Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania Camila M. Adade1,2, Ana Lu´ cia O. Carvalho2,3, Marcelo A. Tomaz4, Tatiana F. R. Costa5, Joseane L. Godinho2,5, Paulo A. Melo4, Ana Paula C. A. Lima5, Juliany C. F. Rodrigues2,4,5,6,7, Russolina B. Zingali2,3, Thaı¨s Souto-Padro´ n1,2* 1 Instituto de Microbiologia Paulo de Go´es, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 2 Instituto Nacional de Cieˆncia e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil, 3 Instituto de Biquı´mica Me´dica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 4 Instituto de Cieˆncias Biome´dicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 5 Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, 6 Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil, 7 Nu´cleo Multidisciplinar de Pesquisa em Biologia (NUMPEX-BIO), Polo Avanc¸ado de Xere´m, Universidade Federal do Rio de Janeiro, Duque de Caxias, Brazil 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. Methodology/Principal Findings: 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 mg/ml). A considerably higher concentration (20 mg/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. Citation: Adade CM, Carvalho ALO, Tomaz MA, Costa TFR, Godinho JL, et al. (2014) Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and Leishmania. PLoS Negl Trop Dis 8(10): e3252. doi:10.1371/journal.pntd.0003252 Editor: Michael P. Pollastri, Northeastern University, United States of America Received May 16, 2014; Accepted September 8, 2014; Published October 16, 2014 Copyright: ß 2014 Adade et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (grant numbers 560931/2010-7; 306967/2011-1), Fundac¸a˜o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) (grant numbers E-26/102.581/2010; E-26/102.874/2012; E-26/110.621/ 2012) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction neglected diseases that have ‘‘fallen below the radar of modern drug discovery’’ [4]. The pathogenic trypanosomatids from the genera Leish- Leishmania parasites cause five different disease forms – mania and Trypanosoma infect over 20 million people cutaneous (CL), mucocutaneous (MCL), diffuse cutaneous leish- worldwide, with an annual incidence of ,3millionnew maniasis (DCL), post-kala-azar dermal leishmaniasis (PKDL) and infections in at least 88 countries. An additional 400 million visceral leishmaniasis (VL, also known as ‘black fever’ or ‘kala- people are at risk of infection by exposure to insect vectors azar’ in India) [5]. VL is the most severe and debilitating form of harboring parasites [1–3]. Leishmania and trypanosome leishmaniasis, and can be fatal if left untreated. First-line treatment infections predominate in poorer nations, and are considered for leishmaniasis is based on pentavalent antimonials such as PLOS Neglected Tropical Diseases | www.plosntds.org 1 October 2014 | Volume 8 | Issue 10 | e3252 Crovirin, a Snake Venom CRISP with Promising Activity against Protozoa Author Summary and thymus [32]. CRISPs belong to the CAP (Crisp, antigen 5, and pathogenesis-related) superfamily of proteins [33]. The pathogenic trypanosomatid parasites of the genera CRISP amino acid sequences have high degree of sequence Leishmania and Trypanosoma infect over 20 million people identity and similarity, and include a highly conserved pattern of 16 worldwide, with an annual incidence of ,3 million new cysteine residues which form 8 disulfide bonds [34]. Ten of these infections. An additional 400 million people are at risk of cysteine residues form an integral part of a well-conserved cysteine- infection by exposure to parasite-infected insects which rich domain at the C-terminus, although CRISP N-terminal act as disease vectors. Trypanosomatid-borne diseases sequences are overall more conserved than other regions of these predominant in poorer nation and are considered neglect- proteins [33–35]. Snake venom CRISPs belong to the CRISP-3 ed, having failed to attract the attention of the pharma- subfamily [36], one of four subgroups of CRISPs, according to ceutical industry. However, novel therapy is sorely needed amino acid sequence homology. Most biological targets of snake for Trypanosoma and Leishmania infections, currently venom CRISPs described to date are ion channels [37–43], treated with ‘dated’ drugs that are often difficult to administer in resource-limiting conditions, have high although the functions and the molecular targets of most snake toxicity and are by no means always successful, partly venom CRISPs remain to be determined. Some snake venom due to the emergence of drug resistance. The last few CRISPs had their biological activities tested on crickets and decades have witnessed a growing interest in examining cockroaches [35]. Snake venom CRISPs have been shown to block 2+ + the potential of bioactive toxins and poisons as drugs or the activity of L-type Ca and/or K -channels and also of cyclic drug leads, as well as for diagnostic applications. In this nucleotide-gated (CNG) ion channels, thereby preventing the context, we isolated and purified crovirin, a protein from contraction of smooth muscle cells [26,37,40–43]. The CRISPs the Crotalus viridis viridis (Cvv) snake venom capable to catrin, piscivorin and ophanin, from the snake Crotalus atrox, inhibiting and/or lysing infective forms of trypanosomatid caused moderate blockage of L-type calcium channels, partially parasites, at concentrations that are not toxic to host cells. inhibiting the contraction of smooth fibers from mouse caudal This feature makes crovirin a promising candidate protein arteries [26]. The Philodryas patagoniensis (green snake) CRISP for the development of novel therapy against neglected patagonin was capable of generating myotoxicity when injected into diseases caused by trypanosomatid pathogens. the gastrocnemius muscle, but did not induce edema formation, haemorrhage or inhibition on platelet aggregation [44]. Despite meglumine antimoniate (Glucantime) and sodium stibogluconate their myotoxicity, there are no reports of CRISP protein lethality to (Pentostan). Amphotericin B and pentamidine are used as second- mice, in concentrations of up to 4.5 mg/kg [35,45], and patagonin line drugs in patients resistant to first-line therapy [1,6]. Recently, did not induce systemic alterations in mice, or histological changes miltefosine has been used in India as part of combination therapy in tissues from the cerebellum, brain, heart, liver and spleen [44]. regimens to treat VL, and the largest increase in miltefosine In a previous publication, we showed that crude venom from the activity was seen in combination with amphotericin B [7,8]. rattlesnake Crotalus viridis viridis had anti-parasitic activity against There are two forms of HAT (also known as sleeping sickness), all forms of T. cruzi, and could be a valuable source of molecules for caused by two subspecies of T. brucei parasites (T.