PeptidylProlyl cis/trans Isomerase Inhibitors of small molecules

PeptidylProlyl cis/trans Isomerase Inhibitors of small molecules

Tadashi Mori and Takafumi Uchida

Molecular Enzymology, Graduate School of Agricultural Science, TohokuUniversity, Sendai, Japan.

Title:Peptidyl-Prolyl Isomerase Inhibitors of small molecules

Abstract

We would like to review the recent study of the inhibitors forpeptidyl-prolyl cis/trans isomerase (PPIase), such as cyclophilin (Cyp),FK506- binding protein (FKBP) and Pin1. The inhibitors of Cyp andFKBP, CsA and FK506 respectively are well known potentimmunosuppressive drugs. However, they cause a variety of side-effects. Therefore efforts are under way to identify PPIase inhibitorswith less side- effects. In this review, efforts of discovering smallmolecule inhibitors are emphasized. While Cyp and FKBP inhibitors havebeen explored fairly thoroughly, the number of efforts to screeninhibitors of the Pin1 is still limited so far. We think that theinhibitor of Pin1 has high potential as a drug.

Keywords

peptidyl-prolyl cis/trans isomerases; cyclophilin; FK-506 binding protein;Pin1; small molecule inhibitors.

INTRODUCTION

The peptidyl-prolyl cis/trans isomerases (PPIases) are a family ofubiquitous enzymes that catalyze the cis/trans isomerization of thepeptide bond on the N-terminal side of proline residues in proteins(Fig. (1)) [1]. PPIases are divided into three classes, cyclophilins(Cyps), FK-506 binding proteins (FKBPs) and the Pin1/parvulin class. Cyps and FKBPs are distinguished by their ability to bind theclinically immunosuppressive drugs cyclosporin and FK506, respectively[1, 2] . Upon binding of these drugs, there are two common outcomes:inhibition of the PPIase activity and inhibition of the common targetcalcineurin. The inhibition of calcineurin phosphatase activityprevents lymphocytes from responding to antigen-induced mitogenicsignals, thus resulting in immunosuppression. However, the inhibitionof the PPIase activity is apparently unrelated to theimmunosuppressive property of the drug/PPIase complexes. Recently, anumber of efforts to search non-immunosuppressive drugs to avoid side-effects and to treat new target class were reported.Members of the Pin1/parvulin class of PPIases bind neither of theseimmunosuppressive drugs, and are structurally unrelated to the othertwo classes of PPIases. Known members of the Pin1/parvulin classinclude Pin1 [3] and Pin4 (Par14) [4]. In general, PPIase activity has been assessed by assays based onprotease-coupled isomer-specific proteolysis using tetrapeptidederivatives, Ala-Xaa-Pro-Yaa-para-nitro-anilide by UV-Visspectroscopy[5] and 4-methylcoumaryl-7-amide (MCA) by fluorescencespectroscopy.[6, 7] The protease-free assay based on minor absorptioncoefficients of cis and trans conformers of tetrapeptide anilides remotefrom far UV peptide absorption has also been described.[8] Theuncatalyzed spontaneous cis/trans isomerization rate is extremely fast,making these activity-based assays not useful for HTS.Recently, we established the high-throughput screening (HTS) methodusing FDSS6000 real-time fluorescence detector [9]. This method wasmodified protease-coupled PPIase assay that had not been adapted HTS.Another assay procedures was a fluorescence polarization-based assayfor CypA[10] and FKBP[11] that can be adapted to HTS were reported.

The technique is based on competition and uses a fluorescein-labeledCsA and FK-506 analog and purified CypA and FKBP to quantitativelymeasure the binding capacity of non-labeled inhibitors. Detection byfluorescence polarization allows real-time measurement of bindingratios without separation steps. In this review, we focus on biological significance screeningstrategiesand a number of efforts to search non-immunosuppressivedrugs to avoid side-effects and to treat new target class rather thanimmunophiline inhibitors like CsA, FK-506 and Rapamicine. There arenumerous efforts to search for conformationally locked prolyl amidesubstrate analogues in the design of PPIase peptide-mimeticinhibitors. But, we would like to introduce the efforts to searchnovel structure of small molecule inhibitors to avoid side-effects andto treat new target class.FKBP INHIBITORS

Interestingly, FK506 and its open chain derivatives were shown todisplay neuroprotective and neuroregenerative effects in a wide rangeof animal models mimicking Parkinson disease, dementia, stroke, andnerve damage [12-19]. For example, FK506 administration resulted inprotection against ischemic brain injury [20], prevention of long termdepression in the rat hippocampus [21], modulation of long termpotentiation [22], prevention of N-methyl-D-aspartate receptordesensitization [23], alteration in neurotransmitter release [24], andattenuation of glutamate neurotoxicity ex vivo [25]. FK506 increasedneurite outgrowth in SH-SY5Y and PC12 cell cultures, and also inprimary cultures of chicken dorsal root ganglion and of hippocampalneurons [16, 26, 27]. However, the molecular mechanism of the FK506-mediated neuroprotection and neuroregeneration remains elusive. In general, the interpretation of effects caused by FK506 in cells isdifficult, because FK506 inhibits not only the enzymatic activity ofFKBPs, but also the protein phosphatase activity of calcineurin (CaN).CaN inhibition is mediated by complex formation with FK506·FKBPcomplexes and is thought to be the initial process leading toimmunosuppression [28-30]. CaN inhibition is used to prevent allograftrejection in transplantation medicine, to treat autoimmune diseases

and to circumvent graft-versus-host diseases. Additionally, inhibitionof the protein phosphatase was proposed basis of FK506-mediatedneuroprotection, because the FKBP ligand rapamycin, which has noeffects on CaN activity, did not exhibit neuroprotective properties[25, 31]. In contrast, monofunctional inhibitors of FKBPs, such as GPI1046,GPI1048, GPI1485 (Guilford Pharmaceuticals and Amgen), and V10,367(Vertex Pharmaceuticals) have been developed (Fig. (2)). Theseinhibitors do not affect CaN activity, while neuroprotective andneuroregenerative effects of FK506 areconserved. In the centralnervous system, GPI1046 promotes protection and sprouting ofserotonin-containing nerve fibers in the somatosensory cortexfollowing parachloramphetamine treatment, induces regenerativesprouting from spared nigro-striatal dopaminergic neurons followingMPTP toxicity in mice or 6-hydroxydopamine toxicity in rats, andalleviates the rotational abnormality in 6-hydroxydopamine-treatedrats [16, 27]. Other monofunctional FK506 derivatives increasebranching from developing dopamine neurons in culture, enhance neuriteoutgrowth of fetal dopamine transplants, increase nerve regeneration,and accelerate functional recovery following peripheral nerve injury[32, 33].

FK506-BINDING DOMAIN ANALOGUES

The greatest number of designed FKBP ligands arose from the dual-domain concept. Schreiber [34] established that FK506 comprises twodistinct binding domains (Fig. (2)). One portion of the molecule bindsat the prolyl isomerase active site of FKBP-12. The remainder of theFK506 molecule forms part of the calcineurin-binding domain of theFK506/FKBP-12 complex, and has been termed the “effector” domain.Compounds capable of binding to the enzymatic active site of FKBP-12but lacking an effector domain would be expected to function asnonimmunosuppressive FKBP-12 ligands. Using the principles ofstructure-based drug design, we synthesized a number of potent,selective small molecule inhibitors of FKBP-12 that are devoid ofimmunosuppressive activity but possess potent neurotrophic activity in

vitro and in vivo. GPI-1046 is typical of this new class of smallmolecule neurotrophic drugs. Among the large number of FKBP inhibitors, V-10,367 is the onlysubnanomolar inhibitor of FKBP12 (Ki = 0.5 nM) identified since FK506(Ki = 0.6 nM)[35, 36]. In agreement with the potency of V-10,367, the1.8 Å resolution crystal structure of the FKBP-ligand complex revealeda strong similarity in conformation of V10,367 and FK506 in the PPIaseactive site.[36] V10,367 presented no immunosuppressive activity, butgreatly increased nerve regeneration in SH-SY5Y neuroblastoma cells.[37]

DESIGNED FKBP INHIBITORS

Several FKBP inhibitors structurally different from FK506 weredesigned. Most of them presented FKBP inhibition in the micromolarrange, although nanomolar inhibition has been attained in severalstudies. These designs were based on the best peptide substrate ofFKBP [38], the crystallographic structures of FKBP unbound or boundwith FK506. Another approach was also performed, that is NMR-basedscreening To discover high-affinity ligands for the FKBP, a linked-fragment approach was applied [39]. Using heteromuclear/protoncorrelation spectra to screen a library of test compounds, a number ofstructurally diverse small molecules which bind to FK506 binding siteor proximal sites on FKBP have been identified. For example, the bestfit compound, compound12 showed high affinity to FKBP (Kd = 19nM) (Fig.(3)).

HTS SCREENING STRATEGIES

There are two HTS screening strategies were reported so far. The FPassay was validated by comparing the values of the dissociationconstants with their PPIase inhibition for a selection of compounds,such as FK506, GPI-1046 and V-10,367. The Ki values found by the twomethods are comparable.[40] Thus it is useful for screening FKBP-binding compounds using a high-throughput fluorescence polarizationassay.

Another high throughput scintillation proximity assay (SPA) was alsodeveloped to identify novel ligands of FKBP-12[41]. Recombinanthistidine-tagged FKBP-12 was immobilized to SPA beads by the antibodythat recognizes the histidine tag of the recombinant protein. Using 1nM [3H] FK506, the well-known macrolide ligand of FKBP-12. Analysis ofsaturation and homologous displacement isotherms indicated theexistence of a single binding site with a KD value of 1.6 nM. Thespecificity of [3H] FK506 binding was demonstrated in displacementexperiments and showed that rapamycin, another macrolide ligand, is aspotent as FK506 (IC50 of 3.5 and 3.2 nM, respectively). The highsignal-to-noise ratio as well as the small standard deviations makesthis novel assay well suited for the automated high throughputscreening. A number of small molecules inhibitors of FKBP werereported. But clinically effective compounds are only analogues of theFKBP binding domain of FK-506.

CYP INHIBITORS

Cyclophilins (Cyps) are abundant proteins with peptidyl-prolylcis/trans isomerase activity [42]. Cyps are present in every organ ata concentration of 1 μg/mg protein. In the blood, most Cyps areassociated with cellular compartments such as erythrocytes andleukocytes [43]. There are seven major Cyps in humans. CypA is foundin the cell cytosol and is the most abundant of the Cyps, representingbetween 0.1 and 0.4% of total cellular proteins [44]. CypA plays arole in the replication of viruses such as vaccinia [45] and HIV-1.CypA is specifically incorporated into nascent HIV-1 viruses bybinding to the capsid region of the Gag precursor protein [46]. Allother Cyp isoforms share > 50% sequence homology with CypA but theydiffer from CypA in their subcellular localizations and bindingaffinities. Human cyclophilins have become interesting drug targets.CypA and CypB are implicated in the life cycle of HIV [47] and [48].Mitochondrial CypD is another possible drug target because of itsapparent gatekeeper function in the transmembrane adenosine nucleotidetranslocase complex [49, 50]. Cell death mediated ischaemia may resultfrom shock or stroke and CypD provides a possible target to preventorgan damage after such injury [51]. A number of cyclosporins,including certain non-immunosuppressive derivatives, are also potentinhibitors of the human malarial parasite Plasmodium falciparum [52, 53].The most potent of these has anti-malarial activity matching that ofestablished anti-malarial drugs. Cyclosporins additionally possesspotent anti-nematode effects [53]. Cyps, such as CypA and CypB, areoverexpressed in some cancer cells and may play a role in cancerpathogenesis [54]. In recent years, it has become clear that Cyps playa significant role in the replication of hepatitis C virus (HCV).Initial anti-HCV in vitro data were obtained with CsA, which was the firstmolecule known with significant Cyp-inhibiting properties. Its anti-HCV activity seemed to be independent of its immune suppressionfunction and to be related to its Cyp-inhibitory action [55]. Theanti-HCV effect of CsA was confirmed in pilot studies in patients[56],[57], but further development of CsA in this indication wasprohibited because of its primary immunosuppressive effect. However,

these findings led to the design of selective Cyp inhibitors such asDebio-025 and NIM-811, which do not bind to CaN and, as a result, haveno further immunosuppressive activity. These drugs have shownimportant anti HCV effect in vitro [48, 58, 59]There are numerous of Cyp inhibitors are reported, but most of theinhibitors has cyclic peptide structure and still has large molecularweight, so that the discoveries of small molecule Cyp inhibitors,which do not bind to CaN, are still required.

STRUCTURE-BASED DESIGN AND VIRTUAL SCREENING

Designing small-molecule cyclophilin inhibitors is challengingbecause of the lack of appropriate non-peptidic ligands forcyclophilin to serve as starting points for rational drug design, aswas the case for FKBP12. CsA and its assorted analogues are all largecyclic peptides that are complex and labor-intensive to synthesize. Asis often the case of a study of Cyp inhibitor screening, thestructure-based rational approach has been implemented in utilizingthe tools of structural biology and computational chemistry inconjunction with combinatorial chemistry and synthetic organicchemistry. [60] Key pieces of information to begin the work were (a) the crystalstructure of CsA bound to CypA (as well as several dipeptides bound toCypA) and (b) the knowledge of the active site of cyclophilin gainedfrom these structures [61-63]. These information were utilized in a“virtual screening” approach for discovery of initial lead structures.These compounds, inhibited the PPIase activity of Cyp with IC50 valuesranging from hundred of nanomolar to several micromolar. [64] Thesecompounds inhibited the PPIase activity of Cyp with IC50 valuesranging from ten nanomolar to several micromolar. Recently Yang, Y. et al reported the database-mining program LIDAEUSand in silico screening to discover the dimedone family of inhibitorswhich show a conserved ‘ball and socket’ binding mode with a dimethylgroup in the hydrophobic binding pocket of human cyclophilin A (CypA)mimicking a key interaction of the natural inhibitor cyclosporin A(CsA). This is the only report to show the X-ray structure of the

cyclophilin–small moleculer complex (Fig. (4)) [65]. A number of clinically effective cyclic peptide inhibitors of CypAwere reported. But clinically effective small molecule inhibitorsobtained from these studies have not been reported so far.

HTS SCREENING STRATEGIES

There are two HTS screening strategies were reported so far. A fluorescence polarization-based assay[66] for CypA is based oncompetition and uses a fluorescein-labeled CsA analog and purifiedCypA to quantitatively measure the binding capacity of nonlabeledinhibitors. Detection by fluorescence polarization allows real-timemeasurement of binding ratios without separation steps. The FP assayuses a fluorescein-labeled CsA analog as probe. The parallel andperpendicular intensities of the probe are linear from 0.01 to 2 nM.The mP signals are between 240 and 70 and reach a plateau at 2 nM. Theprobe binds to CypA at a Kd value of 500 nM. The FP competition assayprovides an accurate activity determination for CypA inhibitors andgives results comparable to those of the coupled spectrophotometric,coupled fluorogenic assays. It is useful for screening CypA-bindingcompounds using a high-throughput fluorescence polarization assay.Recently, we established the high-throughput screening (HTS) methodusing FDSS6000 real-time fluorescence detector[9]. This method wasmodified protease-coupled PPIase assay that has not been adapted HTS.In part because the uncatalyzed spontaneous cis/trans isomerizationrate is extremely fast, with a half-life of 74 s at 10 °C. Measuringthe reaction at low temperature requires prolonged detection time. Although the inhibitors structures has not been reported in these twoassays, but these two high-throughput assay system may be a powerfultool for the discovery of PPIases inhibitors.Pin1 inhibitors

The most widely studied member of the parvulin family, human Pin1,distinguishes itself from Cyp and FKBP through its unique substratespecificity for the phosphorylated Ser/Thr-Pro motif [67-73]. Thisphosphorylation-dependent interaction targets Pin1 to a subset of

phosphorylated substrates, including Cdc25, Wee 1, Myt1, Tau-Rad4, andthe C-terminal domain of RNA polymerase II large domain [69, 71, 74].In addition, Pin1 contains an N-terminal WW domain, which functions asa phosphorylated Ser/Thre-Pro binding module [75]. Another feature isthat, unlike Cyp and FKBP, parvulin is not known to bind anyimmunosuppressant drug. The specificity of Pin1 activity is essential for cell growth;depletion or mutations of Pin1 cause growth arrest, affect cell cyclecheckpoints and induce premature mitotic entry, mitotic arrest andapoptosis in human tumor cells, yeast or Xenopus extracts [76-78]. Inaddition, Pin1 is dramatically misregulated in human cancer samples.Moreover, inhibition of Pin1 by various approaches, including Pin1antisense polynucleotides or genetic depletion, kills human and yeastdividing cells by inducing premature mitotic entry and apoptosis.Further, Pin1 has been shown to be involved with the progression ofneurodegenerative diseases such as Alzheimer's' disease. Thus, Pin1-catalyzed prolyl isomerization regulates the conformationand function of these phosphoprotein substrates and facilitatesdephosphorylation because of the conformational specificity of somephosphatases. Thus, Pin1-dependent peptide bond isomerization is acritical post-phosphorylation regulatory mechanism, allowing cells toturn phosphoprotein function on or off with high efficiency andspecificity during temporally regulated events, including the cellcycle [76].

STRATEGIES TO FIND SMALL MOLECULE INHIBITORS

Pin1 inhibitors have special significance because overexpression ofPin1 was detected in a large number of cancer cell types compared withnormal cell types.[79] A few parvulin inhibitors have been reported,including designed alkene amide bond isosteres,[80] dipeptide or (D)-Ser–Pro peptide inhibitors,[81] pericyclic aromatic compounds,[6] andthe natural product, Juglone.[82] For those inhibitors for which cell-based antiproliferative activity was tested, all were remarkablyactive and their activities were within the same order of magnitude astheir parvulin inhibition results. [6, 80]

NATURAL PRODUCT OF PIN1 INHIBITOR

Unlike Cyp and FKBP, parvulin is not known to bind any cyclic peptideligands. Only the small molecule inhibitor Juglone (5-hydroxy-1,4-naphthoquinone) (Fig. (5)) was reported by the natural product foundby screening a collection of pure secondary metabolites against thePPIase activity of E. coli parvulin. [82] Juglone irreversiblyinhibits the PPIase activity of several parvulins such as E. coliparvulin, the yeast Ess1/Ptf1, and human Pin1, but not of Cyp or FKBP.The inactivation mechanism of E. coli parvulin involves a Michaeladdition of the thiol groups of Cys41 and Cys69 of E. coli parvulin tojuglone, followed by structural alterations of the active site. [82]Although Juglone differentiaties the parvulin family of PPIases fromCyP and FKBP, the nonspecific inhibition activity of juglone towardother enzymes, such as pyruvate decarboxylase, glutathione-S-transferase, and RNA polymerases, [83] precludes it as a reliableanticancer therapy. It is critical to keep in mind that juglone canaffect many other cellular proteins by covalently inactivation activecysteines. Therefore, the specificity of Juglone for Pin1 in the cellis highly questionable.

SCREENED INHIBITORS OF PIN1

We screened 1000 chemically synthesized compounds for Pin1 inhibitionusing the substrate Suc–Ala–Glu–Pro–Phe–7-(4-methylcoumaryl) amide (–MCA). [6] This compound library contains randomly synthesizedcompounds and compounds that were synthesized for potential anticanceror antifungal activities. Among them, several symmetric compoundscontaining fused aromatic rings inhibited Pin1 PPIase activity. Themost potent inhibitors of this series, compounds PiB (Fig. (6A))presented IC50 values of 1.5 μM for Pin1. Molecular modeling revealeda close interaction of the inhibitors with the active site of Pin1 orPar14, suggesting possible competitive inhibition. These inhibitorshad antiproliferative activity against a variety of cancer cell lines.

The sensitivity of various cancer cell lines to those inhibitorscorrelates with the Pin1 and Par14 expression levels in these cancercell lines, again a promising sign for potential anticancer drugs.Cells with low levels of Pin1 were less sensitive to inhibitors thancell expressing high levels of Pin1. Pin1 inhibition by compound PiBcorrelated with its antiproliferative activity. Recently, weinvestigate nanomolar small molecule inhibitor DTM (Fig. (6B)) [84],which is though competitive inhibitor of Pin1.By screening a combinatorial peptide library, a series of nanomolarpeptidec inhibitors of Pin1 were identified Nonproteinogenic aminoacids, incorporated into 5-mer to 8-mer oligopeptides containing d-phosho-othreonine as a central template, yielded selective inhibitorsthat blocked cell cycle progression in HeLa cells in a dose dependentmanner [85].Bayer et al. screend Pepticinnamin E analogs which were inducedapoptosis. In an attempt to identify the possible cellular targets ofthese inducers of apoptosis, the involvement of Pin1 in this processand the established link between Pin1 and the Ras [86] suggested toinvestigate the compound library for possible Pin1 inhibition. Threeof the apoptosis-inducing compounds turned out to be potent Pin1inhibitors displaying IC50 values in the nanomolar to single-digitmicromolar range [87]. However, no further developments were reported regarding to Pin1inhibitors. This lack of efficient inhibitors, the identification anddevelopment of a compound class that might give rise to potent Pin1inhibitors as well as insights into the mechanism that underlies theinhibition are of major importance to current medicinal chemistry andchemical biology research.

CONCLUDING REMARKS

In conclusion, the number of efforts to identify PPIases smallmolecule inhibitors has been reported. But, only FKBP inhibitors havebeen reported to clinically effective. The number of HTS efforts toscreen inhibitors of PPIases is still limited so far. We think thatthe inhibitor of Pin1 also has high potential as a drug.

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Fig. (1). Mechanism of peptidyl-prolyl cis/trans isomerases

Fig. (2). FK506-Binding Domain Analogues, V-10367 and GPI1046

Fig. (3). Comp12 (Kd = 19nM) identified with the NMR-based screening

Fig. (4). Compound5 ; that was reported the X-ray structure of thecyclophilin–small moleculer complex [65]

Fig. (5). Natural product of small molecule Pin1 inhibitor Jugrone

Fig. (6). Pin1 inhibitors derived from PPIase screening .A: PiB and B:DTM