Massive screening yields novel and selective Trypanosoma cruzi triosephosphate isomerase dimer-interface-irreversible inhibitors with anti-trypanosomal activity

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European Journal of Medicinal Chemistry 45 (2010) 5767e5772

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Massive screening yields novel and selective Trypanosoma cruzi triosephosphateisomerase dimer-interface-irreversible inhibitors with anti-trypanosomal activity

Guzmán Álvarez a, Beatriz Aguirre-López b, Javier Varela a, Mauricio Cabrera a, Alicia Merlino a,Gloria V. López a, María Laura Lavaggi a, Williams Porcal a, Rossanna Di Maio a, Mercedes González a,*,Hugo Cerecetto a,*, Nallely Cabrera b, Ruy Pérez-Montfort b,**, Marieta Tuena de Gómez-Puyou b,Armando Gómez-Puyou b,**

aGrupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Montevideo, UruguaybDepartamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

a r t i c l e i n f o

Article history:Received 23 August 2010Received in revised form14 September 2010Accepted 15 September 2010Available online 19 September 2010

Keywords:T. cruziTIMMassive screeningDrug discovery

* Corresponding authors. Iguá 4225, Facultad deUruguay. Tel.: þ598 2 5258618; fax: þ598 2 5250749** Corresponding authors. Universidad Nacional AutPostal 70243, C. P. 04510, México DF, México, Mexicoþ5255 56225630.

E-mail addresses: [email protected] (M. GonzáCerecetto), [email protected] (R. Pérez-Montfort)Gómez-Puyou).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.09.034

a b s t r a c t

Triosephosphate isomerase from Trypanosoma cruzi (TcTIM), an enzyme in the glycolytic pathway thatexhibits high catalytic rates of glyceraldehyde-3-phosphate- and dihydroxyacetone-phosphate-isomer-ization only in its dimeric form, was screened against an in-house chemical library containing nearly 230compounds belonging to different chemotypes. After secondary screening, twenty-six compounds fromeight different chemotypes were identified as screening positives. Four compounds displayed selectivityfor TcTIM over TIM from Homo sapiens and, concomitantly, in vitro activity against T. cruzi.

� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Trypanosoma cruzi (T. cruzi), the causative agent of Chagas’disease, which is widely disseminated in Central and SouthAmerica, represents an endemic disease in 21 countries located inthis geographic region. It has been estimated that this diseaseaffects 9.8e11.0 million people, and 60.0 million are at risk [1,2].Currently available therapies are inadequate due to issues involvingsafety, efficacy, resistance, toxicity, difficulty of administration inimpoverished conditions, and cost. Despite the urgent need for newdrugs, most pharmaceutical companies have neglected this disease,largely due to market policies. An important characteristic in themetabolism of T. cruzi is its dependence on glycolysis as an energysource for cellular survival. Thus, enzymes in this pathway

Ciencias, 11400 Montevideo,.ónoma de México, Apartado. Tel.: þ5255 56225629; fax:

lez), [email protected] (H., [email protected] (A.

son SAS. All rights reserved.

represent excellent targets for the search of small molecules thatinhibit them selectively and affect their metabolic function.

In this sense, triosephosphate isomerase (TIM) has beenproposed as a target for drug design against this disease [3e5]. TIMcatalyzes the isomerization of glyceraldehyde-3-phosphate anddihydroxyacetone phosphate in the fifth step of the glycolyticpathway. Structurally, most of the known TIMs are homodimers,each monomer consisting of eight parallel b-strands, surroundedby eight a-helices and forming a barrel. The interface betweenmonomers occupies a significant portion of the molecular surfacearea of each monomer, around 1496�A2 in TIM from T. cruzi (TcTIM)[6]. Interestingly, TIM is active only in its dimeric form [7,8];therefore the use of small molecules for targeting its interface maypotentially induce structural modifications and alter the integrity ofthe dimer provoking enzyme inactivation. Homo sapiens TIM(HTIM) and the enzymes of the aforementioned parasites have thesame catalytic residues. In contrast, the identity of the approxi-mately 32 interfacial residues of the TIM from either parasite andHTIM is approximately 52%.Whereas, the identity of the amino acidresidues of the interface of TcTIM and the enzyme from Trypano-soma brucei is approximately 82% [9]. Therefore, it is theoreticallypossible to find molecules that exhibit high specificity for theinterface of oligomeric enzymes from parasites.

S

NH2N

S

NNH2 S

H2N

SNH2(I) (II)

Fig. 1. Structures of previously described TcTIM-inhibitors.

G. Álvarez et al. / European Journal of Medicinal Chemistry 45 (2010) 5767e57725768

As part of our ongoing program in the search of molecules thatcould provide leads in the design of a new drug for the treatment ofChagas’ disease [9e11], we undertook a massive screening forinhibitors of TcTIM. Initially we performed a primary screening ofnearly 230 compounds from an in-house library, then TIM-IC50 andTcTIM-selectivity were determined for the best inhibitors, andfinally from these studies some compounds were selected tosubmit to anti-proliferative T. cruzi analyses.

2. Methods, results and discussion

2.1. Chemistry

The studied compounds were selected using the followingcriteria: i) agents belonging to anti-T. cruzi active chemotypes, or/andii) symmetrical and benzo-containing agents, structurally related topreviouslydescribedTcTIMinhibitors (i.e. (I) and (II), Fig.1) [9,10]. Thecompounds belonging to fourteen chemotypes were [11,12]: i)heterocycles with previously described anti-T. cruzi activity, 1. ben-zofuroxanes, 2. furanes and thiophenes, 3. 4-substituted-1,2,6-thia-diazines and 4. synthetic precursors, 5. quinoxaline 1,4-dioxides, 6.phenazine 5,9-dioxides, 7. furoxanes, 8. imidazole N-oxide, 9. inda-zoles; ii) heterocycles without previously described anti-T. cruziactivity, 10. thiazoles, 11. 1,2,4-triazine N-oxides; iii) miscellaneousderivatives, 12. flavonoids, 13. nitroalkenes, and 14. miscellaneous(Fig. 2, Table S1 in Supplementary Content section).

NONO

R1

Chemotype 1

(benzofuroxanes)compd. 1-45

XY R2

Chemotype 2

(furanes and thiophenes)compd. 46-92

NSN

R4

O

O

R3

R3

O

O

Chemotype 3

(4-substituted-1,2,6-thiadiazincompd. 93-101

NON

R12 R11

O

Chemotype 6

(phenazine 5,9-dioxides)compd. 118-121

Chemotype 7

(furoxanes)compd. 122-144

C

(imidco

N

N

O

O

R10 R8

R9 N

N

R

RR16

R15

O

O

N S

NR24 R23

Chemotype 10

(thiazoles)compd. 176-184

Chemotype 1

(1,2,4-triazine N-ocompd. 185-19

N S

ZR25 O

S

NNR26

R27

N

NNH3C

R

O

O

n

Fig. 2. General structures of the different ch

2.2. Biological characterization

2.2.1. Studies against TIMsInitially, the 229 compounds were tested at 400.0 mM using

percentages of irreversible inhibition higher than 70% as an arbi-trary cut-off point. In this first stage of the analysis (see Table S1 inSupplementary Content section), 46 compounds (20%) from all thechemotypes, except 5, 9 and 11, were examined in a second phase.Secondly, compounds were analyzed at 100.0 mM and 50% inacti-vationwas used as the cut-off point. Twenty-six compounds withineight different chemotypes were identified as potential irreversibleinhibitors in these low concentration doses. The highlighted che-motypes, ordered according to the percentage of compounds withinhibitory activity, were thiazoles (67%), 1,2,6-thiadiazines (56%),nitroalkenes (50%), and phenazine 5,9-dioxides (25%).

Twenty-five compounds were selected to determine their IC50.Fig. 3 shows the analyzed compounds, ordered according to therange of concentrations in which they are active. In this study, weobserved that the best irreversible inhibitors belong to chemotypes3 (1,2,6-thiadiazines), 6 (phenazine 5,9-dioxides), and 10 (thia-zoles). Table 1 shows the values for the best irreversible inhibitoragents of TcTIM (IC50 < 30 mM). 1,2,6-Thiadiazine 97, phenazine5,9-dioxide 118, and the two different thiazoles, 176 (1,2,4-thia-diazole) and 177 (1,3,4-oxathiazole), were considered for furtherselectivity studies using HTIM (Table 1).

Clearly, all the studied compounds had selectivity greater than 4against the parasite-enzyme; thiadiazole 176 was the agent withthe lowest discriminatory capacity.

Data analysis revealed that the best irreversible inhibitors ofTcTIM have: i) aromatic systems connected by a single bond, likethe bis(benzothiazole) (I) (Fig. 1), i.e. compounds 125,176e181,195,and 208 (Fig. 2); ii) electrophilic-moieties, like the disulfide (II)(Fig. 1), i.e. Michael-acceptors 2, 8, 48, 93, 95e98, 201, 203, and 205,or thiosemicarbazones 5, 52, and 124 in their structures. It has been

es)

NHSNH O

O NSN

O

O

R3

R3

O

OR3

R3

Chemotype 4

(chemotype 3 synthetic precursors)compd. 102-111

N

N

O

O

R5

R6

R7

Chemotype 5

(quinoxaline 1,4-dioxides)compd. 112-117

hemotype 8

azole N-oxide)mpd. 145-163

Chemotype 9

(indazoles)compd. 164-175

14

13

N

N

R18

R17R19

O

NN

O

R20

CN

NN

O2N

R21

OR22

1

xides)4

Chemotype 12: flavonoids, compd. 195-200Chemotype 13: nitroalkenes, compd. 201-204Chemotype 14: others, compd. 205-229

R3

emotypes studied as TcTIM inhibitors.

Fig. 3. Structures of the best irreversible inhibitors of TcTIM identified herein. The compounds are numbered according to Table S1 (see Supplementary Content section).

G. Álvarez et al. / European Journal of Medicinal Chemistry 45 (2010) 5767e5772 5769

described that the bisaromatic motive, in (I), could interact with theinterface of TcTIM [10,13] and dock into the pocket formed by thearomatic clusters [14]. We observed that not only the absence ofthis motive, but also the absence of a polar group produceda reduction in the inhibition capability (see examples in Fig. 4a). For(II) it has been evidenced [9], by X-ray crystallography, that theelectrophilic disulfide reacts with TcTIM Cys118. We also observedthat the absence of a Michael-acceptor group promotes a completelack of inhibition capability (see examples in Fig. 4b).

2.2.2. Anti-T. cruzi studiesThe irreversible inhibitors of TcTIM 2, 5, 8, 46, 48, 52, 93, 95e98,

and 208 (Fig. 3) were previously evaluated against the parasite, T.cruzi, and with the exception of compound 208 all of them dis-played anti-parasite activity [12]. Besides, to complete the study ofthe anti-proliferative T. cruzi activities, all the compounds shown inTable 1, together with other relevant derivatives, were then testedagainst T. cruzi. The compounds were initially tested in vitro against

Table 1TIM-inhibition results for selected compounds.

Compd. TcTIM IC50 (mM)a HTIM percentage ofinhibition at 100 mMa

93 20.0 � 2.0 nsb

97 13.0 � 1.2 0.0118 26.0 � 2.5 0.0176 3.5 � 0.5 59.0177 10.0 � 1.0 0.0

a Values are means of two experiments.b ns: not studied.

the epimastigote form of T. cruzi, Tulahuen 2 strain. The existence ofthe epimastigote form of T. cruzi as an obligate mammalian intra-cellular stage has been revisited and confirmed [15,16]. Thecompounds were incorporated into the media at 25 mM and theirability to inhibit the parasite growth (PGI, Table 2) was evaluated incomparison to the control (no drug added to the media) at day 5.Then the ID50 doses (50% inhibitory dose) were determined for allof them (Table 2). Bnz was used as the reference trypanosomicidalagents. Some derivatives were also studied against the high viru-lent [17,18] CL Brener clone and against Y strain, a Nifurtimox- andBnz-partially resistant strain [19,20] (Table 2).

All compounds, except 176, displayed anti-T. cruzi activity at theinitial assayed dose, 25.0 mM, with some of them having micro-molar ID50 which is similar to Bnz, the reference drug (Table 2).Phenazine 5,9-dioxide 118 was the best anti-T. cruzi agent for theTulahuen 2 strain and also showed activity against other studiedstrains. Contrarily to a previous description [12], in our assay,derivatives belonging to 1,2,6-thiadiazine-chemotype, 93 and 97,exhibited modest anti-parasite activities. On the other hand,derivatives belonging to thiazole-chemotype,177,178,180, and 181,showed significant anti-T. cruzi activities with sulfone 180 beingone of the most active compounds analyzed. In this sense, o-ben-zoquinone dioximes 205e207 (Table 2) displayed good to excellentanti-T. cruzi behaviour. Consequently, thiazole- and o-benzoqui-none dioxime-chemotypes emerge as new structural motives ofchoice for further chemical modifications in order to improve theiranti-T. cruzi activities.

For compounds 181 and 205 the ID50 values, against T. cruzi,were moderately lower than their IC50 against TcTIM (close to100 mM). However, compounds 118, 180, 203, 206, and 207 werenoticeably more active against the parasite than in their inhibitory

NSN

OO

O O

NO2

HNSHN

O O

N S

N

N

NCl

Cl

OH

O

O

NONO

HN

125

Inh. = 72 %

NON

CH3HN

132

Inh. = 41 %

O

S

NNH3C

HO

181

Inh. = 74 %184

Inh. = 29 %

118

Inh. = 84 %

N

NBr O

O

O

121

Inh. = 50 %

O

Cl

95

Inh. = 85 %

a

b

104

Inh. = 0 %

NSN

OO

O O

111

Inh. = 0 %

Fig. 4. Some observed structureeactivity relationships. The expressed percentage ofTcTIM-inhibitions is at 400 mM. The compounds are numbered according to Table S1(see Supplementary Content section).

Table 2Anti-T. cruzi activity results for selected compounds.

Compd. TcTIM IC50 (mM) T. cruzi straina PGI (%)b ID50 (mM)c

93 IC50 < 30 T2 22.2 50 � 197 T2 20.8 50 � 2118 T2 100.0 2.9 � 0.3

Y 100.0 5.5 � 0.6CLB 100.0 2.0 � 0.2

176 T2 0.0 >25.0177 T2 59.0 22.0 � 1.0178 30 < IC50 < 100 T2 25.5 >25.0124 IC50 w 100 T2 24.7 >25.0125 T2 25.0 >25.0180 T2 100.0 5.0 � 0.5181 T2 50.0 25.0 � 1.0195 T2 19.1 >25.0201 T2 14.6 >25.0203 T2 100.0 5.1 � 0.6205 T2 50.0 25.0 � 2.0206d IC50 ˃ 100 CLB 100.0 4.7 � 0.5207d CLB 100.0 5.0 � 0.5Bnze T2 100.0 7.4 � 0.7

a CLB: CL Brener clone; T2: Tulahuen 2 strain; Y: Y strain.b PGI: percentage of growth inhibition at 25 mM.c Values are means of two experiments.d For chemical structures see Table S1 (Supplementary Content section).e Bnz: benznidazole.

Fig. 5. Lipophilicity, calculated in term of miLogP [21], of some of the best TcTIMirreversible inhibitors and molecular lipophilicity potential (MLP), virtual LogP [22,23],for 176, and 180. Molecules 176 and 180 are represented as tubes with the traditionalatom colors and MLP colors: red/yellow for hydrophilic regions; violet/blue for lipo-philic regions; green for intermediate regions. The dot-arrow shows the polar regionfor 176 and complete-arrows the lipophilic regions for 180 (for interpretation of thereferences to colour in this figure legend, the reader is referred to the web version ofthis article).

G. Álvarez et al. / European Journal of Medicinal Chemistry 45 (2010) 5767e57725770

capacity of TcTIM, perhaps indicating some off-target effects. Aspecial aspect was evidenced with the biological behaviour of thebest irreversible inhibitors of TcTIM 93, 97, 176, and 177, they wereless active against the whole parasite than against the enzyme.According to these derivative structures, the little anti-parasiticactivity could be explained by means of their high hydrophilicity(Fig. 5, [21e23]). This fact could promote low penetration in thewhole organism and consequently low disposability in TcTIM. Onthe other hand, when the activities of thiadiazoles 176, and 180, andoxathiazole 177 are analyzed, some relevant aspects could bepointed out. Being the best TcTIM-inhibitors, the first two are lessactive against the whole parasite, while the third compound wasone of the most active against T. cruzi with moderate TcTIM-inhi-bition activity (IC50 near to 100 mM). Again, their different lip-ophilicities could explain the activity against the parasite, with 180having the highest miLogP, 4.48, and being able to adequatelypenetrate into T. cruzi cells. However,180 is a weak TcTIM-inhibitor.Interestingly, when we analyzed the bio-transformation of sulfone180 inside the parasite we detected derivative 176 as the mainmetabolic product (Fig. S1, Supplementary Content section).Consequently, thiadiazole 180 could act as a pro-drug that, afterparasite intervention, delivers thiadiazolone 176, an excellentTcTIM-inhibitor (Fig. 6).

3. Conclusions

In conclusion, we have performed a massive screening againstTcTIM to identify novel interface-disruptors as potential anti-T. cruzidrugs. We identified four compounds, belonging to three differentchemotypes (1,2,6-thiadiazines, phenazine 5,9-dioxides, and thia-zoles) that displayed selectivity for TcTIM over HTIM, and some ofthem displayed activity against T. cruzi in a whole organism assay.Between these, the phenazine 118 possesses the best anti-T. cruziactivity. It is worth noting that these studies allowed identifyingsulfone 180 as a pro-drug capable of biotransforming inside theparasite to an excellent TcTIM-inhibitor. Focused library synthesisof some of these compound chemotypes, biochemical and QSARstudies is currently in progress.

N S

N SO2H3C

CH3

N S

NH

OH3C

Trypanosoma cruzi

TcTIM

N S

N SO2H3C

CH3

biotransformation

limited penetration

180

176

N S

NH

OH3C

biotransformation

Fig. 6. Speculative mechanism of action of sulfone 180, as pro-drug of the excellent TcTIM-inhibitor 176.

G. Álvarez et al. / European Journal of Medicinal Chemistry 45 (2010) 5767e5772 5771

4. Experimental

4.1. Chemistry

All the studied compounds belonged to our in-house library.They were previously synthesized and structurally characterized byordinary spectroscopic techniques [12].

4.2. Biology

4.2.1. TIM inhibition studiesEnzymes: Both TIMs were recombinant products. TcTIM and

HTIM were expressed in Escherichia coli and purified as described[24,25]. After purification, the enzymes were dissolved in 100 mMtriethanolamine, 10 mM EDTA and 1 mM dithiothreitol (pH 8),precipitated with ammonium sulfate (75% saturation) and stored at4 �C. Before use, the enzymes were extensively dialyzed against100 mM triethanolamine/10 mM EDTA (pH 7.4).

Protein concentration: This was calculated as reported elsewhere[26]. The molar extinction coefficients for TcTIM and HTIM were36,440 and 33,460 M�1 cm�1, respectively.

TIM activity assays: Activity was assayed in the direction ofglyceraldehyde 3-phosphate to dihydroxyacetone phosphate asdescribed elsewhere [3] with 5 ng of the enzymes. For inactivationstudies, TIMs were incubated at a concentration of 5 mg/mL ina buffer containing 100 mM triethanolamine, 10 mM EDTA, pH 7.4and 10% of dimethyl sulfoxide (DMSO) at 36 �C. The mixture alsocontained the compounds at the indicated concentrations.Compoundswere dissolved in DMSO. After 2 h,1 mL was withdrawnan added to 1 mL of reaction mixture for the activity assay. None ofthe molecules tested here affected the activity of a-glycerol phos-phate dehydrogenase, the enzyme used for trapping the product.

4.2.2. Anti-T. cruzi in vitro test [27e29]T. cruzi epimastigotes (Tulahuen 2 and Y strains, or CL Brener

clone) were grown axenically at 28 �C in BHI-Tryptose com-plemented with 5% fetal calf serum. Cells were harvested in late logphase, suspended in fresh medium, counted in a Neubauerchamber and placed in 24-well plates (2�106/mL). Cell growthwasmeasured as the absorbance of the culture at 590 nm, which wasfound to be proportional to the number of cells. Before inoculation,the medium was supplemented with the indicated amount of thecompound to be analyzed from a stock solution in DMSO. The finalconcentration of DMSO in the culture media never exceeded 1% anda control was runwith 1% DMSO and the absence of any compound.No effect on epimastigote growth was observed in the presence of

up to 1% DMSO in the culture media. The percentage of growthinhibition was calculated as follows {1 � [(Ap � A0p)/(Ac � A0c)]} � 100, where Ap ¼ A590 of the culture containing thecompound to be analyzed at day 5; A0p ¼ A590 of the culturecontaining the compound to be analyzed just after addition of theinocula (day 0); Ac ¼ A590 of the culture in the absence of anycompound (control) at day 5; A0c ¼ A590 in the absence of thecompound at day 0. To determine ID50 values, parasite growth wasfollowed in the absence (control) and presence of increasingconcentrations of the corresponding compound. The ID50 valueswere determined as the drug concentrations required to reduce theabsorbance by half that measured for untreated controls.

Acknowledgements

Financial supports from Cooperación Mexicana para el Desar-rollo, from CONACyT (49872) (to R.P.-M.) and from ICyTDF (304/2009) (to R.P.-M.), and from RIDIMEDCHAG-CYTED are acknowl-edged. We thank ANII for scholarships to GA, MC, AM, MLL.

Appendix. Supplementary material

Supplementary data related to this article can be found online atdoi:10.1016/j.ejmech.2010.09.034.

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