Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

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.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.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

Cytotoxicity analyses of crovirin on LLC-MK₂ cells (A) and peritoneal macrophages (B) by trypan blue cell viability and MTS assay, respectively.

<p>No tested concentrations induced significant lost of cell viability in either cell type. Error bars represent standard deviation of the mean of 3 independent experiments.</p

Crovirin activity towards medically important trypanosomes and <i>Leishmania</i>.

a<p>The IC₅₀ and LD₅₀ values are expressed in µg/ml.</p>b<p>Selectivity Index (SI)^e was determined according to the ratio between the LC₅₀^e/IC₅₀ or LD₅₀ values. SI values, where LC₅₀>20 µg/ml.</p>c<p>Treatment period.</p>d<p>parasite/100 cells.</p><p>nd: Not determined.</p><p>Crovirin activity towards medically important trypanosomes and <i>Leishmania</i>.</p

Crovirin, a Snake Venom Cysteine-Rich Secretory Protein (CRISP) with Promising Activity against Trypanosomes and <i>Leishmania - Figure 1 </i>

<p>(<b>A</b>) <b>Crovirin purification from Cvv venom using a reverse phase analytical C₈ column, where the protein was eluted as peak 3.</b> (<b>B</b>) SDS-PAGE analysis of peak 3 (lane B) containing the purified crovirin protein under reducing conditions. The gel was stained with Coomassie blue. Lane A, molecular weight markers. (<b>C</b>) MALDI-TOF mass spectrometry analyses of the intact protein yielded a molecular mass of 24,893.64 Da. The peaks of 12,424.36 and 12,477.62 Da correspond to doubly-charged (z = 2) cationic forms of crovirin.</p