Computational strategies for selective inhibition of falcipain-2
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2020-02-14
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Universidade Estadual Paulista (Unesp)
Resumo
A falcipaína-2 (FP-2) é uma hemoglobinase chave do Plasmodium falciparum, frequentemente selecionada como alvo para o planejamento de fármacos antimaláricos. Apesar dos esforços, nenhum inibidor da FP-2 tem entrado em fase de ensaios clínicos, pois a inibição cruzada de alvos humanos constitui um obstáculo. Neste trabalho, abordamos questões relacionadas à inibição seletiva da FP-2 através de diferentes técnicas computacionais, como simulações de dinâmica molecular, triagem virtual, ancoramento molecular, análise da comunidades e cálculos de volume de cavidades e energia livre. Ademais, realizamos ensaios de inibição in vitro para validar as predições. Apresentamos os compostos HTS07940 e HTS08262, que inibem a FP-2 e culturas de P. falciparum com boa seletividade respeito à catepsina K humana (hCatK) e à linha celular HeLa. Os inibidores foram identificados por meio de uma estratégia de triagem virtual que descartou ligantes com alta afinidade pela hCatK. Outras formas de atingir inibição seletiva são exploradas, como a busca por inibidores alostéricos, que ligam tipicamente sítios menos conservados. Analisamos uma região da FP-2 equivalente a um sítio alostérico da hCatK previamente caracterizado, o sítio 6, e predizemos vários ligantes potenciais. Após avaliação experimental, dois compostos, ZINC03225317 e ZINC72290660, são confirmados como inibidores não competitivos da FP-2, o que reforça a relevância deste sítio para o planejamento de fârmacos. A busca de cavidades alostéricas é expandida a outros sítios, baseada em informações experimentais prévias relacionadas a uma E-chalcona identificada acidentalmente como inibidor não competitivo da FP-2. Os resultados revelam a ocorrência de uma cavidade transiente numa região denominada sítio 3, que permanece maioritariamente ocluída pela cadeia lateral do resíduo K34. O modo de ligação predito da E-chalcona é consistente com os dados experimentais disponíveis e fornece informação sobre o mecanismo alostérico a nível molecular. Por fim, estudamos os determinantes moleculares da alta seletividade pela FP-2 de nitrilos que contêm substituintes de 3-piridina em P2, previamente estudados. Segundo as nossas predições, pontes de água envolvendo os resíduos I85 e D234 da FP-2 e o nitrogênio da piridina explicam os perfis experimentais de atividade. Portanto, inibidores seletivos da FP-2 podem ser planejados promovendo a formação de pontes de água no fundo do subsítio S2 e/ou introduzindo grupos químicos que substituem a molécula de água envolvida.
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.
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.
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Palavras-chave
Malária, Plasmodium falciparum, Falcipaína-2, Inibidores, Seletividade, Alosteria, Dinâmica molecular, Triagem virtual, Energia livre, Ensaios de inibição, Cavidades transientes, Modo de ligação, Comunidade, Rota de sinalização, Ponte de água, Falcipain-2, Inhibitors, Selectivity, Allostery, Molecular dynamics simulation, Virtual screening, Free energy, Inhibition assays, Transient pockets., Binding mode, Community, Signaling Pathway, Water bridge