Computational Reverse-Engineering of a Spider-Venom Derived Peptide Active Against Plasmodium falciparum SUB1

Giacomo Bastianelli,Dung Le-Nguyen,Michael Nilges,Elodie Crublet,Stéphane Pêtres,Christophe Nguyen,Jean-Christophe Barale,Olivier Gorgette,Anthony Bouillon,Annalisa Pastore

doi:10.1371/journal.pone.0021812

Abstract

BackgroundPsalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC in the low micromolar range. PcFK1 was proposed to act as an ion channel inhibitor, although experimental validation of this mechanism is lacking. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity.FindingsMolecular modeling and docking calculations showed that one loop could interact with the binding site of PfSUB1. The calculated free energy of binding averaged −5.01 kcal/mol, corresponding to a predicted low-medium micromolar constant of inhibition. PcFK1 inhibited the enzymatic activity of the recombinant PfSUB1 enzyme and the in vitro P.falciparum culture in a range compatible with our bioinformatics analysis. Using contact analysis and free energy decomposition we propose that residues A14 and Q15 are important in the interaction with PfSUB1.ConclusionsOur computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity. This outlines the usefulness of advanced bioinformatics tools to predict the function of a protein structure. The structural features of PcFK1 represent an interesting protein scaffold for future protein engineering.

Highlights

Despite recent scaling up of control efforts, malaria remains a major public health problem [1]
Our computational reverse engineering supported the hypothesis that PcFK1 targeted PfSUB1, and this was confirmed by experimental evidence showing that PcFK1 inhibits PfSUB1 enzymatic activity
Like other subtilisin-like proteases, PfSUB1 recognizes a broad range of sequences, but shows a pattern of preferred amino acids with hydrophobic aliphatic side chains in P4 while polar or positively charged residues are often present in P3

Summary

Introduction

Despite recent scaling up of control efforts, malaria remains a major public health problem [1]. Psalmopeotoxin I (PcFK1) is composed of 33 residues, while Psalmopeotoxin II (PcFK2) has 28 residues; both have three disulfide bridges and belong to the cystine knot motif superfamily These two peptides inhibit the growth of the parasite with an IC50 in the low micromolar range, show no antifungal or antibacterial activity and are neither hemolytic nor do they affect the growth or viability of human epithelial cells. Psalmopeotoxin I (PcFK1), a protein of 33 aminoacids derived from the venom of the spider Psalmopoeus Cambridgei, is able to inhibit the growth of Plasmodium falciparum malaria parasites with an IC50 in the low micromolar range. The surface loops of PcFK1 have some sequence similarity with the parasite protein sequences cleaved by PfSUB1, a subtilisin-like protease essential for egress of Plasmodium falciparum merozoites and invasion into erythrocytes. As PfSUB1 has emerged as an interesting drug target, we explored the hypothesis that PcFK1 targeted PfSUB1 enzymatic activity

Methods

Results

Conclusion