title: Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.
abstract: BACKGROUND
  The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania.
  
  
  RESULTS
  Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10-2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells.
  
  
  CONCLUSIONS
  This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.
authors.forename: Camila M
authors.initials: CM
authors.lastname: Adade
authors.affiliation: Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil; Instituto de Biquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana Lúcia O
authors.initials: AL
authors.lastname: Carvalho
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Tomaz
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Tatiana F R
authors.initials: TF
authors.lastname: Costa
authors.affiliation: Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Joseane L
authors.initials: JL
authors.lastname: Godinho
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Paulo A
authors.initials: PA
authors.lastname: Melo
authors.affiliation: Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana Paula C A
authors.initials: AP
authors.lastname: Lima
authors.affiliation: Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil; Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil; Núcleo Multidisciplinar de Pesquisa em Biologia (NUMPEX-BIO), Polo Avançado de Xerém, Universidade Federal do Rio de Janeiro, Duque de Caxias, Brazil.
authors.forename: Juliany C F
authors.initials: JC
authors.lastname: Rodrigues
authors.affiliation: Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil; Instituto de Biquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Russolina B
authors.initials: RB
authors.lastname: Zingali
authors.affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.
authors.forename: Thaïs
authors.initials: T
authors.lastname: Souto-Padrón
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D002352:Carrier Proteins; D014355:Chagas Disease / Q000188:drug therapy; D003435:Crotalid Venoms / Q000494:pharmacology*; D017839:Crotalus / Q000378:metabolism; D003593:Cytoplasm; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D060588:LIM Domain Proteins; D007891:Leishmania; D007894:Leishmania mexicana / Q000187:drug effects* / Q000254:growth & development; D051379:Mice; D058069:Neglected Diseases / Q000188:drug therapy / Q000469:parasitology; D021261:Parasitic Sensitivity Tests; D030162:Reptilian Proteins / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry; D016833:Trypanosoma brucei rhodesiense / Q000187:drug effects* / Q000254:growth & development; D014349:Trypanosoma cruzi / Q000187:drug effects* / Q000254:growth & development
chemical_list: D000981:Antiprotozoal Agents; C486181:CRIP1 protein, human; D002352:Carrier Proteins; D003435:Crotalid Venoms; D060588:LIM Domain Proteins; D030162:Reptilian Proteins; C000605113:crovirin protein, Crotalus viridis