Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design
Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs. Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs. Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1Balakrishnan I. Gillespie S.H. Zuckerman J.N. Principles and Practice of Travel Medicine. John Wiley & Sons, New York2002: 91-124Crossref Google Scholar). Chagas' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2Docampo R. Moreno S.N. Parasitol Res. 2003; 90 (Suppl. 1): S10-S13Crossref PubMed Scopus (102) Google Scholar, 3Laufer M.K. Djimdé A.A. Plowe C.V. Am. J. Trop. Med. Hyg. 2007; 77: 160-169Crossref PubMed Scopus (58) Google Scholar, 4Ouellette M. Trop. Med. Int. Health. 2001; 6: 874-882Crossref PubMed Scopus (59) Google Scholar). T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5Caffrey C.R. Scory S. Steverding D. Curr. 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In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2) 6The abbreviations used are: FP-2falcipain-2FP-3falcipain-3SAMStanford Automated MountingSSRLStanford Synchrotron Radiation Lightsourcer.m.s.d.root mean square distanceVSPhphenyl vinyl sulfoneHphhomophenylalanylMumorpholino ureaN-MpipN-methylpiperazineBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolZbenzyloxycarbonylAMCaminomethylcoumarin. and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15Rosenthal P.J. McKerrow J.H. Aikawa M. Nagasawa H. Leech J.H. J. Clin. Invest. 1988; 82: 1560-1566Crossref PubMed Scopus (311) Google Scholar, 16Sijwali P.S. Koo J. Singh N. Rosenthal P.J. Mol. Biochem. Parasitol. 2006; 150: 96-106Crossref PubMed Scopus (137) Google Scholar, 17Sijwali P.S. Rosenthal P.J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4384-4389Crossref PubMed Scopus (254) Google Scholar). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18Schulz F. Gelhaus C. Degel B. Vicik R. Heppner S. Breuning A. Leippe M. Gut J. Rosenthal P.J. Schirmeister T. ChemMedChem. 2007; 2: 1214-1224Crossref PubMed Scopus (44) Google Scholar). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17Sijwali P.S. Rosenthal P.J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 4384-4389Crossref PubMed Scopus (254) Google Scholar), FP-3 has proved recalcitrant to gene knock-out (16Sijwali P.S. Koo J. Singh N. Rosenthal P.J. Mol. Biochem. Parasitol. 2006; 150: 96-106Crossref PubMed Scopus (137) Google Scholar) suggesting a critical function for this enzyme and underscoring its potential as a drug target. falcipain-2 falcipain-3 Stanford Automated Mounting Stanford Synchrotron Radiation Lightsource root mean square distance phenyl vinyl sulfone homophenylalanyl morpholino urea N-methylpiperazine 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol benzyloxycarbonyl aminomethylcoumarin. Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19Chen Y.T. Lira R. Hansell E. McKerrow J.H. Roush W.R. Bioorg. Med. Chem. Lett. 2008; 18: 5860-5863Crossref PubMed Scopus (42) Google Scholar, 20Fujii N. Mallari J.P. Hansell E.J. Mackey Z. Doyle P. Zhou Y.M. Gut J. Rosenthal P.J. McKerrow J.H. Guy R.K. Bioorg. 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This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28McKerrow J.H. Rosenthal P.J. Swenerton R. Doyle P. Curr. Opin. Infect. Dis. 2008; 21: 668-672Crossref PubMed Scopus (109) Google Scholar, 29Renslo A.R. McKerrow J.H. Nat. Chem. Biol. 2006; 2: 701-710Crossref PubMed Scopus (296) Google Scholar). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds. Recombinant cruzain was expressed in Escherichia coli and purified as described previously (8Eakin A.E. Mills A.A. Harth G. McKerrow J.H. Craik C.S. J. Biol. Chem. 1992; 267: 7411-7420Abstract Full Text PDF PubMed Google Scholar, 30Eakin A.E. McGrath M.E. McKerrow J.H. Fletterick R.J. Craik C.S. J. Biol. Chem. 1993; 268: 6115-6118Abstract Full Text PDF PubMed Google Scholar, 31Gillmor S.A. Craik C.S. Fletterick R.J. Protein Sci. 1997; 6: 1603-1611Crossref PubMed Scopus (169) Google Scholar). Activated cruzain was incubated overnight with molar excess amounts of inhibitor dissolved in DMSO to prevent further proteolytic activity. Complete enzymatic inhibition was confirmed via fluorometric assay with the substrate Z-Phe-Arg-AMC. Excess inhibitor was removed by anion-exchange chromatography. Fractions containing pure, inhibited cruzain were pooled and concentrated to 8 mg/ml, with tandem buffer exchange to 2 mm Bis-Tris, pH 5.8, using a Viva-Spin (Viva Science) column (molecular mass of 15 kDa). Crystals of maximum size were obtained after ∼4 days via the hanging drop method, from a solution of 1.25 m ammonium sulfate and 100 mm HEPES, pH 7.5, at 22 °C. Crystals were cryoprotected in mother liquor containing 20% ethylene glycol, mounted in standard cryo loops, and loaded into a sample cassette used with the Stanford Automated Mounting (SAM) system (32Cohen A.E. Ellis P.J. Deacon A.M. Miller M.D. Phizackerley R.P. J. Appl. Crystallogr. 2002; 35: 720-726Crossref PubMed Scopus (251) Google Scholar). All diffraction data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) Beamline 9-1, Menlo Park, CA, after selecting an optimal crystal from screening performed with the robotic SAM system (32Cohen A.E. Ellis P.J. Deacon A.M. Miller M.D. Phizackerley R.P. J. Appl. Crystallogr. 2002; 35: 720-726Crossref PubMed Scopus (251) Google Scholar). Data processing in the HKL2000 package (33Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38773) Google Scholar) showed that crystals belonged to space group C2, and the structure was solved by molecular replacement using a model derived from cruzain bound to the vinyl sulfone, K11002 (PDB ID 1F29). Using MOLREP (34Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4224) Google Scholar), two independent molecules were located with translation function scores of 14.49 and 14.03. Rigid body of this solution an of and for the of both inhibitor molecules in the was at the model was by of model in P. K. Crystallogr. Biol. Crystallogr. 2004; PubMed Scopus Google Scholar) and space in A.A. E.J. Crystallogr. Biol. Crystallogr. 1997; PubMed Scopus Google Scholar). were with and of the ethylene and the ammonium sulfate were also in and with This structure has been in the Protein Data All for data structure and are in diffraction data and structure Cell is the diffraction and the all by is the diffraction and the all by A. J.N. J. D.C. Res. 2007; 35: PubMed Scopus Google Scholar). in a new the between cathepsin was expressed in P. and purified as described previously (7Caffrey C.R. Hansell E. Lucas K.D. Brinen L.S. Alvarez Hernandez A. Cheng J. Gwaltney 2nd, S.L. Roush W.R. Stierhof Y.D. Bogyo M. Steverding D. McKerrow J.H. Mol. Biochem. Parasitol. 2001; 118: 61-73Crossref PubMed Scopus (154) Google Scholar) with a at of the to a site from the was incubated with molar excess of the dissolved in of activity was confirmed by fluorometric assay with the was concentrated to using in for Crystals of maximum size were obtained after days via the hanging drop method, from a solution of 100 mm pH and m at °C. data were collected at a using at and a Data processing was performed in space group with the HKL2000 package (33Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38773) Google Scholar). structure was solved via molecular replacement in J. Crystallogr. A. Scopus Google Scholar), using cruzain (PDB ID as a solution a of and an of inhibitor was and to the using and for the of the inhibitor molecule was at the molecules were with P. K. Crystallogr. Biol. Crystallogr. 2004; PubMed Scopus Google Scholar) and of were with A.A. E.J. Crystallogr. Biol. Crystallogr. 1997; PubMed Scopus Google Scholar). This structure has been in the Protein Data All for data structure and are in FP-3 was expressed in E. coli with the and were to P.S. B.R. Gut J. Singh A. Rosenthal P.J. Biochem. J. 2001; PubMed Scopus Google Scholar). activity of FP-3 was with the substrate as described P.S. McKerrow J.H. Rosenthal P.J. Proc. Natl. Acad. Sci. 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Fletterick R.J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) of some of this binding to cruzain, and the of the inhibitor is therefore to the with the enzyme that absence of these in the complex for the for inhibition by also a for inhibition by are a of the and structures that the to in is to the that be to to with in the makes to of the and provide for the although is to with this be to the In to cruzain, and the closely related FP-3 is active and to inhibition by small molecules P.S. B.R. Gut J. Singh A. Rosenthal P.J. Biochem. J. 2001; PubMed Scopus Google Scholar, J. J. R.K. J. Fletterick R.J. Rosenthal P.J. M. Brinen L.S. A. A. McKerrow J.H. 2007; 15: Full Text Full Text PDF PubMed Scopus Google Scholar). These are by that FP-3 is to inhibition by both and cruzain or We have previously that the in FP-3 site is for a cathepsin L-like protease a of two and and the at the of the Lee J.H. A. Sajid M. Rosenthal P.J. Brinen L.S. J. Med. Chem. PubMed Scopus Google Scholar). Our structure independent of the complex in the and in at the to the to be the structural data with studies that the enzyme has a and a for with the at the P.S. B.R. Gut J. Singh A. Rosenthal P.J. Biochem. J. 2001; PubMed Scopus Google Scholar). with this substrate FP-3 was therefore to Our and structural data that cruzain and be targeted for inhibition by the vinyl sulfones. has been shown in to be to be with an pharmacokinetic and to in models of and disease in both and the of these results a for an New Drug is in to the inhibitor to in In with the enzymes, of the vinyl sulfones as effective FP-3 inhibitors in of the structural the However, this to a of selectivity that be in the of both cruzain and We that cysteine proteases are promising drug and are that structural insights in the design of small molecules that these We for at the We also and Fletterick for critical of the and for with with