Computational strategies for selective inhibition of falcipain-2

Author(s)

Jorge Enrique Hernández González

• Document type: Doctoral Thesis
• Press: São José do Rio Preto. 2020-03-13.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
• Defense date: 2020-02-14
• Advisor: Vitor Barbanti Pereira Leite; Pedro Geraldo Pascutti

Abstract

Falcipain-2 (FP-2) is a key hemoglobinase of Plasmodium falciparum that has been extensively targeted in antimalarial drug discovery projects. Despite the efforts, none of the existing FP-2 inhibitors has entered the clinical trials yet, as cross-inhibition of human off-targets remains a major concern. Here, we tackle issues related to selective FP-2 inhibition by employing different computational techniques, such as molecular dynamics simulations, virtual screening, docking, community analysis and pocket volume and free energy calculations. We also perform in vitro inhibition assays to validate the predictions. We report two compounds, HTS07940 and HTS08262, displaying inhibition against FP-2 and P. falciparum cultures with good selectivity with respect to human cathepsin K (hCatK) and HeLa cell line. The inhibitors were identified through a virtual screening strategy that ruled out ligands with high affinity for hCatK. Moreover, other ways to achieve selective inhibition are explored, such as the search for allosteric inhibitors, which bind to typically less conserved sites. We analyze an FP-2 region equivalent to a previously-characterized allosteric site of hCatK, termed site 6, and predicted various potential ligands. After experimental evaluation, two compounds, ZINC03225317 and ZINC72290660, are confirmed as non-competitive inhibitors of FP-2, thus reinforcing the suitability of site 6 as a druggable allosteric cavity. The search for potential cavities is expanded to other sites on the basis of previous experimental information related to a serendipitously-identified E-chalcone displaying non-competitive inhibition against FP-2. Our results reveal the occurrence of a transient pocket in a region termed site 3, which remains occluded most of the simulation time by the side-chain of residue K34. The predicted binding mode of the E-chalcone is consistent with the available experimental data and sheds light upon important features of the allosteric mechanism at molecular level. Finally, we study the molecular determinants of the high selectivity for FP-2 of previously-reported nitriles containing 3-pyridine substituents at P2. Our results reveal that water bridges involving residues I85 and D234 of FP-2, and the pyridine nitrogen, explain the experimental activity profiles. Therefore, selective FP-2 inhibitors can be designed by promoting the formation of water bridges at the bottom of the S2 subsite and/or by introducing substituents that replace the bridging water molecule. (AU)

• FAPESP's process: 16/24587-9 - In silico identification of novel competitive and alosteric inhibitors of falcipains 2 and 3
• Grantee: Jorge Enrique Hernández González
• Support Opportunities: Scholarships in Brazil - Doctorate