP-1 are increasedin these animals A DPP IV inhibitor improved glucosetolerance in wild type but not in DPP IV(--) mice [191]Comparable results were obtained in DPP IV-deficient rats[192] The same DPP IV-deficient rats showed also animprovement in insulin resistance induced by a high fat diet[193] A further study with DPP IV(--) mice shows thatthey are protected against obesity and insulin resistancewhen fed on a high fat diet [194] They are refractory to thedevelopment of obesity and adiposity resulting from

GLP-2 is co-secreted along with GLP-1 from intestinalendocrine cells The principal role of GLP-2 appears to bethe maintenance of growth and absorptive function of theintestinal mucosal villus epithelium (for a recent review see[170199]) GLP-2 administration to rodents enhances villusgrowth and increases small bowel mass with weaker trophiceffects observed in the large bowel and stomach GLP-2 alsorapidly up-regulates hexose transport and nutrientabsorption GLP-2 administration produces beneficial effectsin rodent models of intestinal disease It ameliorates shortbowel syndrome secondary to intestinal resection reversesmucosal hypoplasia associated with parenteral nutritionattenuates inflammatory disease in the small and largeintestine and decreases mucosal damage secondary tovascular ischemia and chemotherapy-induced enteritis Invitro GLP-2 is easily inactivated by cleavage of His-Ala byDPP IV [200201] Also in vivo GLP-2 exhibits a very shorthalf-life principally due to enzymatic inactivation by DPP


IV [200202] Protease resistant GLP-2 analogues have beendeveloped that exhibit longer durations of action and greaterpotency in vivo [200203204] A pilot study of GLP-2administration in humans with short bowel syndromedemonstrated significant improvements in energyabsorption bone density increased body weight and leanbody mass which correlated with increased crypt plus villusheight [205206]

83 Functions of DPP IV in the Immune System andHIV-1 Entry

The presence of DPP IVCD26 on cells of thehematopoietic system and the tight regulation of itsexpression on T cells suggests that DPP IVCD26 has a keyrole in T cell function and immune regulation The optimalactivation of T cells requires at least two signals the firstsignal is provided by stimulation of the T cell receptor(TcR)CD3 complex by a specific peptide antigen or an anti-CD3 mAb the second signal can be delivered by triggeringco-stimulatory surface molecules such as CD26 Severalexcellent and recent reviews describe the role of CD26 in theimmune system [214215216217218219] An importantquestion related to the potential use of DPP IV inhibitors iswhether or not the enzymatic activity of CD26 is requiredfor its co-stimulatory action This question has beenaddressed by the use of Jurkat T-cells (human acuteleukaemia T cell line) transfected with wild-type DPPIVCD26 (CD26+DPP IV+) and with enzymatically inactiveDPP IVCD26 (CD26+DPP IV-) Following stimulationCD26+DPP IV+ cells consistently produce higher levels ofinterleukin-2 than the CD26+DPP IV- cells and the CD26-cells This suggests that enzymatic activity of DPP IVmight contribute to but is not essential for signaltransduction [220] However other authors were unable toreproduce these results and concluded that enzymatic activityis not required [221222] DPP IV inhibitors were also usedto investigate the role of DPP IVCD26 in the immunesystem DPP IV inhibitors suppress in vitro T cellproliferation and cytokine production after stimulation[90119219223-226] The reasons for the differing resultsregarding the role of DPP IV enzymatic activity in CD26-mediated T cell function are not yet clearly elucidatedRelative specificity and non-specific toxicity of DPP IVinhibitors are potential confounding factors In addition thebiological effect of DPP IV activity may depend on theparticular experimental model and to particular growthconditions [218] Also in vivo DPP IV inhibitors canmodify the immune response (vide infra) Apart from itscatalytic activity DPP IVCD26 can associate with othermolecules such as adenosine deaminase CD45 and thechemokine receptor CXCR4 These associations are alsoimportant for the role of DPP IVCD26 in the immunesystem The above mentioned truncation of chemokines canalso contribute to possible immunological functions

The regulation of somatic growth is under complexhormonal control Pituitary growth hormone synthesis andsecretion is regulated by direct neuroendocrine signals fromthe brain as well as numerous peripheral feedback signalsThe predominant neuroendocrine peptides regulating growthhormone secretion are GRF which stimulates growthhormone synthesis and secretion and somatostatin whichsuppresses growth hormone secretion Stable GRF analoguesare under active investigation for conditions characterised byrelative or absolute growth hormone deficiency The plasmaprotease responsible for primary proteolytic cleavage of GRFwas shown to be DPP IV liberating Tyr-Ala to give GRF(3-44)-NH2 This degradation is responsible for the inactivationof GRF both in vitro and in vivo [207208] and could beovercome by inhibition with diprotin A or by substitutionof the first or second amino acid with a D-amino acid Adetailed kinetic analysis of DPP IV proteolysis of GRF andanalogues showed some interesting results concerning thesubstrate specificity at the P1 position [209] The detailedinvestigation of GRF degradation led to the development ofGRF analogues with enhanced metabolic stability[210211212]

824 Chemokines

Chemokines or lsquochemotactic cytokinesrsquo form a largefamily of small secretory proteins produced by leukocytesepithelium endothelium and tissue cells eitherconstitutively or after induction Chemokines playfundamental roles in the development homeostasis andfunction of the immune system They act as regulatorymolecules in leukocyte maturation and traffic and in homingof lymphocytes and the development of lymphoid tissuesThese chemotactic cytokines not only attract leukocytes tosites of inflammation but can also activate their respectivetarget cells A great number of chemokines have proline attheir penultimate position and are truncated in vitro by DPPIV [15368] The processing by DPP IV has an importantimpact on the biological activity of several chemokinesDPP IV truncation reduces the inflammatory properties ofmost chemokines and enhance those of LD78β reduces theredundancy in their target cell specificity and influences theantiviral response (for a review see [68]) In vitro severalchemokines were shown to be excellent substrates for DPPIV with preference for SDF-1 MDC I-TAC and IP-10 Theselectivity constants for SDF-1α and MDC are much higherthan those of the incretins GLP-1 and GIP [68213150]However in vivo the situation is not so clear It is possiblethat modulation of one chemokine is corrected for by anotherpathway This is consistent with the observation that knock-out mice lacking either a specific chemokine or achemokine-receptor are generally viable with a normalphenotype and only show a partially impaired immuneresponse

A possible role for DPP IVCD26 in the process of HIV-1 entry has been suggested (reviewed in [68]) The earlyselective reduction of CD4+CD26+ T cells is remarkableThis might be explained by clustering of DPP IVCD26with the CXCR4 chemokine receptor that is a co-receptor forHIV-1 by DPP IV mediated truncation of chemokines or byinteractions between DPP IVCD26 and the HIV-1 derivedproteins Tat and gp120

84 Possible Role of DPP IV in PsychoneuroendocrineFunctions

Based on the fact that neuropeptides such as neuropeptideY and substance P are substrates for DPP IV it is possiblethat DPP IV is involved in psychoneuroendocrine functionsMoreover changes in DPP IV activity have been reported in


sera from patients with psychiatric or psychosomaticdiseases [71] Patients with major depression andschizophrenia show a decrease in serum DPP IV activity[227228229230] Patients with anorexia nervosa show anincrease in DPP IV serum activity [231232233]

of incretins or other substrates has not yet been investigatedAlso the selectivity of the reported DPP IV inhibitors withrespect to these enzymes is an important issue whenconsidering possible long-term unwanted effects

91 DPP IV Inhibitors for the Treatment of Type 2Diabetes85 Role in Cancer Progression and Cell Adhesion

Diabetes is a chronic disease that features abnormalglucose homeostasis About 10 of the patients have type 1diabetes whereas 90 have type 2 diabetes also known asnon-insulin dependent diabetes mellitus (NIDDM) Type 2diabetes develops in middle or later life and is largelyassociated with obesity Insulin resistance is an early featureof type 2 diabetes which is initially compensated in part byincreased production of insulin by pancreatic β-cellsSubsequently as these cells become exhausted theproduction of insulin decreases The combined effects ofinsulin resistance and impaired insulin secretion reduceinsulin-mediated glucose uptake and utilisation by skeletalmuscle and prevent insulin-mediated suppression of hepaticglucose output This leads to abnormal high glucose valuesin blood Chronic hyperglycaemia causes non-enzymaticglycation of proteins osmotic effects and metabolicconsequences but is in itself not sufficient to explain thechronic complications of the disease Importantly alsoinsulin resistance is implicated in major diseases includingatherosclerotic cardiovascular disease and dyslipidaemia(metabolic syndrome) Chronic complications such asretinopathy nephropathy neuropathy and atherosclerosis arethe major problems for type 2 diabetic patients sufferingfrom poor glycaemic control

DPP IVCD26 binding to the extracellular matrix is anaspect of its biology with potential implications inneoplastic diseases CD26 binds to collagen and fibronectinin a variety of experimental conditions [234235236237]This interaction may play a role in the clinical behaviour andprogression of selected cancers For example CD26molecules found on lung endothelial cells specificallyinteract with fibronectin assembled on breast cancer cellsurface potentially promoting tumour cell adhesion andmetastasis [5253238] This polymeric fibronectin is buildup on metastatic cells during their journey in the bloodcirculation A novel consensus motif in fibronectin has beenfound that mediates DPP IV adhesion [239] Also DPPIVCD26 is expressed in several tumours suggesting a rolefor DPP IV in their development [240] Besides its role inthe pathophysiology of certain solid tumours CD26 mayalso have a role in the development of some haematologicalmalignancies [218] The addition of an anti-CD26monoclonal antibody inhibits the growth of human T celllymphoma cells both in vitro and in an in vivo mouse model[241]

Recently it was reported that DPP IV and seprase formcomplexes on invadopodia of activated cells becomingpotent extracellular matrix-degrading proteases [24224321]

Currently type 2 diabetic patients are treated by acombination of diet and exercise or with insulin and variousoral pharmacological agents These agents have severaltargets to reverse several aspects of the disease Exogenousinsulin to supplement endogenous insulin supplies is usedwhen other treatment options fail Sulfonylureas andglinides stimulate the residual insulin secretion Biguanidessuch as metformin and thiazolidinediones counter insulinresistance respectively by decreasing hepatic glucose outputand increasing muscle insulin sensitivityThiazolidinediones exert their action by binding to thenuclear peroxisome proliferator activated receptor γ (PPAR-γ ) The mechanism of action of metformin is not yetcompletely understood Acarbose is an α-glucosidaseinhibitor that reduces the rate of intestinal carbohydratedigestion and therefore reduces absorption In patients wherethe disease is more advanced such drugs are frequently usedin combination to achieve better glycaemic control Each ofthe above oral agents suffers from inadequate efficacy and anumber of serious adverse effects Insulin and sulfonylureascan cause hypoglycaemia metformin sometimes causeslactic acidosis and acarbose is responsible for gastro-intestinal disturbances Hypoglycaemia is less pronouncedwith the thiazolidinediones but several adverse effects suchas oedema headache fatigue weight gain and rarely livertoxicity have been reported As a consequence therecontinues to be a high demand for new antidiabetic agents[244245]

Although DPP IV is considered as a useful diagnosticand prognostic marker there are no reports on the use ofDPP IV inhibitors as a therapeutic approach for cancer

9 EVIDENCE FOR THE THERAPEUTICPOTENTIAL OF DPP IV INHIBITORS

Most reports to date on the therapeutic potential of DPPIV inhibitors focus on their use in treatment of type 2diabetes based on their stabilising effect on the incretinhormones Some papers mention the use of DPP IVinhibitors as immunosuppressants or for the treatment ofother diseases However selectivity is an issue that needsfurther investigation in order to determine the value of DPPIV inhibitors in the treatment of type 2 diabetes DPP IVitself has multiple substrates many of which are probablynot yet known As mentioned before apart from theincretins GLP-1 and GIP several neuropeptides peptidehormones and chemokines are substrates for DPP IV Theimportance of DPP IV inhibition in these other systems isnot always known but a major consideration would bewhether DPP IV is the primary inactivating pathway forthese substrates in vivo Another potential problem arisesfrom the function of CD26 (DPP IV) in the immune systemFurthermore the validation of the therapeutic potential ofDPP IV inhibitors in vivo is complicated by a multiplicityof enzymes reported to exhibit DPP IV-like activityincluding DPP II DPP IVβ DPP 8 DPP 9 FAPα [35]The physiological role of these proteases in the metabolism

Currently several DPP IV inhibitors are in preclinical andclinical studies for the treatment of type 2 diabetes [246] In


the literature data on the following compounds were found N-isoleucylthiazolidide (2 Ile-Thia P3298) N-valylpyrrolidide (3 Val-Pyrr) FE 999011 (15) NVP-DAP728 (17) NVP-LAF237 (18)

999011 dose-dependently attenuated glucose excursionduring an oral glucose tolerance test and increased GLP-1(7-36) release in response to intraduodenal glucoseAdministration of FE 999011 twice-a-day for 7 daysimproved glucose tolerance and insulin sensitivity even inthe absence of inhibitor given at the time of the glucoseload Encouraged by these results FE 999011 was tested inZucker diabetic fatty (ZDF) rats This is a model for type IIdiabetes since these rats become diabetic after 8 weeks of ageif fed a diet containing 65 fat Prevention of the diabeticsituation was possible only with the twice-a-dayadministration suggesting that continuous inhibition ofDPP IV is required for optimal efficacy The onset ofhyperglycaemia was delayed by 21 days In addition foodand water intake were stabilised to prediabetic levels andhypertriglyceridaemia was reduced while preventing the risein circulating free fatty acids Also basal plasma levels ofGLP-1 were increased and pancreatic gene expression for theGLP-1 receptor was upregulated All these data suggest thatFE 999011 (15) could be of clinical value to delay theprogression from impaired glucose tolerance to type 2diabetes

Inhibition of circulating DPP IV [247] with orallyadministered Ile-Thia (2) enhanced insulin secretion andimproved glucose tolerance in response to an oral glucosechallenge in lean and obese Zucker rats The enhancedincretin response was greater in obese than in lean animalswith a more profound improvement in glucose tolerance[248] This encouraging result on the acute effects oftreatment with 1 was followed by a study on the long-termeffects [249] Vancouver diabetic fatty (VDF) rats weretreated for 3 months with 2 (10 mgkg orally twice daily)VDF rats are a substrain of the fatty (fafa) Zucker rat whichdisplay abnormalities characteristic of type 2 diabetes Oralglucose tolerance tests revealed a sustained improvement inglucose tolerance in the animals after 3 months treatmentThe tests were performed after drug washout indicating thatthe observation was a chronic effect rather than an acuteeffect Concomitant insulin determinations showed anincreased early-phase insulin response in the treated group(43 increase) Furthermore improvements in β-cell glucoseresponsiveness and peripheral insulin sensitivity were novelobservations that provide further support for the use of DPPIV inhibitors in the treatment of type 2 diabetes Thisimprovement in hepatic and peripheral insulin sensitivitywas confirmed in a euglycemic-hyperinsulinemic clampstudy [250] A recent study in streptozotocin-induceddiabetic rats shows that treatment with 2 stimulates β-cellsurvival and islet neogenesis This provides evidence tosupport the potential utility of DPP IV inhibitors in thetreatment of type 1 diabetes and possibly late-stage type 2diabetes This is probably related to GLP-1 stimulation of β-cell growth differentiation and cell survival [251] A smallclinical study showed that an oral dose of 2 (60 mg)improved postprandial blood glucose tolerance in patientswith type 2 diabetes Compound 2 is currently undergoingphase 2 clinical trials for the treatment of type 2 diabetes[252]

The nitrile of Novartis (17 NVP-DPP728) has also beeninvestigated extensively Acute inhibition of DPP IV inZucker fatty rats (fafa) with an oral dose of 17 (10micromolkg) improved insulin secretion and glucose tolerance[255] Similar short-term effects of NVP-DPP728 wereobserved in cynomologus monkeys [113] and humans [256]DPP IV inhibition with NVP-DPP728 prevented N-terminaldegradation of endogenous incretins in dogs resulting inincreased plasma concentrations of intact biologically activeGIP and GLP-1 However total incretin secretion wasreduced by DPP IV inhibition suggesting the possibility ofa feedback mechanism [257] A long-term study in miceshows an improvement in glucose tolerance and isletfunction [258] This inhibitor improved glucose tolerance inwild type rats but not in DPP IV-deficient rats Theseresults indicate that the amelioration of glucose tolerance byNVP-DPP728 in the wild type rats was directly due toinhibition of plasma DPP IV activity [259260]

Another inhibitor of this class (Val-pyrrolidide Val-Pyrr3) reduces the degradation of GLP-1 thereby potentiating itsinsulinotropic effect in the anaesthetised pig [190] Morerecently it was observed that Val-Pyrr similarly potentiatesthe insulinotropic effect of another incretin hormone GIP[253] This inhibitor also improved glucose tolerance andinsulin secretion in high fat-fed glucose-intolerant mice[254]

Pharmacokinetic evaluation of NVP-DPP728 in rats andmonkeys shows a high absolute bioavailability In humansa 100 mg oral dose provided a half-life of 085 h a morethan 80 inhibition of plasma DPP IV activity forapproximately 4 h and a significant increase in active GLP-1 levels [113] This compound was selected for a 4 weekstudy in a clinical setting for type 2 diabetes Comparedwith placebo NVP-DPP728 at 100 mg three times dailyreduced fasting glucose levels prandial glucose excursionsand mean 24 h glucose levels in diet-controlled type 2diabetic patients Laboratory safety and tolerability was goodin all groups This showed for the first time that inhibitionof DPP IV is a feasible approach to the treatment of type 2diabetes in humans in the early stage of the disease [256]

Oral administration of the pyrrolidine-2-nitrile 15 (FE999011) to Zucker fatty rats produced an immediatesuppression of plasma DPP IV activity In the same studyalso the effect of nitrile 17 (NVP-DPP728) was investigated[108] Maximal inhibition of DPP IV activity was obtained30 min after oral administration of NVP-DPP728 (10mgkg) and 1 h after oral administration of FE 999011 (10mg kg) NVP-DPP728 significantly reduced DPP IVactivity for 6 h with return to control values after 12 h FE999011 has a longer duration of action with a significantDPP IV inhibition for 12 h and return to control values after24 h This means that twice-a-day oral administrationcontinuously inhibits DPP IV activity In the same rats FE

The short duration of action of NVP-DPP728 (17) hasresulted in the need for frequent (with-meal) administrationlimiting the useful duration of GLP-1 potentiation to theimmediate prandial period [108254] NVP-LAF237 (18) hasbeen identified as an outcome of a search for DPP IVinhibitors with the potential for once-daily administrationOral administration of NVP-LAF237 (10 micromolkg) to


Zucker fatty rats (fafa) results in a fast DPP IV inhibitionand increased levels of intact GLP-1 Relative to Val-Pyrrand NVP-DPP728 NVP-LAF237 displayed markedlyincreased duration of action in normal Sprague-Dawley ratsevidenced by increased potency 4 h post-dose (ED50 valuesof 19 14 and 1 micromolkg) A single 1 micromolkg dose ofNVP-LAF237 inhibited plasma DPP IV activity by morethan 50 for approximately 10 h in Cynomologus monkeys[261] Because of this longer duration of action it seemsthat Novartis changed their development of DPP IVinhibitors from NVP-DPP728 (17) to NVP-LAF237 (18)[106]

have anti-arthritic effects although the precise mechanism oftheir action remains to be elucidated

Commencing experimental cardiac allograft rejection wasreflected by increased DPP IV enzymatic serum activity Theirreversible diphenyl phosphonate inhibitor 35 couldabrogate acute and accelerated rejection in this modelInhibition abolished circulating IgM titers and delayed IgGisotype switching These data provide evidence that DPPIVCD26 plays a central role in T cell-mediated immuneresponses toward allo-antigens [267268269270]

Lys[Z(NO2)]-pyrrolidide (4a) and Lys[Z(NO2)]-thiazolidide (4b) were used in a murine experimentalautoimmune encephalomyelitis model In this model theseverity and incidence during the acute phase of theautoimmune inflammatory disease was significantly reducedand the onset of clinical signs was variably delayed Theinhibitors inhibited T cell proliferation The authors suggestthat this therapeutic effect may be mediated at least in partby upregulation of the immunosuppressive cytokine TGF-β1in situ and the inhibition of T cell effector functions [271]

92 Other Therapeutic Perspectives

Apart from the well-documented use of DPP IVinhibitors in the treatment of type 2 diabetes some otherindications have been suggested Some are related to theprotection of bioactive peptides by inhibition of DPP IV Inthese cases DPP IV inhibitors could be used alone or incombination with the bioactive peptide Other indicationsare related to the function of DPP IV in the immune system DPP IV is capable of metabolising neuropeptides and

inhibition of DPP IV induces an increased secretion of latenttransforming growth factor-β1 (TGF-β1) [226271] Sinceenhancement of TGF-β signalling is associated withneuroprotective effects it was suggested that DPP IVinhibitors could be novel neuroprotective agents Indeedpyrrolidine-2-nitriles have neuroprotective andneuroregenerative effects in vitro and in vivo and couldtherefore be useful for the treatment of neurodegenerativedisorders [272]

A DPP IV inhibitor targeted to the central nervoussystem could improve the sedative and anxiolytic effects ofneuropeptide Y [156]

These CNS targeted DPP IV inhibitors could alsoprolong the effects of endomorphin-1 and ndash2 and hencepotentiate analgesia as shown by the intracerebroventricularadministration of Ala-pyrrolidine-2-nitrile or diprotin A (13)[165168169]

DPP IV inhibitors could prevent the degradation ofPACAP and could therefore not only be used to controlglucose metabolism but also lipid metabolism and adaptivethermogenesis [195]

The DPP IV inhibitors Ile-thiazolidide (2) and Ile-pyrrolidine-2-nitrile abrogate stress-induced abortions and anenhanced production of interferon-γ in mice [273274]

The DPP IV inhibitor Val-boroPro (23) stimulates thegrowth of hematopoietic progenitor cells in vivo andaccelerates neutrophil and erythrocyte regeneration in mousemodels of neutropenia and acute anaemia Hematopoieticstimulation by 23 correlated with increased cytokine levelsin vivo However 23 was also active in the absence of DPPIV and the authors suggest that FAPα is the target enzymeInteraction of 23 with the catalytic site seems to be requiredsince acetylation of the N-terminal aminofunction abrogatesDPP IV inhibition and hematopoietic stimulation [275]This is an example that the development of highly specificinhibitors is very important for the determination of thephysiological role of DPP IV Furthermore Val-boroPro(23) is also reported as a DPP II (QPP) inhibitor and causesapoptosis in quiescent lymphocytes [35]

A combination of GLP-2 and a DPP IV inhibitor willresult in an enhanced intestinotrophic effect possibly usefulin intestinal disorders such as short bowel syndromeIndeed the combination of GLP-2 and Val-Pyrr (3) provedto be effective in rats and mice [262]

A combination of GRF and a DPP IV inhibitor could beuseful to treat diseases characterised by relative or absolutegrowth hormone deficiency

The function of DPP IV in the immune system suggeststhe potential usefulness of DPP IV inhibitors in immune-related disorders such as autoimmune diseases arthritis andtransplant rejection

In vivo the administration of a DPP IV inhibitorsuppresses the antibody production in mice after stimulationwith bovine serum albumin [263] Although CD26(--) micedisplay an apparently normal phenotype the deficiency ofCD26 results in a change of cytokine and immunoglobulinsecretion after stimulation by pokeweed mitogen [264]

10 CONCLUSION

Concludingly we can state that DPP IV inhibitors haveproven their therapeutic potential during short-term studiesin normal and diabetic rodents pigs monkeys healthyvolunteers and type 2 diabetic patients Longer studies inrodents not only indicate an improvement in glucosetolerance but also an amelioration of other diabeticcomplications It was suggested that DPP IV inhibitorscould be of clinical value to delay the progression fromimpaired glucose tolerance to type 2 diabetes [108] In this

DPP IV inhibitors were also investigated for inhibitionof collagen- or alkyldiamine-induced hind paw swelling inrats as an arthritis model [265266] The following DPP IVinhibitors were effective in a dose-dependent manner Ala-boroPro Lys[Z(NO2)]-thiazolidide (4b) Ala-Pro-4-nitrobenzoylhydroxylamine (33) TMC-2A (26) and TSL-225 (27) The authors concluded that DPP IV inhibitors


same report it was also described that a continuousinhibition of DPP IV at a certain level is required to obtainan optimal effect In this respect the development of potentand long-acting DPP IV inhibitors will be required A goodexample is the longer duration of action of the Novartiscompound NVP-LAF237 (13) compared to NVP-DPP728(12) However long-term studies are absolutely necessary toassess the value of DPP IV inhibitors in a chronic diseasesuch as type 2 diabetes Because actions of GLP-1 areglucose-dependent GLP-1 metabolism represents an idealtarget for stimulating insulin secretion without causinghypoglycaemia Therefore DPP IV inhibitors for thetreatment of type 2 diabetes could have an advantage overexogenous insulin and sulfonylureas that frequently causehypoglycaemia Recent results from animal studies alsoindicate the potential usefulness of DPP IV inhibitors for thetreatment of type 1 diabetes and late-stage type 2 diabetes[194251]

certainly will result in new classes of inhibitors that have anon-peptidic nature

ACKNOWLEDGEMENTS

This work received support from The Fund for ScientificResearch ndash Flanders (Belgium) (FWO) and The SpecialFund for Research ndash University of Antwerp (BOF UA) PVan der Veken is a fellow of the Institute for the Promotionof Innovation by Science and Technology in Flanders (IWT)

REFERENCES

[1] Augustyns K Bal G Thonus G Belyaev A Zhang XMBollaert W Lambeir AM Durinx C Goossens F HaemersA Curr Med Chem 1999 6 311

[2] Vanhoof G Goossens F de Meester I Hendriks D ScharpeS FASEB J 1995 9 736

[3] Rosenblum JS Kozarich JW Curr Opinion Chem Biol 20037 496

It is too early to comment on the other suggestedtherapeutic applications of DPP IV inhibitors in the field ofimmune-related disorders and other diseases

[4] Barrett AJ Rawlings ND Woessner JF Handbook ofProteolytic Enzymes 1998

[5] Sedo A Malik R Biochim Biophys Acta 2001 1550 107Since type 2 diabetes is a chronic disease carefulinvestigation of unwanted effects due to selectivity problemswill become an important issue As described above DPPIV might be involved in the degradation of severalneuropeptides peptide hormones and chemokines Anotherpotential problem arises from the function of CD26 (DPPIV) in the immune system Furthermore the validation ofthe therapeutic potential of DPP IV inhibitors in vivo iscomplicated by a multiplicity of enzymes reported to exhibitDPP IV-like activity including dipeptidyl peptidase II(DPP II) or quiescent cell proline dipeptidase (QPP)dipeptidyl peptidase IVβ (DPP IVβ) dipeptidyl peptidase 8(DPP 8) dipeptidyl peptidase 9 (DPP 9) and fibroblast-activation protein α (FAPα) The physiological role of theseproteases in the metabolism of incretins or other substrateshas not yet been investigated but the selectivity of thereported DPP IV inhibitors with respect to these enzymesmight be an important issue when considering possiblelong-term unwanted effects In general aminoacylpyrrolidides and thiazolidides have a low selectivity indexwhen DPP IV inhibition is compared with DPP II inhibition[92] Eg Ile-Thia (2 P3298) has a selectivity index of 65in favour of DPP IV [95] Val-Pyrr (3) is a little bit moreselective with a selectivity index of 102 [95] or 56 [92] infavour of DPP IV However administration of thesecompounds in diabetic animal models does not seem tocause any signs of unwanted effects Furthermore CD26knockout mice (CD26(--) mice) and DPP IV-deficient ratsare fertile and healthy The pyrrolidine-2-nitrile inhibitors(15 17 18) are more favourable when considering potencyand selectivity A disadvantage of this class of compoundscompared to the aminoacyl pyrrolidides and thiazolididesmight be the lower chemical stability of the nitrile functionAt this moment there are not sufficient data to evaluate thevalue of the other reported DPP IV inhibitors

[6] Cunningham DF OConnor B Biochim Biophys Acta 19971343 160

[7] Polgar L Cell Mol Life Sci 2002 59 349[8] Abbott CA Yu D McCaughan GW Gorrell MD Adv Exp

Med Biol 2000 477 103[9] Chen T Ajami K McCaughan GW Gorrell MD Abbott

CA Adv Exp Med Biol 2003 524 79[10] Pineiro-Sanchez ML Goldstein LA Dodt J Howard L

Yeh Y Chen WT J Biol Chem 1997 272 7595[11] Sun S Albright CF Fish BH George HJ Selling BH

Hollis GF Wynn R Protein Expr Purif 2002 24 274[12] Niedermeyer J Scanlan MJ Garin-Chesa P Daiber C

Fiebig HH Old LJ Rettig WJ Schnapp A Int J Cancer1997 71 383

[13] Goldstein LA Ghersi G Pineiro-Sanchez ML Salamone MYeh Y Flessate D Chen WT Biochim Biophys Acta 19971361 11

[14] Kelly T Kechelava S Rozypal TL West KW KorourianS Mod Pathol 1998 11 855

[15] Park JE Lenter MC Zimmermann RN Garin-Chesa POld LJ Rettig WJ J Biol Chem 1999 274 36505

[16] Levy MT McCaughan GW Abbott CA Park JECunningham AM Muller E Rettig WJ Gorrell MDHepatology 1999 29 1768

[17] Goodman JD Rozypal TL Kelly T Clin Exp Metastasis2003 20 459

[18] Huber MA Kraut N Park JE Schubert RD Rettig WJPeter RU Garin-Chesa P J Invest Dermatol 2003 120 182

[19] Iwasa S Jin X Okada K Mitsumata M Ooi A Cancer Lett2003 199 91

[20] Jin X Iwasa S Okada K Mitsumata M Ooi A AnticancerRes 2003 23 3195

[21] Chen WT Adv Exp Med Biol 2003 524 197[22] Cheng JD Dunbrack R-LJ Valianou M Rogatko A

Alpaugh RK Weiner LM Cancer Res 2002 62 4767[23] Scott AM Wiseman G Welt S Adjei A Lee FT Hopkins

W Divgi CR Hanson LH Mitchell P Gansen DN LarsonSM Ingle JN Hoffman EW Tanswell P Ritter G CohenLS Bette P Arvay L Amelsberg A Vlock D Rettig WJOld LJ Clin Cancer Res 2003 9 1639

[24] Hofheinz RD al Batran SE Hartmann F Hartung G JagerD Renner C Tanswell P Kunz U Amelsberg A KuthanH Stehle G Onkologie 2003 26 44

The recent publication of several crystal structures ofDPP IV in complex with inhibitors will greatly enhance thedevelopment of new DPP IV inhibitors Computer-assistedrational design of inhibitors and virtual screening most

[25] Niedermeyer J Kriz M Hilberg F Garin-Chesa PBamberger U Lenter MC Park J Viertel B Puschner HMauz M Rettig WJ Schnapp A Mol Cell Biol 2000 201089


[26] Abbott CA Yu DM Woollatt E Sutherland GRMcCaughan GW Gorrell MD Eur J Biochem 2000 2676140

[62] Fulop V Szeltner Z Polgar L EMBO Rep 2000 1 277[63] Szeltner Z Rea D Renner V Fulop V Polgar L J Biol

Chem 2002 277 42613[27] Olsen C Wagtmann N Gene 2002 299 185 [64] Szeltner Z Rea D Juhasz T Renner V Mucsi Z Orosz G

Fulop V Polgar L J Biol Chem 2002 277 44597[28] Qi SY Riviere PJ Trojnar J Junien JL Akinsanya KOBiochem J 2003 373 179 [65] Wrenger S Reinhold D Hoffmann T Kraft M Frank R

Faust J Neubert K Ansorge S FEBS Lett 1996 383 145[29] Yokotani N Doi K Wenthold RJ Wada K Hum MolGenet 1993 2 1037 [66] Wrenger S Hoffmann T Faust J Mrestani-Klaus C Brandt

W Neubert K Kraft M Olek S Frank R Ansorge SReinhold D J Biol Chem 1997 272 30283

[30] Hough RB Lengeling A Bedain V Lo C Bucan M ProcNatl Acad Sci U S A 1998 95 13800

[31] Nadal MS Ozaita A Amarillo Y de Miera EV Ma YMo W Goldberg EM Misumi Y Ikehara Y Neubert TARudy B Neuron 2003 37 449

[67] Brandt W Ludwig O Thondorf I Barth A Eur J Biochem1996 236 109

[68] Lambeir AM Durinx C Scharpe S De M I Crit Rev ClinLab Sci 2003 40 209[32] Leiting B Pryor KD Wu JK Marsilio F Patel RA Craik

CS Ellman JA Cummings RT Thornberry NA BiochemJ 2003 371 525

[69] Antczak C De M I Bauvois B Bioessays 2001 23 251[70] Antczak C De M I Bauvois B Journal of Biological

Regulators and Homeostatic Agents 2001 15 130[33] McDonald JK Reilly TJ Zeitman BB Ellis S J BiolChem 1968 243 2028 [71] Hildebrandt M Reutter W Arck P Rose M Klapp BF Clin

Sci (Lond) 2000 99 93[34] Araki H Li Y Yamamoto Y Haneda M Nishi KKikkawa R Ohkubo I J Biochem (Tokyo) 2001 129 279 [72] Slimane TA Lenoir C Bello V Delaunay JL Goding JW

Chwetzoff S Maurice M Fransen JA Trugnan G Exp CellRes 2001 270 45

[35] Chiravuri M Schmitz T Yardley K Underwood R DayalY Huber BT J Immunol 1999 163 3092

[36] Underwood R Chiravuri M Lee H Schmitz T KabcenellAK Yardley K Huber BT J Biol Chem 1999 274 34053

[73] Slimane TA Lenoir C Sapin C Maurice M Trugnan GExp Cell Res 2000 258 184

[37] Chiravuri M Lee H Mathieu SL Huber BT J Biol Chem2000 275 26994

[74] Darmoul D Voisin T Couvineau A Rouyer-Fessard CSalomon R Wang Y Swallow DM Laburthe M BiochemBiophys Res Commun 1994 203 1224[38] Chiravuri M Agarraberes F Mathieu SL Lee H Huber

BT J Immunol 2000 165 5695 [75] Zweibaum A Hauri HP Sterchi E Chantret I Haffen KBamat J Sordat B Int J Cancer 1984 34 591[39] Chiravuri M Huber BT Apoptosis 2000 5 319

[40] Blanco J Nguyen C Callebaut C Jacotot E Krust BMazaleyrat JP Wakselman M Hovanessian AG Eur JBiochem 1998 256 369

[76] Vanhoof G de Meester I van Sande M Scharpe S YaronA Eur J Clin Chem Clin Biochem 1992 30 333

[77] Wilson MJ Ruhland AR Pryor JL Ercole C Sinha AAHensleigh H Kaye KW Dawkins HJ Wasserman NFReddy P Ahmed K J Urol 1998 160 1905

[41] Fulop V Bocskei Z Polgar L Cell 1998 94 161[42] Fulop V Szeltner Z Renner V Polgar L J Biol Chem

2001 276 1262 [78] Wilson MJ Haller R Slaton JW Wasserman NF SinhaAA Adv Exp Med Biol 2003 524 257[43] Rasmussen HB Branner S Wiberg FC Wagtmann N

Nature Structural Biology 2003 10 19 [79] IwakiEgawa S Watanabe Y Kikuya Y Fujimoto Y JBiochem 1998 124 428[44] Durinx C Lambeir AM Bosmans E Falmagne JB

Berghmans R Haemers A Scharpe S de Meester I Eur JBiochem 2000 267 5608

[80] Durinx C Neels H Van der Auwera JC Naelaerts KScharpe S de Meester I Clin Chem Lab Med 2001 39 155

[45] Engel M Hoffmann T Wagner L Wermann M Heiser UKiefersauer R Huber R Bode W Demuth HUBrandstetter H Proc Natl Acad Sci U S A 2003 100 5063

[81] de Meester I Korom S Van Damme J Scharpe S ImmunolToday 1999 20 367

[82] Gorrell MD Wickson J McCaughan GW Cell Immunol1991 134 205[46] Thoma R Loffler B Stihle M Huber W Ruf A Hennig M

Structure 2003 11 947 [83] Buhling F Kunz D Reinhold D Ulmer AJ Ernst M FladHD Ansorge S Nat Immun 1994 13 270[47] Rahfeld J Schierhorn M Hartrodt B Neubert K Heins J

Biochim Biophys Acta 1991 1076 314 [84] Buhling F Junker U Reinhold D Neubert K Jager LAnsorge S Immunol Lett 1995 45 47[48] Fischer G Heins J Barth A Biochim Biophys Acta 1983

742 452 [85] Vanham G Kestens L de Meester I Vingerhoets J PenneG Vanhoof G Scharpe S Heyligen H Bosmans ECeuppens JL et al J Acquir Immune Defic Syndr 1993 6749

[49] Heins J Welker P Schonlein C Born I Hartrodt BNeubert K Tsuru D Barth A Biochim Biophys Acta 1988954 161

[50] Abbott CA McCaughan GW Gorrell MD FEBS Lett 1999458 278

[86] Ogata S Misumi Y Tsuji E Takami N Oda K Ikehara YBiochemistry 1992 31 2582

[51] Ajami K Abbott CA Obradovic M Gysbers V Kahne TMcCaughan GW Gorrell MD Biochemistry 2003 42 694

[87] Villhauer EB Coppola GM Hughes TE Ann Rep MedChem 2001 36 191

[52] Cheng HC Abdel-Ghany M Elble RC Pauli BU J BiolChem 1998 273 24207

[88] Yaron A Naider F Crit Rev Biochem Mol Biol 1993 28 31[89] Brandt W Lehmann T Thondorf I Born I Schutkowski M

Rahfeld JU Neubert K Barth A Int J Pept Protein Res1995 46 494

[53] Abdel-Ghany M Cheng H Levine RA Pauli BU InvasionMetastasis 1998 18 35

[54] Scanlan MJ Raj BK Calvo B Garin-Chesa P Sanz-Moncasi MP Healey JH Old LJ Rettig WJ Proc NatlAcad Sci U S A 1994 91 5657

[90] Schon E Born I Demuth HU Faust J Neubert KSteinmetzer T Barth A Ansorge S Biol Chem Hoppe Seyler1991 372 305

[55] Kameoka J Tanaka T Nojima Y Schlossman SF MorimotoC Science 1993 261 466

[91] Yoshimoto T Yamamoto N Ogawa H Furukawa S TsuruD Agric Biol Chem 1991 55 1135

[56] Abbott CA McCaughan GW Levy MT Church WBGorrell MD Eur J Biochem 1999 266 798

[92] Senten K Van der Veken P Bal G de Meester I LambeirAM Scharpe S Bauvois B Haemers A Augustyns KBioorg Med Chem Lett 2002 12 2825[57] Ludwig K Fan H Dobers J Berger M Reutter W

Bottcher C Biochem Biophys Res Commun 2004 313 223 [93] Senten K Van der Veken P de Meester I Lambeir AMScharpe S Haemers A Augustyns K J Med Chem 2003 465005

[58] Rahfeld J Schutkowski M Faust J Neubert K Barth AHeins J Biol Chem Hoppe Seyler 1991 372 313

[59] Puschel G Mentlein R Heymann E Eur J Biochem 1982126 359

[94] Augustyns KJL Lambeir AM Borloo M DeMeester IVedernikova I Vanhoof G Hendriks D Scharpe SHaemers A Eur J Med Chem 1997 32 301[60] Kaspari A Diefenthal T Grosche G Schierhorn A Demuth

HU Biochim Biophys Acta 1996 1293 147 [95] Stockel-Maschek A Mrestani-Klaus C Stiebitz B DemuthH Neubert K Biochim Biophys Acta 2000 1479 15[61] Schutkowski M Neubert K Fischer G Eur J Biochem 1994

221 455


[96] Ashworth DM Atrash B Baker GR Baxter AJ JenkinsPD Jones DM Szelke M Bioorg Med Chem Lett 1996 61163

[134] Akiyama T Abe M Harada S Kojima F Sawa RTakahashi Y Naganawa H Homma Y Hamada MYamaguchi A Aoyagi T Muraoka Y Takeuchi T JAntibiot (Tokyo) 2001 54 744[97] Parmee ER He J Mastracchio A Edmondson SD Colwell

L Eiermann G Feeney WP Habulihaz B He H KilburnR Leiting B Lyons K Marsilio F Patel RA Petrov A DiSalvo J Wu JK Thornberry NA Weber AE Bioorg MedChem Lett 2004 14 43

[135] Eisai Co LTD 2002 EP01258480[136] Fischer G Demuth HU Barth A Pharmazie 1983 38 249[137] Demuth HU Baumgrass R Schaper C Fischer G Barth A

J Enzyme Inhib 1988 2 129[98] Novartis AG 2000 US6107317 [138] Demuth HU Neumann U Barth A J Enzyme Inhib 1989 2

239[99] Probiodrug 1999 WO9967278[100] Umezawa H Aoyagi T Ogawa K Naganawa H Hamada

M Takeuchi T J Antibiot 1984 37 422[139] Lin J Toscano PJ Welch JT Proc Natl Acad Sci U S A

1998 95 14020[101] Subramanyam M Gutheil WG Bachovchin WW Huber

BT J Immunol 1993 150 2544[140] Boduszek B Oleksyszyn J Kam CM Selzler J Smith RE

Powers JC J Med Chem 1994 37 3969[102] Gutheil WG Subramanyam M Flentke GR Sanford DG

Munoz E Huber BT Bachovchin WW Proc Natl Acad SciU S A 1994 91 6594

[141] Lambeir AM Borloo M de Meester I Belyaev AAugustyns K Hendriks D Scharpe S Haemers A BiochimBiophys Acta 1996 1290 76

[103] Wrenger S Faust J Mrestani-Klaus C Fengler A Stockel-Maschek A Lorey S Kahne T Brandt W Neubert KAnsorge S Reinhold D J Biol Chem 2000 275 22180

[142] Belyaev A Borloo M Augustyns K Lambeir AM DeMeester I Scharpeacute S Blaton N Peeters OM De Ranter CHaemers A Tetrahedron Lett 1995 36 3755

[104] Lorey S Stockel-Maschek A Faust J Brandt W Stiebitz BGorrell MD Kahne T Mrestani-Klaus C Wrenger SReinhold D Ansorge S Neubert K Eur J Biochem 2003270 2147

[143] de Meester I Belyaev A Lambeir AM De Meyer GR VanOsselaer N Haemers A Scharpe S Biochem Pharmacol1997 54 173

[144] Senten K Daniels L Van d V De M I Lambeir AMScharpe S Haemers A Augustyns K J Comb Chem 2003 5336

[105] Li J Wilk E Wilk S Arch Biochem Biophys 1995 323 148[106] Villhauer EB Brinkman JA Naderi GB Burkey BF

Dunning BE Prasad K Mangold BL Russell ME HughesTE J Med Chem 2003 46 2774

[145] Belyaev A Zhang X Augustyns K Lambeir AM deMeester I Vedernikova I Scharpe S Haemers A J MedChem 1999 42 1041[107] Ferring BV 1995 WO9515309

[108] Sudre B Broqua P White RB Ashworth D Evans DMHaigh R Junien JL Aubert ML Diabetes 2002 51 1461

[146] Senten K VanderVeken P Bal G Haemers A AugustynsK Tetrahedron Lett 2001 42 9135


[147] Tiruppathi C Miyamoto Y Ganapathy V Roesel RAWhitford GM Leibach FH J Biol Chem 1990 265 1476

[148] Tiruppathi C Miyamoto Y Ganapathy V Leibach FH AmJ Physiol 1993 265 G81[110] Novartis AG 2000 US6124305

[111] Novartis AG 2000 US6110949 [149] Morita A Chung YC Freeman HJ Erickson RHSleisenger MH Kim YS J Clin Invest 1983 72 610[112] Hughes TE Mone MD Russell ME Weldon SC

Villhauer EB Biochemistry 1999 38 11597 [150] de Meester I Lambeir AM Proost P Scharpe S Adv ExpMed Biol 2003 524 3[113] Villhauer EB Brinkman JA Naderi GB Dunning BE

Mangold BL Mone MD Russell ME Weldon SC HughesTE J Med Chem 2002 45 2362

[151] de Meester I Durinx C Bal G Proost P Struyf S GoossensF Augustyns K Scharpe S Adv Exp Med Biol 2000 477 67

[114] Willand N Joossens J Gesquiere JC Tartar AL EvansDM Roe MB Tetrahedron 2002 58 5741

[152] Mentlein R Regul Pept 1999 85 9[153] Lambeir AM Proost P Durinx C Bal G Senten K

Augustyns K Scharpe S Van Damme J de Meester I J BiolChem 2001 276 29839

[115] Novartis AG 2000 US6166063[116] Ferring BV 2001 WO0140180A2[117] Zhao K Lim DS Funaki T Welch JT Bioorg Med Chem

2003 11 207[154] Lambeir AM Durinx C Proost P Van Damme J Scharpe

S de Meester I FEBS Lett 2001 507 327[118] Van der Veken P Kertesz I Senten K Haemers A

Augustyns K Tetrahedron Lett 2003 44 6231[155] Mentlein R Dahms P Grandt D Kruger R Regul Pept

1993 49 133[119] Flentke GR Munoz E Huber BT Plaut AG Kettner CA

Bachovchin WW Proc Natl Acad Sci U S A 1991 88 1556[156] Karl T Hoffmann T Pabst R von Horsten S Pharmacol

Biochem Behav 2003 75 869[120] Gutheil WG Bachovchin WW Biochemistry 1993 32 8723 [157] Zukowska Z Neuropeptides 2002 36 437[121] Kelly TA Adams J Bachovchin WW Barton RW

Campbell SJ Coutts SJ J Am Chem Soc 1993 115 12637[158] Karl T Hoffman T Pabst R von Horsten S Physiology and

Behavior 2003 80 123[122] Sudmeier JL Gunther UL Gutheil WG Coutts SJ Snow

RJ Barton RW Bachovchin WW Biochemistry 1994 3312427

[159] Batterham RL Bloom SR Ann N Y Acad Sci 2003 994 162[160] Grouzmann E Monod M Landis B Wilk S Brakch N

Nicoucar K Giger R Malis D Szalay-Quinodoz I CavadasC Morel DR Lacroix JS FASEB J 2002 16 1132[123] Snow RJ Bachovchin WW Barton RW Campbell SJ

Coutts SJ Freeman DM Gutheil WG Kelly TA KennedyCA Krolikowski DA Leonard SF Pargellis CA Tong LAdams J J Am Chem Soc 1994 116 10860

[161] Ahmad S Wang L Ward PE J Pharmacol Exp Ther 1992260 1257

[162] Palmieri FE Ward PE Biochim Biophys Acta 1983 755 522[124] Point Therapeutics Inc 1999 WO9962914 [163] Heymann E Mentlein R FEBS Lett 1978 91 360[125] Coutts SJ Kelly TA Snow RJ Kennedy CA Barton RW

Adams J Krolikowski DA Freeman DM Campbell SJKsiazek JF Bachovchin WW J Med Chem 1996 39 2087

[164] Mussap CJ Geraghty DP Burcher E J Neurochem 199360 1987

[165] Shane R Wilk S Bodnar RJ Brain Res 1999 815 278[126] Novo Nordisk AS 2002 WO0202560 [166] Bird AP Faltinek JR Shojaei AH J Control Release 2001

73 31[127] Drug Data Report 2002 24 620[128] Boehringer Ingelheim Pharma KG 2002 WO02068420 [167] Tomboly C Peter A Toth G Peptides 2002 23 1573[129] Yamada M Okagaki C Higashijima T Tanaka S Ohnuki

T Sugita T Bioorg Med Chem Lett 1998 8 1537[168] Ronai AZ Timar J Mako E Erdo F Gyarmati Z Toth G

Orosz G Furst S Szekely JI Life Sciences 1998 64 145[130] Ohnuki T Yamada M Sugita T Drugs of the Future 1999 24

665[169] Sakurada C Sakurada S Hayashi T Katsuyama S Tan

NK Sakurada T Biochem Pharmacol 2003 66 653[131] Coppola GM Zhang YL Schuster HF Russell ME

Hughes TE Bioorg Med Chem Lett 2000 10 1555[170] Mayo KE Miller LJ Bataille D Dalle S Goke B Thorens

B Drucker DJ Pharmacol Rev 2003 55 167[132] Takeda Chemical Industries LTD 2002 WO02062764 [171] Pospisilik JA Hinke SA Pederson RA Hoffmann T

Rosche F Schlenzig D Glund K Heiser U McIntosh CHDemuth H Regul Pept 2001 96 133

[133] Shimazawa R Takayama H Kato F Kato M Hashimoto YBioorg Med Chem Lett 1999 9 559


[172] Hinke SA Pospisilik JA Demuth HU Mannhart S Kuhn-Wache K Hoffmann T Nishimura E Pederson RAMcIntosh CH J Biol Chem 2000 275 3827

[209] Bongers J Lambros T Ahmad M Heimer EP BiochimBiophys Acta 1992 1122 147

[210] Kubiak TM Friedman AR Martin RA Ichhpurani AKAlaniz GR Claflin WH Goodwin MC Cleary DL KellyCR Hillman RM et al J Med Chem 1993 36 888

[173] Holst JJ Orskov C Scand J Clin Lab Invest Suppl 2001 75[174] Creutzfeldt W Exp Clin Endocrinol Diabetes 2001 109 Suppl

2 S288 [211] Campbell RM Stricker P Miller R Bongers J Liu WLambros T Ahmad M Felix AM Heimer EP Peptides1994 15 489

[175] Nauck MA Stockmann F Ebert R Creutzfeldt WDiabetologia 1986 29 46

[176] Nauck MA Heimesaat MM Orskov C Holst JJ Ebert RCreutzfeldt W J Clin Invest 1993 91 301

[212] Cervini LA Donaldson CJ Koerber SC Vale WW RivierJE J Med Chem 1998 41 717

[177] Gault VA Flatt PR OHarte FP Biochem Biophys ResCommun 2003 308 207

[213] Proost P Struyf S Schols D Opdenakker G Sozzani SAllavena P Mantovani A Augustyns K Bal G Haemers ALambeir AM Scharpe S Van Damme J de Meester I JBiol Chem 1999 274 3988

[178] Perry T Greig NH Trends Pharmacol Sci 2003 24 377[179] Holst JJ Diabetes Metabolism Research and Reviews 2002 18

430 [214] DeMeester I Korom S VanDamme J Scharpe S ImmunolToday 1999 20 367[180] Rachman J Barrow BA Levy JC Turner RC Diabetologia

1997 40 205 [215] Gorrell MD Gysbers V McCaughan GW Scand JImmunol 2001 54 249[181] Nauck MA Wollschlager D Werner J Holst JJ Orskov

C Creutzfeldt W Willms B Diabetologia 1996 39 1546 [216] Morimoto C Schlossman SF Immunol Rev 1998 161 55[182] Knudsen LB Pridal L Eur J Pharmacol 1996 318 429 [217] von Bonin A Huhn J Fleischer B Immunol Rev 1998 161

43[183] Mentlein R Gallwitz B Schmidt WE Eur J Biochem 1993214 829 [218] Dang NH Morimoto C Histol Histopathol 2002 17 1213

[184] Kieffer TJ McIntosh CH Pederson RA Endocrinology1995 136 3585

[219] Reinhold D Kahne T Steinbrecher A Wrenger S NeubertK Ansorge S Brocke S Biol Chem 2002 383 1133

[185] Hansen L Deacon CF Orskov C Holst JJ Endocrinology1999 140 5356

[220] Tanaka T Kameoka J Yaron A Schlossman SF MorimotoC Proc Natl Acad Sci U S A 1993 90 4586

[186] Deacon CF Nauck MA Toft-Nielsen MB Pridal LWillms B Holst JJ Diabetes 1995 44 1126

[221] Hegen M Mittrucker HW Hug R Demuth HU NeubertK Barth A Fleischer B Immunobiology 1993 189 483

[187] Holst JJ Curr Med Chem 1999 6 1005 [222] Steeg C Hartwig U Fleischer B Cell Immunol 1995 164311[188] Holz GG Chepurny OG Curr Med Chem 2003 10 2471

[189] Drucker DJ Curr Pharm Des 2001 7 1399 [223] Schon E Jahn S Kiessig ST Demuth HU Neubert KBarth A von Baehr R Ansorge S Eur J Immunol 1987 171821

[190] Deacon CF Hughes TE Holst JJ Diabetes 1998 47 764[191] Marguet D Baggio L Kobayashi T Bernard AM Pierres

M Nielsen PF Ribel U Watanabe T Drucker DJWagtmann N Proc Natl Acad Sci U S A 2000 97 6874

[224] Reinhold D Bank U Buhling F Neubert K Mattern TUlmer AJ Flad HD Ansorge S Immunobiology 1993 188403[192] Nagakura T Yasuda N Yamazaki K Ikuta H Yoshikawa

S Asano O Tanaka I Biochem Biophys Res Commun 2001284 501

[225] Reinhold D Bank U Buhling F Kahne T Kunt D Faust JNeubert K Ansorge S Immunobiology 1994 192 121

[193] Yasuda N Inoue T Nagakura T Yamazaki K Kira KSaeki T Tanaka I Biochem Biophys Res Commun 2002 298779

[226] Reinhold D Bank U Buhling F Lendeckel U Faust JNeubert K Ansorge S Immunology 1997 91 354

[227] Maes M de Meester I Vanhoof G Scharpe S Bosmans EVandervorst C Verkerk R Minner B Suy E Raus J BiolPsychiatry 1991 30 577

[194] Conarello SL Li Z Ronan J Roy RS Zhu L Jiang G LiuF Woods J Zycband E Moller DE Thornberry NAZhang BB Proc Natl Acad Sci U S A 2003 100 6825 [228] Maes M de Meester I Scharpe S Desnyder R Ranjan R

Meltzer HY Acta Psychiatr Scand 1996 93 1[195] Zhu L Tamvakopoulos C Xie D Dragovic J Shen XFenyk-Melody JE Schmidt K Bagchi A Griffin PRThornberry NA Sinha-Roy R J Biol Chem 2003 278 22418

[229] Maes M de Meester I Verkerk R De Medts P Wauters AVanhoof G Vandoolaeghe E Neels H Scharpe SPsychoneuroendocrinology 1997 22 65[196] Jamen F Persson K Bertrand G Riodriguez-Henche N

Puech R Bockaert J Ahren B Brabet P J Clin Invest 2000105 1307

[230] Elgun S Keskinege A Kumbasar HPsychoneuroendocrinology 1999 24 823

[197] Gray SL Cummings KJ Jirik FR Sherwood NM MolEndocrinol 2001 15 1739

[231] Hildebrandt M Rose M Mayr C Schuler C Reutter WSalama A Klapp BF Scand J Immunol 1999 50 536

[198] Gray SL Yamaguchi N Vencova P Sherwood NMEndocrinology 2002 143 3946

[232] Hildebrandt M Rose M Mayr C Arck P Schuler CReutter W Salama A Klapp BF Adv Exp Med Biol 2000477 197[199] Lovshin J Drucker DJ Regul Pept 2000 90 27

[200] Drucker DJ Shi Q Crivici A Sumner-Smith M TavaresW Hill M DeForest L Cooper S Brubaker PL NatBiotechnol 1997 15 673

[233] Hildebrandt M Rose M Monnikes H Reutter W Keller WKlapp BF Nutrition 2001 17 451

[234] Bauvois B Biochem J 1988 252 723[201] Lambeir AM Proost P Scharpe S de Meester I Biochem

Pharmacol 2002 64 1753[235] Piazza GA Callanan HM Mowery J Hixson DC Biochem

J 1989 262 327[202] Hartmann B Harr MB Jeppesen PB Wojdemann M

Deacon CF Mortensen PB Holst JJ J Clin EndocrinolMetab 2000 85 2884

[236] Dang NH Torimoto Y Schlossman SF Morimoto C J ExpMed 1990 172 649

[237] Loster K Zeilinger K Schuppan D Reutter W BiochemBiophys Res Commun 1995 217 341[203] DaCambra MP Yusta B Sumner-Smith M Crivici A

Drucker DJ Brubaker PL Biochemistry 2000 39 8888 [238] Cheng HC Abdel-Ghany M Zhang S Pauli BU Clin ExpMetastasis 1999 17 609[204] Drucker DJ J Clin Endocrinol Metab 2001 86 1759

[205] Jeppesen PB Hartmann B Thulesen J Graff J Lohmann JHansen BS Tofteng F Poulsen SS Madsen JL Holst JJMortensen PB Gastroenterology 2001 120 806

[239] Cheng HC Abdel-Ghany M Pauli BU J Biol Chem 2003278 24600

[240] Antczak C De M I Bauvois B Bioessays 2001 23 251[206] Haderslev KV Jeppesen PB Hartmann B Thulesen J

Sorensen HA Graff J Hansen BS Tofteng F Poulsen SSMadsen JL Holst JJ Staun M Mortensen PB Scand JGastroenterol 2002 37 392

[241] Ho L Aytac U Stephens LC Ohnuma K Mills GBMcKee KS Neumann C LaPushin R Cabanillas FAbbruzzese JL Morimoto C Dang NH Clin Cancer Res2001 7 2031

[207] Frohman LA Downs TR Williams TM Heimer EP PanYCE Felix AM J Clin Invest 1986 78 906

[242] Ghersi G Dong H Goldstein LA Yeh Y Hakkinen LLarjava HS Chen WT J Biol Chem 2002 277 29231

[208] Frohman LA Downs TR Heimer EP Felix AM J ClinInvest 1989 83 1533

[243] Ghersi G Dong H Goldstein LA Yeh Y Hakkinen LLarjava HS Chen WT Adv Exp Med Biol 2003 524 87


[244] Bailey CJ Trends Pharmacol Sci 2000 21 259 [262] Hartmann B Thulesen J Kissow H Thulesen S Orskov CRopke C Poulsen SS Holst JJ Endocrinology 2000 1414013

[245] Zhang BB Moller DE Curr Opin Chem Biol 2000 4 461[246] Augustyns K Van der Veken P Senten K Haemers A Exp

Opin Ther Pat 2003 13 499 [263] Kubota T Flentke GR Bachovchin WW Stollar BD ClinExp Immunol 1992 89 192[247] Pauly RP Demuth HU Rosche F Schmidt J White HA

Lynn F McIntosh CH Pederson RA Metabolism 1999 48385

[264] Yan S Marguet D Dobers J Reutter W Fan H Eur JImmunol 2003 33 1519

[248] Pederson RA White HA Schlenzig D Pauly RP McIntoshCH Demuth HU Diabetes 1998 47 1253

[265] Tanaka S Murakami T Horikawa H Sugiura MKawashima K Sugita T Int J Immunopharmacol 1997 1915[249] Pospisilik JA Stafford SG Demuth HU Brownsey R

Parkhouse W Finegood DT McIntosh CH Pederson RADiabetes 2002 51 943

[266] Tanaka S Murakami T Nonaka N Ohnuki T Yamada MSugita T Immunopharmacology 1998 40 21

[250] Pospisilik JA Stafford SG Demuth HU McIntosh CHPederson RA Diabetes 2002 51 2677

[267] Korom S de Meester I Onodera K Stadlbauer TH BorlooM Lambeir AM Kupiec-Weglinski JW Transplant Proc1997 29 1274[251] Pospisilik JA Martin J Doty T Ehses JA Pamir N Lynn

FC Piteau S Demuth HU McIntosh CH Pederson RADiabetes 2003 52 741

[268] Korom S de Meester I Stadlbauer TH Chandraker ASchaub M Sayegh MH Belyaev A Haemers A ScharpeS Kupiec-Weglinski JW Transplantation 1997 63 1495[252] Sorbera LA Revel L Castaner J Drugs of the Future 2001

26 859 [269] Korom S de Meester I Coito AJ Graser E Pratschke JKonig S Grimm H Volk HD Scharpe S Kupiec-WeglinskiJW Transplant Proc 1999 31 873

[253] Deacon CF Danielsen P Klarskov L Olesen M Holst JJDiabetes 2001 50 1588

[254] Ahren B Holst JJ Martensson H Balkan B Eur JPharmacol 2000 404 239

[270] Korom S de Meester I Belyaev A Schmidbauer GSchwemmle K Adv Exp Med Biol 2003 524 133

[255] Balkan B Kwasnik L Miserendino R Holst JJ Li XDiabetologia 1999 42 1324

[271] Steinbrecher A Reinhold D Quigley L Gado A Tresser NIzikson L Born I Faust J Neubert K Martin R AnsorgeS Brocke S J Immunol 2001 166 2041[256] Ahren B Simonsson E Larsson H Landin-Olsson M

Torgeirsson H Jansson PA Sandqvist M Bavenholm PEfendic S Eriksson JW Dickinson S Holmes D DiabetesCare 2002 25 869

[272] Wu YQ Limburg DC Wilkinson DE Jackson P SteinerJP Hamilton GS Belyakov SA Adv Exp Med Biol 2003524 351

[257] Deacon CF Wamberg S Bie P Hughes TE Holst JJ JEndocrinol 2002 172 355

[273] Hildebrandt M Arck PC Kruber S Demuth HU ReutterW Klapp BF Scand J Immunol 2001 53 449

[258] Reimer MK Holst JJ Ahren B Eur J Endocrinol 2002146 717

[274] Ruter J Hoffmann T Heiser U Demuth HU Arck PCKlapp BF Hildebrandt M Cell Immunol 2002 220 150

[259] Mitani H Takimoto M Hughes TE Kimura M Jpn JPharmacol 2002 88 442

[275] Jones B Adams S Miller GT Jesson MI Watanabe TWallner BP Blood 2003 102 1641

[260] Mitani H Takimoto M Kimura M Jpn J Pharmacol 200288 451

[276] Oefner C D Arcy A Mac SA Pierau S Gardiner R DaleGE Acta Crystallographica Section D Biological Crystallography2003 59 Part 7 1206[261] Hughes TE Russell ME Bolognese L Diabetes 2002 62

272-OR


N

O

NH2

O

R1

HO

N

O

N

O

N

O

NH2

O

R1

HN

NH

R3

O

R

O

NH

HN

O

RO

R

HN

OH

Rn

O

PCP

POP

DPP IVFAP αDPP8DPP9DPP II

Aminopept idase P2X-Pro dipeptidase

DPP IV = dipeptidyl peptidase IV FAP α = fibroblast activation protein α subunit DPP8 = dipept idyl peptidase8 DPP9 = dipept idyl peptidase 9 DPP II = dipept idyl peptidase II POP = prolyl oligopeptidase PCP = lysosomal Pro-X carboxypept idase

Fig (1) Human proline-specific peptidases

in tumours but not in normal tissues [12-20] Thisrestricted expression and the gelatinasecollagenase activitypoint to the possibility that FAPα might be a key cellsurface protease involved in promoting extracellular matrixdegradation in cancer invasion and wound healing [21] Arecent study showed the promotion of tumour growth byFAPα in an animal model [22] Moreover antibodies toFAPα were shown to inhibit tumour growth in this modelHowever a humanised anti-FAPα antibody showed noefficacy in a Phase II clinical trial for metastatic colorectalcancer [2324] Still the highly restricted expression andlack of overt developmental defects in FAPα-deficient mice[25] make FAPα a valuable target for the development oftherapeutics for cancer

nucleophilic serine is replaced with Asp and so it lackspeptidase activity [29] Despite the absence of this catalyticactivity DPP6 exerts an important developmental functionThe mouse white rump mutation which lacks expression ofthe DPP6 gene is embryonic lethal in homozygotes andcauses a pigmentation defect in heterozygotes [30] It isrecently established that DPP6 is a critical component ofneuronal A-type potassium channels [31] This biologicalrole of DPP6 possibly indicates that the other members ofthis subfamily have additional functions apart from theirpeptidase activity This is already shown for DPP IV (videinfra)

Dipeptidylpeptidase homologue DPP10 (DPP10) has asignificant sequence identity with DPP6 (51) DPP IV(32) and FAPα (29) Like DPP6 it has no peptidaseactivity due to a mutation of the catalytic serine to glycine[28] DPP10 is mainly expressed in the brain and pancreasIt has a predicted transmembrane region If this is confirmedDPP10 is a type II membrane protein like DPP IV FAPαand DPP6

Dipeptidyl peptidase 8 (DPP8) shares 27 amino acididentity and 51 amino acid similarity with the proteinsequences of DPP IV and FAPα and this increases to 35amino acid identity and 57 amino acid similarity in thehydrolase domain [9] Like DPP IV DPP8 hydrolyses thep-nitroanilide substrates Ala-Pro Arg-Pro and Gly-Pro witha neutral pH optimum and its tissue expression isubiquitous Unlike DPP IV and FAPα DPP8 is a solublemonomeric protein localised in the cytoplasm [26]

So the subfamily S9B contains next to the 4 peptidases(DPP IV FAPα DPP8 and DPP9) also two enzymes withno peptidase activity (the dipeptidylpeptidase homologuesDPP6 and DPP10) Phylogenetic analysis suggests thatDPP IV is clustered with FAPα DPP8 with DPP9 andDPP6 with DPP10 It is assumed that DPP8 and DPP9 arediverged from the others earlier in evolution DPP8 andDPP9 seem to be more closely related to ancient DPP IVenzymes found in bacteria nematodes and arthropods [28]After bioinformatic search of the human genome it isbelieved that all members of this subfamily have now beenidentified [927]

Dipeptidyl peptidase 9 (DPP9) shares 60 amino acididentity and 77 amino acid similarity with DPP8 DPP9is also ubiquitously expressed and was found in a solubleputative cytosolic form DPP9 is predicted to have DPP IV-like enzymatic activity because of the presence of theGWSYG serine protease motif and a Ser Asp His catalytictriad [27] This was later confirmed by the hydrolysis of afluorogenic Ala-Pro substrate [28]

Dipeptidylpeptidase homologue DPP6 (DPP6 DPP X)contains two of the catalytic residues (Asp His) but the





Mername AA0072


























H-XaaPro-Yaa- Yes






H-Xaa-ProYaa- No


H-ProXaa- No





























O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

O

N

O O

N O

O

O

O

Tyr54 7

N

NHis7 40

Asp708

3127

28

27

28

2526

26

28

32

33

31

27

31

Glu206

Glu2 05

Tyr631

Ser6 30

O O

O

N

N O

O

O

N

NHis7 40

Asp708

O

Tyr54 7

N O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

27

26

28

2830

27

30

37

28

Glu206

Glu2 05

Tyr631

Ser6 30

O

Tyr54 7

O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

N O

O

O

N

NHis7 40

Asp708

N

I3225

26

30

33

2732

25

30

31

28

Glu206

Glu2 05

Tyr631

Ser6 30

Arg643

N

N

N

O O

N

ON

O

N

N

O

R

N O

O

O

Tyr473

N

NHis680

Asp64 1

ON

3027

2927

38

28

Asn555

Ala55 4

A B

C

D
































O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR





Mername AA0072


























H-XaaPro-Yaa- Yes






H-Xaa-ProYaa- No


H-ProXaa- No





























O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

O

N

O O

N O

O

O

O

Tyr54 7

N

NHis7 40

Asp708

3127

28

27

28

2526

26

28

32

33

31

27

31

Glu206

Glu2 05

Tyr631

Ser6 30

O O

O

N

N O

O

O

N

NHis7 40

Asp708

O

Tyr54 7

N O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

27

26

28

2830

27

30

37

28

Glu206

Glu2 05

Tyr631

Ser6 30

O

Tyr54 7

O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

N O

O

O

N

NHis7 40

Asp708

N

I3225

26

30

33

2732

25

30

31

28

Glu206

Glu2 05

Tyr631

Ser6 30

Arg643

N

N

N

O O

N

ON

O

N

N

O

R

N O

O

O

Tyr473

N

NHis680

Asp64 1

ON

3027

2927

38

28

Asn555

Ala55 4

A B

C

D
































O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR




























O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

O

N

O O

N O

O

O

O

Tyr54 7

N

NHis7 40

Asp708

3127

28

27

28

2526

26

28

32

33

31

27

31

Glu206

Glu2 05

Tyr631

Ser6 30

O O

O

N

N O

O

O

N

NHis7 40

Asp708

O

Tyr54 7

N O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

27

26

28

2830

27

30

37

28

Glu206

Glu2 05

Tyr631

Ser6 30

O

Tyr54 7

O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

N O

O

O

N

NHis7 40

Asp708

N

I3225

26

30

33

2732

25

30

31

28

Glu206

Glu2 05

Tyr631

Ser6 30

Arg643

N

N

N

O O

N

ON

O

N

N

O

R

N O

O

O

Tyr473

N

NHis680

Asp64 1

ON

3027

2927

38

28

Asn555

Ala55 4

A B

C

D
































O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR









O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

O

N

O O

N O

O

O

O

Tyr54 7

N

NHis7 40

Asp708

3127

28

27

28

2526

26

28

32

33

31

27

31

Glu206

Glu2 05

Tyr631

Ser6 30

O O

O

N

N O

O

O

N

NHis7 40

Asp708

O

Tyr54 7

N O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

27

26

28

2830

27

30

37

28

Glu206

Glu2 05

Tyr631

Ser6 30

O

Tyr54 7

O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

N O

O

O

N

NHis7 40

Asp708

N

I3225

26

30

33

2732

25

30

31

28

Glu206

Glu2 05

Tyr631

Ser6 30

Arg643

N

N

N

O O

N

ON

O

N

N

O

R

N O

O

O

Tyr473

N

NHis680

Asp64 1

ON

3027

2927

38

28

Asn555

Ala55 4

A B

C

D
































O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR


O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

O

N

O O

N O

O

O

O

Tyr54 7

N

NHis7 40

Asp708

3127

28

27

28

2526

26

28

32

33

31

27

31

Glu206

Glu2 05

Tyr631

Ser6 30

O O

O

N

N O

O

O

N

NHis7 40

Asp708

O

Tyr54 7

N O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

27

26

28

2830

27

30

37

28

Glu206

Glu2 05

Tyr631

Ser6 30

O

Tyr54 7

O ON O

Asn710

O

O

O

N

O

NO N

N

N

Arg125

O

N

ON

O

N

N O

O

O

N

NHis7 40

Asp708

N

I3225

26

30

33

2732

25

30

31

28

Glu206

Glu2 05

Tyr631

Ser6 30

Arg643

N

N

N

O O

N

ON

O

N

N

O

R

N O

O

O

Tyr473

N

NHis680

Asp64 1

ON

3027

2927

38

28

Asn555

Ala55 4

A B

C

D
































O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR






























O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR
















O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR


O

N O

O

OH

H

OH

NH

N

NHis740

H

Asp708

O O

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NH

R

HH H

N

N

NH2

Arg12 5

H

H

H

H

N O

Asn710

H

H

-

- +

-Glu206

Glu205

Tyr631

Ser630

N

NHis74 0

H

Asp708

O O

H

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

N

O NHHH H

N

N

NH2

Arg125

H

H

H

HR

N O

Asn710

H

H

-

- +

-

-

+

Glu206

Glu20 5

Tyr63 1

Ser63 0

O

N O

O

OH

H

O

NH

O

Tyr547

H

O

O

O

HN

O

NHO

O

N

N+

R

O

HH H

N

N

NH2

Arg125

H

H

H N O

Asn710

H

H

N

NHis74 0

H

Asp708

O O

N

O NH

RH

H

-

- +

-Glu206

Glu20 5

Tyr63 1

Ser63 0

A

B

C


























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR
























NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR









NHN

OR

O

NH2N

B

OR

HOOH










N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR


N SH2 N

O

NH2N

O

NH2N

O COOH

NH2N

O

N XH2 N

O

NHR

NH2 N

O

F

N SH2N

SN S

H2 N

O

H

NH2

N XH2 N

O

H

NHS

O

O

R1 R2

NH2 N

O

R

HO

N SNH

O

N

O

N SNH

O

NH2

O

NH2N

OHN O

OH

O

2 (Ile-Thia P3298)

8

1 3 (Val-Pyrr)



7

9


11


13


















N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR














N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR


N NNH

O CN

HN

NC

NHHO

N

O CN

NNH

O CN

OO

OO

H2 N

F CN

NH2N

O CN

NH2N

O CN

NH2 N

O CN

HN O

O

O

H2N

CN

NH

N

O BHO OH

NO

BNH2

+

OH

OH

17 (NVP-DPP728)

18 (NVP-LAF237) 19

21

14 15 (FE 999011)

16

20

22 23










N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR


N NH

N

NN

N

O

O

N

HN

OH

H2N

OO N

HO

OH

O

HO

OH

OH N

HN

H2N

OO OH

N

O

O

O O

NH2

N

S

O

N

P

O

NH2

NHSO3H

NH2

O

NN

NN

N

O

OO

NH2

NH2

N

HN

O

O

NH2

N

NN

O

NS

O O

24

26 (TMC-2A) 27 (TSL-225)

28

30 31

25

29

32


















NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR











NH2N

OO N

H

O

O

NO2

H2 N

FO N

H

O

O

NO2

NH

N

O PO

O

O

NH

N

O PO

O

O

R

R

3334























































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR





















































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR






































824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR






















824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR






824 Chemokines











































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR







































10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR



























10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR


















10 CONCLUSION






ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR




ACKNOWLEDGEMENTS


REFERENCES




















































































221 455






















































































































































272-OR

























































221 455






















































































































































272-OR






















































































































































272-OR


























































































272-OR































272-OR

ChemInform Abstract: The Unique Properties of Dipeptidyl-Peptidase IV (DPP IV / CD26) and the Therapeutic Potential of DPP IV Inhibitors

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