Background Cruzain, the major cysteine protease of Trypanosoma cruzi, is an essential enzyme for the parasite life cycle and has been validated as a viable target to treat Chagas' disease. As a proof-of-concept, K11777, a potent inhibitor of cruzain, was found to effectively eliminate T. cruzi infection and is currently a clinical candidate for treatment of Chagas' disease. Methodology/Principal Findings WRR-483, an analog of K11777, was synthesized and evaluated as an inhibitor of cruzain and against T. cruzi proliferation in cell culture. This compound demonstrates good potency against cruzain with sensitivity to pH conditions and high efficacy in the cell culture assay. Furthermore, WRR-483 also eradicates parasite infection in a mouse model of acute Chagas' disease. To determine the atomic-level details of the inhibitor interacting with cruzain, a 1.5 ？ crystal structure of the protease in complex with WRR-483 was solved. The structure illustrates that WRR-483 binds covalently to the active site cysteine of the protease in a similar manner as other vinyl sulfone-based inhibitors. Details of the critical interactions within the specificity binding pocket are also reported. Conclusions We demonstrate that WRR-483 is an effective cysteine protease inhibitor with trypanocidal activity in cell culture and animal model with comparable efficacy to K11777. Crystallographic evidence confirms that the mode of action is by targeting the active site of cruzain. Taken together, these results suggest that WRR-483 has potential to be developed as a treatment for Chagas' disease.
References
[1]
Lockman JW, Hamilton AD (2005) Recent developments in the identification of chemotherapeutics for Chagas disease. Curr Med Chem 12: 945–959. doi: 10.2174/0929867053507289
[2]
Kirchhoff LV (1996) American trypanosomiasis (Chagas' disease). Gastroenterol Clin North Am 25: 517–533. doi: 10.1016/S0889-8553(05)70261-2
[3]
Rossi MA, Bestetti RB (1995) The challenge of chagasic cardiomyopathy. Cardiology 86: 1–7. doi: 10.1159/000176822
[4]
Van den Bossche H (1978) Chemotherapy for parasitic infections. Nature 273: 626–630. doi: 10.1038/273626a0
[5]
Linares GEG, Ravaschino EL, Rodriguez JB (2006) Progresses in the field of drug design to combat tropical protozoan parasitic diseases. Curr Med Chem 13: 335–360. doi: 10.2174/092986706775476043
[6]
McKerrow JH, Sun E, Rosenthal PJ, Bouvier J (1993) The proteases and pathogenicity of parasitic protozoa. Ann Rev Microbiol 47: 821–853. doi: 10.1146/annurev.mi.47.100193.004133
[7]
Engel JC, Doyle PS, Hsieh I, McKerrow JH (1998) Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection. J Exp Med 188: 725–734. doi: 10.1084/jem.188.4.725
[8]
Barr SC, Warner KL, Kornreic BG, Piscitelli J, Wolfe A, et al. (2005) A cysteine protease inhibitor protects dogs from cardiac damage during infection by Trypanosoma cruzi. Antimicrob Agents Chemother 49: 5160–5161. doi: 10.1128/AAC.49.12.5160-5161.2005
[9]
Doyle PS, Zhou YM, Engel JC, McKerrow JH (2007) A cysteine protease inhibitor cures Chagas' disease in an immunodeficient-mouse model of infection. Antimicrob Agents Chemother 51: 3932–3939. doi: 10.1128/AAC.00436-07
[10]
Huang L, Brinen LS, Ellman JA (2003) Crystal structures of reversible ketone-based inhibitors of the cysteine protease cruzain. Bioorg Med Chem 11: 21–29. doi: 10.1016/S0968-0896(02)00427-3
[11]
Mott BT, Ferreira RS, Simeonov A, Jadhav A, Ang KKH, et al. (2010) Identification and Optimization of Inhibitors of Trypanosomal Cysteine Proteases: Cruzain, Rhodesain, and TbCatB. J Med Chem 53: 52–60. doi: 10.1021/jm901069a
[12]
McGrath ME, Eakin AE, Engel JC, McKerrow JH, Craik CS, et al. (1995) The crystal structure of cruzain: a therapeutic target for Chagas' disease. J Mol Biol 247: 251–259. doi: 10.1006/jmbi.1994.0137
[13]
Gillmor SA, Craik CS, Fletterick RJ (1997) Structural determinants of specificity in the cysteine protease cruzain. Protein Sci 6: 1603–1611. doi: 10.1002/pro.5560060801
[14]
Brinen LS, Hansell E, Cheng J, Roush WR, McKerrow JH, et al. (2000) A target within the target: probing cruzain's P1′ site to define structural determinants for the Chagas' disease protease. Structure 8: 831–840. doi: 10.1016/S0969-2126(00)00173-8
[15]
Choe Y, Brinen LS, Price MS, Engel JC, Lange M, et al. (2005) Development of α-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease. Bioorg Med Chem 13: 2141–2156. doi: 10.1016/j.bmc.2004.12.053
[16]
Kerr ID, Lee JH, Farady CJ, Marion R, Rickert M, et al. (2009) Vinyl sulfones as antiparasitic agents and a structural basis for drug design. J Biol Chem 284: 25697–24703. doi: 10.1074/jbc.M109.014340
[17]
Bryant C, Kerr ID, Debnath M, Ang KKH, Ratnam J, et al. (2009) Novel non-peptidic vinylsulfones targeting the S2 and S3 subsites of parasite cysteine proteases. Bioorg Med Chem Lett 19: 6218–6221. doi: 10.1016/j.bmcl.2009.08.098
[18]
Serveau C, Lalmanach G, Juliano MA, Scharfstein J, Juliano L, et al. (1996) Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors. Biochem J 313: 951–956.
[19]
Powers JC, Asgian JL, Ekici OD, James KE (2002) Irreversible inhibitors of serine, cysteine, and threonine proteases. Chem Rev 12: 4639–4750. doi: 10.1021/cr010182v
[20]
Alvarez-Hernandez A, Roush WR (2002) Recent advances in the synthesis, design and selection of cysteine protease inhibitors. Curr Opin Chem Biol 6: 459–465. doi: 10.1016/S1367-5931(02)00345-9
[21]
Leung-Toung R, Zhao Y, Li W, Tam R, Karimian K, et al. (2006) Thiol proteases: inhibitors and potential therapeutic targets. Curr Med Chem 13: 547–581. doi: 10.2174/092986706776055733
[22]
Santos MMM, Moreira R (2007) Michael acceptors as cysteine protease inhibitors. Mini Rev Med Chem 7: 1040–1050. doi: 10.2174/138955707782110105
[23]
Liu S, Hanzlik RP (1992) Structure-activity relationships for inhibition of papain by peptide Michael acceptors. J Med Chem 35: 1067–1075. doi: 10.1021/jm00084a012
[24]
Palmer JT, Rasnick D, Klaus JL, Bromme D (1995) Vinyl sulfones as mechanism-based cysteine protease inhibitors. J Med Chem 38: 3193–3196. doi: 10.1021/jm00017a002
[25]
Roush WR, Gwaltney SL II, Cheng J, Scheidt KA, McKerrow JH, et al. (1998) Vinyl sulfonate esters and vinyl sulfonamides: potent, irreversible inhibitors of cysteine proteases. J Am Chem Soc 120: 10994–10995. doi: 10.1021/ja981792o
[26]
Roush WR, Cheng J, Knapp-Reed B, Alvarez-Hernandez A, McKerrow JH, et al. (2001) Potent second generation vinyl sulfonamide inhibitors of the trypanosomal cysteine protease cruzain. Bioorg Med Chem Lett 11: 2759–2762. doi: 10.1016/S0960-894X(01)00566-2
[27]
Eakin AE, McGrath ME, McKerrow JH, Fletterick RJ, Craik CS (1993) Production of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi. J Biol Chem 268: 6115–6118.
[28]
Eakin AE, Mills AA, Harth G, McKerrow JH, Craik CS (1992) The sequence, organization, and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. J Biol Chem 267: 7411–7420.
[29]
Caffrey CR, Hansell E, Lucas KD, Brinen LS, Alvarez-Hernandez A, et al. (2001) Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense. Mol Biochem Parasitol 118: 61–73. doi: 10.1016/S0166-6851(01)00368-1
[30]
Mackey ZB, O'Brien TC, Greenbaum DC, Blank RB, McKerrow JH (2004) A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei. J Biol Chem 279: 48426–48433. doi: 10.1074/jbc.M402470200
[31]
Mallari JP, Shelat AA, O'Brien T, Caffrey CR, Kosinski A, et al. (2008) Development of potent purine-derived nitrile inhibitors of the trypanosomal protease TbCatB. J Med Chem 51: 545–552. doi: 10.1021/jm070760l
[32]
Beith JG (1995) Theoretical and practical aspects of proteinase inhibition kinetics. Methods Enzymol 248: 59–84. doi: 10.1016/0076-6879(95)48007-2
[33]
Engel JC, Dvorak JA, Segura EL, Crane MS (1982) J Protozool 29: 550–560.
[34]
Engel JC, Ang KKH, Chen S, Arkin MR, McKerrow JH, et al. (2010) Image-based high throughput drug screening targeting the intracellular stage of Trypanosoma cruzi, the agent of Chagas' Disease. Antimicrob Agents Chemother 54: 3326–3334. doi: 10.1128/AAC.01777-09
[35]
Cohen AE, Ellis PJ, Miller MD, Deacon , AM , Phizackerley RP (2002) An automated system to mount cryo-cooled protein crystals on a synchrotron beamline, using compact sample cassetes and a small-scale robot. J Appl Cryst 35: 720–726. doi: 10.1107/s0021889802016709
[36]
Leslie AGW (1992) Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ES-EAMCB Newsletter on Protein Crystallography 26:
[37]
Collaborative Computational Project, N. (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760–763. doi: 10.1107/S0907444994003112
[38]
Nowick JS, Holmes DL, Noronha G, Smith EM, Nguyen TM, et al. (1996) Synthesis of peptide isocyanates and isothiocyanates. J Org Chem 61: 3929–3934. doi: 10.1021/jo960038n
[39]
Meléndez-López SG, Herdman S, Hirata K, Choi MH, Choe Y, et al. (2007) Use of recombinant Entamoeba histolytica cysteine proteinase 1 (EhCP1) to identify a potent inhibitor of amebic invasion in a human colonic model. Eukaryot Cell 6: 1130–1136. doi: 10.1128/EC.00094-07
[40]
Garcia MP, Nóbrega OT, Teixeira AR, Sousa MV, Santana JM (1998) Characterisation of a Trypanosoma cruzi acidic 30 kDa cysteine protease. Mol Biochem Parasitol 91: 263–72. doi: 10.1016/S0166-6851(97)00205-3
[41]
dos Reis FC, Júdice WA, Juliano MA, Juliano L, Scharfstein J, et al. (2006) The substrate specificity of cruzipain 2, a cysteine protease isoform from Trypanosoma cruzi. FEMS Microbiol Lett 259: 215–220. doi: 10.1111/j.1574-6968.2006.00267.x
[42]
Aparicio IM, Scharfstein J, Lima APCA (2004) A new cruzipain-mediated pathway of human cell invasion by Trypanosoma cruzi requires trypomastigote membranes. Infect Immun 72: 5892–5902. doi: 10.1128/IAI.72.10.5892-5902.2004
