Advertisement

Cruzain

The Path from Target Validation to the Clinic
  • Mohammed Sajid
  • Stephanie A. Robertson
  • Linda S. Brinen
  • James H. McKerrow
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 712)

Abstract

Cruzain is the major papain-like cysteine protease of Trypanosoma cruzi, the etiological agent causing Chagas’ disease in humans in South America. Cruzain is indispensable for the survival and propagation of this protozoan parasite and therefore, it has attracted considerable interest as a potential drug target. This chapter charts the path from the initial identification of this proteases activity and its validation as a bone fide drug target to the arduous task of the discovery of an inhibitor targeting this protease and finally the path towards the clinic.

Keywords

Cysteine Protease Trypanosoma Cruzi Target Validation Vinyl Sulfone Sulfone Moiety 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Chagas C. Nova entidade morbida do homen. Resumo greal dos estudos estiologicos e clinicos. Mem Inst Oswaldo Cruz 1911; 3:219–275.Google Scholar
  2. 2.
    Wilson LS, Strosberg AM, Barrio K. Cost-effectiveness of Chagas disease interventions in latin america and the Caribbean: Markov models. Am J Trop Med Hyg 2005; 73:901–910.PubMedGoogle Scholar
  3. 3.
    Rodriques Coura J, de Castro SL. CoMFA and HQSAR of acylhydrazide cruzain inhibitors. Mem Inst Oswaldo Cruz 2002; 97:3–24.PubMedCrossRefGoogle Scholar
  4. 4.
    Duschak VG, Couto AS. An insight on targets and patented drugs for chemotherapy of Chagas disease. Recent Pat Antiinfect Drug Discov 2007; 2:19–51.PubMedCrossRefGoogle Scholar
  5. 5.
    Rivera G, Bocanegra-Garcia V, Ordaz-Pichardo C et al. New therapeutic targets for drug design against Trypanosoma cruzi, advances and perspectives. Curr Med Chem 2009; 16:3286–3293.PubMedCrossRefGoogle Scholar
  6. 6.
    Moreira DR, Leite AC, dos Santos RR et al. Approaches for the development of new anti-Trypanosoma cruzi agents. Curr Drug Targets 2009; 10:212–231.PubMedCrossRefGoogle Scholar
  7. 7.
    Itow S, Camargo EP. Proteolytic activites in Cell extracts of Trypanosoma cruzi. J Protozool 1977; 24:591–595.PubMedGoogle Scholar
  8. 8.
    Avila JL, Casanova MA, Avila A et al. Acid and neutral hydrolases in Trypanosoma cruzi. Characterization and assay J Protozool 1979; 26:304–311.Google Scholar
  9. 9.
    Bontempi E, Martinez J, Cazzulo JJ. Subcellular localization of a cysteine proteinase from Trypanosoma cruzi. Mol Biochem Parasitol 1989; 33:43–47.PubMedCrossRefGoogle Scholar
  10. 10.
    Cazzulo JJ, Couso R, Raimondi A et al. further characterization and partial amino acid sequence of a cysteine proteinase from Trypanosoma cruzi. Mol Biochem Parasitol 1989; 33:33–41.PubMedCrossRefGoogle Scholar
  11. 11.
    Rangel HA, Araujo PM, Repka D et al. Trypanosoma cruzi: isolation and characterization of a proteinase. Exp Parasitol 1981; 52:199–209.PubMedCrossRefGoogle Scholar
  12. 12.
    Lalmanach G, Lecaille F, Chagas JR et al. Inhibition of trypanosomal cysteine proteinases by their propeptides. J Biol Chem 1998; 273:25112–25116.PubMedCrossRefGoogle Scholar
  13. 13.
    Huete-Perez JA, Engel JC, Brinen LS et al. Protease trafficking in two primitive eukaryotes is mediated by a prodomain protein motif. J Biol Chem 1999; 274:16249–16256.PubMedCrossRefGoogle Scholar
  14. 14.
    Cazzulo JJ, Hellman U, Couso R et al. Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi. Mol Biochem Parasitol 1990; 38:41–48.PubMedCrossRefGoogle Scholar
  15. 15.
    Duschak VG, Couto AS. Cruzipain, the major cysteine protease of Trypanosoma cruzi: a sulfated glycoprotein antigen as relevant candidate for vaccine development and drug target. a review. Curr Med Chem 2009; 16:3174–3202.PubMedCrossRefGoogle Scholar
  16. 16.
    Barboza M, Duschak VG, Fukuyama Y et al. Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi. Identification of sulfated high-mannose type oligosaccharides. Febs J 2005; 272:3803–3815.PubMedCrossRefGoogle Scholar
  17. 17.
    Labriola C, Sousa M, Cazzulo JJ. Purification of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi by affinity chromatography. Biol Res 1993; 26:101–107.PubMedGoogle Scholar
  18. 18.
    Barboza M, Duschak VG, Cazzulo JJ et al. Presence of sialic acid in N-linked oligosaccharide chains and O-linked N-acetylglucosamine in cruzipain, the major cysteine proteinase of Trypanosoma cruzi. Mol Biochem Parasitol 2003; 127:69–72.PubMedCrossRefGoogle Scholar
  19. 19.
    Cazzulo JJ, Martinez J, Parodi AJ et al. On the posttranslational modifications at the C-terminal domain of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. FEMS Microbiol Lett 1992; 79:411–416.PubMedGoogle Scholar
  20. 20.
    Parodi AJ, Labriola C, Cazzulo JJ. The presence of complex-type oligosaccharides at the C-terminal domain glycosylation site of some molecules of cruzipain. Mol Biochem Parasitol 1995; 69:247–255.PubMedCrossRefGoogle Scholar
  21. 21.
    Martinez J, Campetella O, Frasch AC et al. The reactivity of sera from chagasic patients against different fragments of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, suggests the presence of defined antigenic and catalytic domains. Immunol Lett 1993; 35:191–196.PubMedCrossRefGoogle Scholar
  22. 22.
    Yomas AM, Kelly JM. Stage-regulated expression of cruzipain, the major cysteine protease of Trypanosoma cruzi is independent of the level of RNA1. Mol Biochem Parasitol 1996; 76:91–103.CrossRefGoogle Scholar
  23. 23.
    Fampa P, Lisboa CV, Jansen AM et al. Protease expression analysis in recently field-isolated strains of Trypanosoma cruzi: a heterogeneous profile of cysteine protease activities between TC I and TC II major phylogenetic groups. Parasitology 2008; 135:1093–1100.PubMedCrossRefGoogle Scholar
  24. 24.
    Campetella O, Henriksson J, Aslund L et al. The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes. Mol Biochem Parasitol 1992; 50:225–234.PubMedCrossRefGoogle Scholar
  25. 25.
    Martinez J, Cazzulo JJ. Anomalous electrophoretic behaviour of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi in relation to its apparent molecular mass. FEMS Microbiol Lett 1992; 74:225–229.PubMedCrossRefGoogle Scholar
  26. 26.
    Cazzulo JJ. Proteinases of Trypanosoma cruzi: patential targets for the chemotherapy of Changas desease. Curr Top Med Chem 2002; 2:1261–1271.PubMedCrossRefGoogle Scholar
  27. 27.
    Sajid M, McKerrow JH. Cysteine proteases of parasitic organisms. Mol Biochem Parasitol 2002; 120:1–21.PubMedCrossRefGoogle Scholar
  28. 28.
    Martinez J, Henriksson J, Rydaker M et al. Genes for cysteine proteinases from Trypanosoma rangeli. FEMS Microbiol Lett 1995; 129:135–141.PubMedCrossRefGoogle Scholar
  29. 29.
    Lima AP, Tessier DC, Thomas DY et al. Identification of new cysteine protease gene isoforms in Trypanosoma cruzi. Mol Biochem Parasitol 1994; 67:333–338.PubMedCrossRefGoogle Scholar
  30. 30.
    Eakin AE, Mills AA, Harth G et al. The sequence, organization and expression of the major cysteine protease (cruzain) from Trypanosoma cruzi. J Biol Chem 1992; 267:7411–7420.PubMedGoogle Scholar
  31. 31.
    McGrath ME, Eakin AE, Engel JC et al. The crystal structure of cruzain: a therapeutic target for Chagas’ disease. J Mol Biol 1995; 247:251–259.PubMedCrossRefGoogle Scholar
  32. 32.
    Judice WA, Cezari MH, Lima AP et al. Comparison of the specificity, stability and individual rate constants with respective activation parameters for the peptidase activity of cruzipain and its recombinant form, cruzain, from Trypanosoma cruzi. Eur J Biochem 2001; 268:6578–6586.PubMedCrossRefGoogle Scholar
  33. 33.
    Alves LC, Melo RL, Cezari MH et al. Analysis of the S(2) subsite specificities of the recombinant cysteine proteinases CPB of Leishmania mexicana and cruzain of Trypanosoma cruzi, using fluorescent substrates containing nonnatural basic amino acids. Mol Biochem Parasitol 2001; 117:137–143.PubMedCrossRefGoogle Scholar
  34. 34.
    Harris JL, Backes BJ, Leonetti F et al. Rapidandgeneral profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci USA 2000; 97:7754–7759.PubMedCrossRefGoogle Scholar
  35. 35.
    Del Nery E, Juliano MA, Lima AP et al. Kininogenase activity by the major cysteinyl proteinase (cruzipain) from Trypanosoma cruzi. J Biol Chem 1997; 272:25713–25718.PubMedCrossRefGoogle Scholar
  36. 36.
    Eakin AE, McGrath ME, Mckerrow JH et al. Production of crystallizable cruzain, the major cysteine protease from Trypanosoma cruzi. J Biol Chem 1993; 268:6115–6118.PubMedGoogle Scholar
  37. 37.
    Souto-Padron T, Campetella OE, Cazzulo JJ et al. Cysteine proteinase in Trypanosoma cruzi: immunocytochemical localization and involvement in parasite-host Cell interaction. J Cell Sci 1990; 96:485–490.PubMedGoogle Scholar
  38. 38.
    Soares MJ, Souto-Padron T, De Souza W. Identification of a large prelysosomal compartment in the pathogenic protozoon Trypanosoma cruzi. J Cell Sci 1992; 102:157–167.PubMedGoogle Scholar
  39. 39.
    Parussini F, Duschak VG, Cazzulo JJ. Membrane-bound cysteine proteinase isoforms in different developmental stages of Trypanosoma cruzi. Cell Mol Biol 1998; 44:513–519.PubMedGoogle Scholar
  40. 40.
    Nascimento AE, de Souza W. High resolution localization of cruzipain and Ssp4 in Trypanosoma cruzi by replica staining label fracture. Biol Cell 1996; 86:53–58.PubMedCrossRefGoogle Scholar
  41. 41.
    Aparicio IM, Scharfstein J, Lima AP. A new cruzipain-mediated pathway of human Cell invasion by Trypanosoma cruzi requires trypomastigote membranes. Infect Immun 2004; 72:5892–5902.PubMedCrossRefGoogle Scholar
  42. 42.
    Bontempi E, Cazzulo JJ. Digestion of human immunoglobulin G by the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. FEMS Microbiol Lett 1990; 58:337–341.PubMedGoogle Scholar
  43. 43.
    Krettli AU, Weisz-Carrington P, Nussenzweig RS. Membrane-bound antibodies to bloodstream Trypanosoma cruzi in mice: strain differences in susceptibility to complement-mediated lysis. Clin Exp Immunol 1979; 37:416–423.PubMedGoogle Scholar
  44. 44.
    Scharfstein J, Monteiro AC, Schmitz V et al. Angiotensin-converting enzyme limits inflammation elicited by Trypanosoma cruzi cysteine proteases: a peripheral mechanism regulating adaptive immunity via the innate kinin pathway. Biol Chem 1998; 389:1015–1024.CrossRefGoogle Scholar
  45. 45.
    Scharfstein J, Lima AP. Roles of naturally occurring protease inhibitors in the modulation of host cell signaling and cellular invasion by Trypanosoma cruzi. Subcell Biochem 2008; 47:140–154.PubMedCrossRefGoogle Scholar
  46. 46.
    Scharfstein J. Parasite cysteine proteinase interactions with alpha 2-macroglobulin or kininogens: differential pathways modulating inflammation and innate immunity in infection by pathogenic trypanosomatids. Immunobiology 2006; 211:117–125.PubMedCrossRefGoogle Scholar
  47. 47.
    Yokoyama-Yasunaka JK, Pral EM, Oliveira Junior OC et al. Trypanosoma cruzi: identification of proteinases in shed components of trypomastigote forms. Acta Trop 1994; 57:307–315.PubMedCrossRefGoogle Scholar
  48. 48.
    Harth G, Andrews N, Mills AA et al. Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi. Mol Biochem Parasitol 1993; 58:17–24.PubMedCrossRefGoogle Scholar
  49. 49.
    Meirelles MN, Juliano L, Carmona E et al. Inhibitors of the major cysteinyl proteinase (GP57/51) impair host Cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro. Mol Biochem Parasitol 1992; 52:175–184.PubMedCrossRefGoogle Scholar
  50. 50.
    Engel JC, Doyle PS, Hsieh I et al. Cysteine protease inhibitors alter Golgi complex ultrastructure and function in Trypanosoma cruzi. J Exp Med 1998; 188:725–734.PubMedCrossRefGoogle Scholar
  51. 51.
    Caffrey CR, Scory S, Steverding D. Cysteine proteinases of trypanosome parasites: novel targets for chemotherapy. Curr Drug Targets 2000; 1:155–162.PubMedCrossRefGoogle Scholar
  52. 52.
    Campetella O, Martinez J, Cazzulo JJ. A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi. FEMS Microbiol Lett 1990; 55:145–149.PubMedCrossRefGoogle Scholar
  53. 53.
    Judice WA, Puzer L, Cotrin SS et al. Carboxydipeptidase activities of recombinant cysteine peptidases. cruzain of Trypanosoma cruzi and CPB of Leishmania mexicana. Eur J Biochem 2004; 271:1046–1053.PubMedCrossRefGoogle Scholar
  54. 54.
    Cazzulo JJ, Cazzulo Franke MC, Martinez J et al. Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi. Biochim Biophys Acta 1990; 1037:186–191.PubMedCrossRefGoogle Scholar
  55. 55.
    Reis FC, Costa TF, Sulea T et al. The propeptide of cruzipain—a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain and of the human enzyme cathepsin F. Febs J 2007; 274:1224–1234.PubMedCrossRefGoogle Scholar
  56. 56.
    Santos CC, Sant’anna C, Terres A et al. Use of proteolytic enzymes as an additional tool for trypanosomatid identification. J Cell Sci 2005; 1118:901–915.CrossRefGoogle Scholar
  57. 57.
    Santos CC, Scharfstein J, Lima AP. Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion. Parasitol Res 2006; 99:323–324.PubMedCrossRefGoogle Scholar
  58. 58.
    Ramos AM, Duschak VG, Gerez de Burgos NM et al. Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein. Exp Parasitol 2002; 100:121–130.PubMedCrossRefGoogle Scholar
  59. 59.
    Cazzulo JJ, Stoka V, Turk V. Cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi. Biol Chem 1997; 378:1–10.PubMedCrossRefGoogle Scholar
  60. 60.
    Lima AP, Almeida PC, Tersariol IL et al. Heparan sulfate modulates kinin release by Trypanosoma cruzi through the activity of cruzipain. J Biol Chem 2002; 277:5875–5881.PubMedCrossRefGoogle Scholar
  61. 61.
    Guinazu N, Pellegrini A, Carrera-Silva EA et al. Immunisation with a major Trypanosoma cruzi antigen promotes pro-inflammatory cytokines, nitric oxide production and increases TLR2 expression. Int J Parasitol 2007; 37:1243–1254.PubMedCrossRefGoogle Scholar
  62. 62.
    Schnapp AR, Eickhoff CS, Scharfstein J et al. Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice. Microbes Infect 2002; 4:805–813.PubMedCrossRefGoogle Scholar
  63. 63.
    Cazorla SI, Becker PD, Frank FM et al. Oral vaccination with Salmonella enterica as a cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi. Infect Immun 2008; 76:324–333.PubMedCrossRefGoogle Scholar
  64. 64.
    Schnapp AR, Eickhoff CS, Sizemore D et al. Cruzipain induces both mucosal and systemic protection against Trypanosoma cruzi in mice. Infect Immun 2002; 70:5065–5074.PubMedCrossRefGoogle Scholar
  65. 65.
    Cazorla SI, Frank FM, Malchiodi EL. Vaccination approaches against Trypanosoma cruzi infection. Expert Rev Vaccines 2009; 8:921–935.PubMedCrossRefGoogle Scholar
  66. 66.
    Engel JC, Doyle PS, Palmer J et al. Cysteine protease inhibitors cure an experimental Trypanosoma cruzi infection. J Cell Sci 1998; 111:597–606.PubMedGoogle Scholar
  67. 67.
    Kerr ID, Lee JH, Farady CJ et al. Vinyl sulfones as antiparasitic agents and a structural basis for drug design. J Biol Chem 2009; 284:25697–25703.PubMedCrossRefGoogle Scholar
  68. 68.
    Choe Y, Brinen LS, Price MS et al. Development of alpha-keto-based inhibitors of cruzain, a cysteine protease implicated in chagas disease. Bioorg Med Chem 2005; 13:2141–2156.PubMedCrossRefGoogle Scholar
  69. 69.
    Huang L, Brinen LS, Ellman JA. Crystal structures of reversible ketone-Based inhibitors of the cysteine protease cruzain. Bioorg Med Chem 2003; 11:21–29.PubMedCrossRefGoogle Scholar
  70. 70.
    Gillmor SA, Craik CS, Fletterick RJ. Structural determinants of specificity in the cysteine protease cruzain. Protein Sci 1997; 6:1603–1611.PubMedCrossRefGoogle Scholar
  71. 71.
    Brak K, Kerr ID, Barrett KT et al. Nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for chagas disease chemotherapy. J Med Chem 2010; 53:1763–1773.PubMedCrossRefGoogle Scholar
  72. 72.
    Bryant C, Kerr ID, Debnath M et al. Novel nonpeptidic vinylsulfones targeting the S2 and S3 subsites of parasite cysteine proteases. Bioorg Med Chem Lett 2009; 19:6218–6221.PubMedCrossRefGoogle Scholar
  73. 73.
    Choe Y, Leonetti F, Greenbaum DC et al. Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities. J Biol Chem 2006; 281:12824–12832.PubMedCrossRefGoogle Scholar
  74. 74.
    Brinen LS, Hansell E, Cheng J et al. A target within the target: probing cruzain’s P1’ site to define structural determinants for the chagas’ disease protease. Structure 2000; 8:831–840.PubMedCrossRefGoogle Scholar
  75. 75.
    Kerr ID, Lee JH, Pandey KC et al. Structures of falcipain-2 and falcipain-3 bound to small molecule inhibitors: implications for substrate specificity. J Med Chem 2009; 52:852–857.PubMedCrossRefGoogle Scholar
  76. 76.
    Krantz A, Copp LJ, Coles PJ et al. Peptidyl (acyloxy)methyl ketones and the quiescent affinity label concept: the departing group as a variable structural element in the design of inactivators of cysteine proteinases. Biochemistry 1991; 30:4678–4687.PubMedCrossRefGoogle Scholar
  77. 77.
    Smith RA, Coles PJ, Spencer RW et al. Peptidyl O-acyl hydroxamates: potent new inactivators of cathepsin B. Biochem Biophys Res Commun 1998; 155:1201–1206.CrossRefGoogle Scholar
  78. 78.
    Powers JC, Asgian JL, Ekici OD et al. Irreversible inhibitors of serine, cysteine and threonine proteases. Chem Rev 2002; 102:4639–4750.PubMedCrossRefGoogle Scholar
  79. 79.
    Baskin-Bey ES, Washburn K, Feng S et al. Clinical Trial of the Pan-caspase Inhibitor, IDN-6556, in Human liver Preservation Injury. Am J Transplant 2007; 7:218–225.PubMedCrossRefGoogle Scholar
  80. 80.
    Brady KD. Bimodal inhibition of caspase-1 by aryloxymethyl and acyloxymethyl ketones. Biochemistry 1998; 37:8508–8515.PubMedCrossRefGoogle Scholar
  81. 81.
    Brady KD, Giegel DA, Grinnell C et al. A catalytic mechanism for caspase-1 and for bimodal inhibition of caspase-1 by activated aspartic ketones. Bioorg Med Chem 1999; 7:621–631.PubMedCrossRefGoogle Scholar
  82. 82.
    Palmer JT, Rasnick D, Klaus JL et al. Vinyl sulfones as mechanism-based cysteine protease inhibitors. J Med Chem 1995; 38:3193–3196.PubMedCrossRefGoogle Scholar
  83. 83.
    Engel JC, Doyle PS, McKerrow JH. Trypanocidal effect of cysteine protease inhibitors in vitro and in vivo in experimental chagas disease. Medicina (B Aires) 1999; 59:171–175.Google Scholar
  84. 84.
    Barr SC, Warner KL, Kornreic BG et al. A cysteine protease inhibitor protects dogs from cardiac damage during infection by Trypanosoma cruzi. Antimicrob Agents Chemother 2005; 49:5160–5161.PubMedCrossRefGoogle Scholar
  85. 85.
    Mckerrow JH, Doyle PS, Engel JC et al. Two approaches to discovering and developing new drugs for chagas disease. Mem Inst Oswaldo Cruz 2009; 104:263–269.PubMedCrossRefGoogle Scholar
  86. 86.
    Marin-Neto JA, Rassi A, Morillo CA et al. BENEFIT Investigators Am Heart J 2009; 156:37–43.CrossRefGoogle Scholar
  87. 87.
    Sterverding D, Caffrey C, Sajid M. Cysteine Proteinase Inhibitors as Therapy for Parasitic Diseases: Advances in Inhibitor Design. Mini Rev Med Chem 2006; 6:1025–1032.CrossRefGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Mohammed Sajid
    • 1
  • Stephanie A. Robertson
    • 2
  • Linda S. Brinen
    • 2
    • 3
  • James H. McKerrow
    • 2
  1. 1.Afd. ParasitologieLeiden University Medical CenterLeidenThe Netherlands
  2. 2.Sandler Center for Drug DiscoveryUniversity of California— San FranciscoSan FranciscoUSA
  3. 3.Department of Cellular and Molecular PharmacologyUniversity of California— San FranciscoSan FranciscoUSA

Personalised recommendations