RESUMO
Aim: A structural model of chorismate synthase (CS) from the pathogenic fungus Candida albicans was used for virtual screening simulations. Methods: Docking, molecular dynamics, cell growth inhibition and protein binding assays were used for search and validation. Results: Two molecules termed CS8 and CaCS02 were identified. Further studies of the minimal inhibitory concentration demonstrated fungicidal activity against Paracoccidioides brasiliensis with a minimal inhibitory concentration and minimal fungicidal concentration of 512 and 32 µg·ml-1 for CS8 and CaCS02, respectively. In addition, CaCS02 showed a strong synergistic effect in combination with amphotericin B without cytotoxic effects. In vitro studies using recombinant CS from P. brasiliensis showed IC50 of 29 µM for CaCS02 supporting our interpretation that inhibition of CS causes the observed fungicidal activity.
Assuntos
Antifúngicos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Paracoccidioides/efeitos dos fármacos , Fósforo-Oxigênio Liases/antagonistas & inibidores , Sequência de Aminoácidos , Anfotericina B/farmacologia , Animais , Antifúngicos/química , Antifúngicos/metabolismo , Candida albicans/enzimologia , Chlorocebus aethiops , Sinergismo Farmacológico , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Paracoccidioides/enzimologia , Fósforo-Oxigênio Liases/química , Fósforo-Oxigênio Liases/metabolismo , Ligação Proteica , Células VeroRESUMO
Paracoccidioidomycosis (PCM), caused by Paracoccidioides, is a systemic mycosis with granulomatous character and a restricted therapeutic arsenal. The aim of this work was to search for new alternatives to treat largely neglected tropical mycosis, such as PCM. In this context, the enzymes of the shikimate pathway constitute excellent drug targets for conferring selective toxicity because this pathway is absent in humans but essential for the fungus. In this work, we have used a homology model of the chorismate synthase (EC 4.2.3.5) from Paracoccidioides brasiliensis (PbCS) and performed a combination of virtual screening and molecular dynamics testing to identify new potential inhibitors. The best hit, CP1, successfully adhered to pharmacological criteria (adsorption, distribution, metabolism, excretion, and toxicity) and was therefore used in in vitro experiments. Here we demonstrate that CP1 binds with a dissociation constant of 64 ± 1 µM to recombinant chorismate synthase from P. brasiliensis and inhibits enzymatic activity, with a 50% inhibitory concentration (IC50) of 47 ± 5 µM. As expected, CP1 showed no toxicity in three cell lines. On the other hand, CP1 reduced the fungal burden in lungs from treated mice, similar to itraconazole. In addition, histopathological analysis showed that animals treated with CP1 displayed less lung tissue infiltration, fewer yeast cells, and large areas with preserved architecture. Therefore, CP1 was able to control PCM in mice with a lower inflammatory response and is thus a promising candidate and lead structure for the development of drugs useful in PCM treatment.