Molecular interaction assays in silico of crotapotin from Crotalus durissus terrificus against the molecular target trypanothione reductase from Leishmania braziliensis.

Abstract

Background: Leishmaniasis is a neglected disease that mainly affects impoverished populations and receives limited attention from governments and research institutions. Current treatments are based on antimonial therapies, which present high toxicity and cause significant side effects, such as cardiotoxicity and hepatotoxicity. This study proposes using crotapotin, isolated from Crotalus durissus terrificus venom, as a potential inhibitor of the enzyme trypanothione reductase from Leishmania braziliensis (LbTR).

Methods: In silico assays were conducted to evaluate the interaction of crotapotin with LbTR using molecular docking and molecular dynamics techniques. Recombinant LbTR was expressed in E. coli, and its enzymatic activity was confirmed. The inhibitory action of crotapotin on LbTR was then tested in enzymatic assays.

Results: The stability of these interactions was confirmed over 200 ns molecular dynamics simulations, with a clustering analysis using the GROMACS method revealing a total of 12 distinct clusters. The five most representative clusters showed low RMSD values, indicating high structural stability of the LbTR-crotapotin complex. In particular, cluster 1, with 3,398 frames and an average RMSD of 0.189 nm from the centroid, suggests a dominant stable conformation of the complex. Additional clusters maintained average RMSD values between 0.173 nm and 0.193 nm, further reinforcing the robustness of the complex under physiological conditions. Recombinant LbTR expression was successful, yielding 4.8 mg/L with high purity, as verified by SDS-PAGE. In the enzymatic assays, crotapotin partially inhibited LbTR activity, with an IC₅₀ of 223.4 μM.

Conclusion: The in silico findings suggest a stable and structured interaction between crotapotin and LbTR, with low structural fluctuation, although the inhibition observed in in vitro assays was moderate. These results indicate the potential of crotapotin as a promising basis for developing specific LbTR inhibitors, contributing to the bioprospecting of new antiparasitic agents.