Towards the Development of Anti-virulence Approaches for Candidiasis: A Novel Series of Small Molecule Inhibitors of Candida albicans Filamentation and Biofilm Formation

Candida albicans remains the main etiological agent of candidiasis, the most common fungal infection and now the third most frequent infection in US hospitals. The limited arsenal of antifungal agents and their limited efficacy contribute to the unacceptably high morbidity and mortality rates associated with these infections. The yeast-to-hypha transition represents the main virulence factor associated with the pathogenesis of C. albicans infections. In addition, filamentation is pivotal for robust biofilm development, which represents another major virulence factor during candidiasis further complicating treatment. Targeting pathogenic mechanisms rather than growth represents an attractive, yet clinically unexploited approach in the development of novel antifungal agents. Here we have performed high content phenotypic screening assays to identify small molecule inhibitors of C. albicans filamentation, leading to the identification of a novel series of bioactive compounds with a common biaryl amide core structure. The leading compound of this series, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide (also referred to as 9029936), was able to prevent filamentation under all liquid and solid media conditions tested, suggesting that it impacts a common, core component of the cellular machinery mediating hypha formation. The lead compound also demonstrated in vivo activity in three different clinically-relevant murine models of invasive candidiasis, oral candidiasis and catheter-related candidemia. This compound also displayed activity against a panel of azole resistant clinical isolates and gain of function strains. Moreover, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide displayed potent filament and biofilm inhibitory activity against C. dubliniensis, a species closely related to C. albicans. It demonstrated indifference when used in combinatorial assays with currently available antifungals. Serial passage experiments showed no development of resistance against our lead compound. Furthermore, the lead compound displayed a safe profile based on CEREP Safety Panel and CC50 (toxicity) studies, underscoring its excellent "drug-like" characteristics. Image flow cytometry analysis revealed that N-[3-(allyloxy)-phenyl]-4-methoxybenzamide not only affects hyphal length, but also cell wall and vacuole integrity. RNA-Sequencing analyses revealed the differential expression of key filamentation genes in cells treated with this compound. Based on its in vitro and in vivo activities, this leading compound represents a promising candidate for the development of novel anti-virulence strategies against C. albicans infections.