RESUMEN
AIMS: CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. METHODS AND RESULTS: In our work, we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus and two other fungi, Aspergillus niger and Pleurotus ostreatus. To elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15. CONCLUSION: We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development.
Asunto(s)
Antifúngicos/farmacología , Benzoato 4-Monooxigenasa/antagonistas & inhibidores , Cinamatos/farmacología , Inhibidores Enzimáticos/farmacología , Antifúngicos/química , Antifúngicos/metabolismo , Ascomicetos/efectos de los fármacos , Benzoato 4-Monooxigenasa/química , Benzoato 4-Monooxigenasa/metabolismo , Dominio Catalítico , Cinamatos/química , Cinamatos/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Hongos/enzimología , Pleurotus/efectos de los fármacosRESUMEN
Fungal CYP53 enzymes are highly conserved proteins, involved in phenolic detoxification, and have no homologues in higher eukaryotes, rendering them favorable drug targets. Aiming to discover novel CYP53 inhibitors, we employed two parallel virtual screening protocols and evaluated highest scoring hit compounds by analyzing the spectral binding interactions, by surveying the antifungal activity, and assessing the inhibition of catalytic activity. On the basis of combined results, we selected 3-methyl-4-(1H-pyrrol-1-yl)benzoic acid (compound 2) as the best candidate for hit-to-lead follow-up in the antifungal drug discovery process.
Asunto(s)
Antifúngicos/química , Ascomicetos/química , Benzoato 4-Monooxigenasa/antagonistas & inhibidores , Benzoatos/química , Inhibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inhibidores , Pirroles/química , Rhodotorula/química , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Diseño de Fármacos , Descubrimiento de Drogas , Isoenzimas/química , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas Recombinantes/química , Homología Estructural de ProteínaRESUMEN
A novel cytochrome P450, CYP53A15, was identified in the pathogenic filamentous ascomycete Cochliobolus lunatus. The protein, classified into the CYP53 family, was capable of para hydroxylation of benzoate. Benzoate is a key intermediate in the metabolism of aromatic compounds in fungi and yet basically toxic to the organism. To guide functional analyses, protein structure was predicted by homology modeling. Since many naturally occurring antifungal phenolic compounds are structurally similar to CYP53A15 substrates, we tested their putative binding into the active site of CYP53A15. Some of these compounds inhibited CYP53A15. Increased antifungal activity was observed when tested in the presence of benzoate. Some results suggest that CYP53A15 O-demethylation activity is important in detoxification of other antifungal substances. With the design of potent inhibitors, CYP53 enzymes could serve as alternative antifungal drug targets.