ABSTRACT Fluconazole (FLC) is widely used to prevent and treat invasive fungal infections. However, FLC is a fungistatic agent, allowing clinical FLC-susceptible isolates to tolerate FLC. Making FLC fungicidal in combination with adjuvants is a promising strategy to avoid FLC resistance and eliminate the persistence and recurrence of fungal infections. Here, we identify a new small molecule compound, CZ66, that can make FLC fungicidal. The mechanism of action of CZ66 is targeting the C-4 sterol methyl oxidase, encoded by the ERG251 gene, resulting in decreased content of sterols with the 14α-methyl group and ultimately eliminating FLC tolerance of Candida albicans. CZ66 most likely interacts with Erg251 through residues Glu195, Gly206, and Arg241. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance. IMPORTANCE Fluconazole (FLC) tolerance increases the frequency of acquired FLC resistance, and a high FLC tolerance level is associated with persistent candidemia. Multiple functional proteins, such as calcineurin, heat shock protein 90 (Hsp90), and ADP ribosylation factor, are essential for the survival of C. albicans exposed to FLC, but how these factors increase the fungicidal activity of FLC remains to be determined. In this study, we found that 14α-methylsterols replace ergosterol to allow C. albicans to survive FLC, but Erg251 inactivated by CZ66 results in loss of 14α-methylsterol synthesis and cell death of C. albicans treated with FLC. Establishing Erg251 as a synergistic lethal target protein of FLC should direct research to identify specific small molecule inhibitors of 14α-methylsterol synthesis and open the way to abolishing fungal FLC tolerance.
