Structure-Guided Discovery of the Novel Covalent Allosteric Site and Covalent Inhibitors of Fructose-1,6-Bisphosphate Aldolase to Overcome the Azole Resistance of Candidiasis

Fructose-1,6-bisphosphate aldolase
(FBA) represents an attractive
new antifungal target. Here, we employed a structure-based optimization
strategy to discover a novel covalent binding site (C292 site) and
the first-in-class covalent allosteric inhibitors of FBA from Candida albicans (CaFBA). Site-directed mutagenesis,
liquid chromatography–mass spectrometry, and the crystallographic
structures of APO–CaFBA, CaFBA–G3P, and
C157S–2a4 revealed that S268 is an essential pharmacophore
for the catalytic activity of CaFBA, and L288 is an allosteric regulation
switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors
exhibited good inhibitory activity against azole-resistant C. albicans, and compound 2a11 can inhibit
the growth of azole-resistant strains 103 with the MIC80 of 1 μg/mL. Collectively, this work identifies a new covalent
allosteric site of CaFBA and discovers the first generation of covalent
inhibitors for fungal FBA with potent inhibitory activity against
resistant fungi, establishing a structural foundation and providing
a promising strategy for the design of potent antifungal drugs.