Virtual Screening Yields Inhibitors of Novel Antifungal Drug Target, Benzoate 4-Monooxygenase

Abstract

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.

Antifungal activity of cinnamic acid derivatives involves inhibition of benzoate 4-hydroxylase (CYP53)

“…in Berne et al . ), there was an essential difference in their shape: in contrast to the previous study, the sloped asymptotes of curves in the presence of increasing inhibitor concentrations in this study are all parallel (see Fig. ).…”

“…The spectral dissociation constant for interaction between cinnamic acid and CYP53A15 ( K s = 96 μ mol l −1 ) was comparable with interactions between the structurally similar benzoic acid and CYP53A15 ( K s of 88 μ mol l −1 ) (Berne et al . ) and was more than three times higher than the K s (330 μ mol l −1 ) described for benzoate 4‐monooxygenase from basidiomycete R. minuta (Fukuda et al . ).…”

“…The characteristic kinetic constants for the inhibition of BA hydroxylation by CYP53A15 in the presence of cinnamic acid and the three most effective (inhibitory) cinnamic acid derivatives were evaluated according to the reaction model for BA hydroxylation by CYP53A15 proposed in our recent work (Berne et al . ): the experimental curves for the time course of para‐hydroxy benzoic acid formation, which followed the characteristic double character, were submitted to the web solver ENZO (Bevc et al . ).…”

“…Furthermore, two cinnamic acid derivatives were among the highest scoring compounds in our recent virtual screening of possible inhibitors of CYP53 catalytic activity (Berne et al . ).…”

In Silico Screening

“…While binding poses are generally well predicted, the accuracy in identifying the crystallized binding mode is where most programs vary [88]. Several compounds were identified as micromolar fungal CYP53 inhibitors by relying on FlexX [96]. Several compounds were identified as micromolar fungal CYP53 inhibitors by relying on FlexX [96].…”