Identification and mechanism of a highly selective, antifungal acetyl CoA synthetase inhibitor

Fungal infections pose significant health challenges, especially for immunocompromised individuals, with limited antifungal treatments exacerbated by increasing resistance. Acetyl-CoA synthetase (ACS) has emerged as a promising antifungal target due to its critical role in fungal metabolism, particularly in species like Cryptococcus neoformans where ACS activity is essential under host-like conditions. This study identifies a novel ACS inhibitor, isoxazole 1, through a chemical-genetic screen exploiting the synthetic lethality of ACS and ACL mutants. Isoxazole 1 selectively inhibits C. neoformans ACS (CnAcs1) and demonstrates synergy with fluconazole in vitro.Structural and biochemical analyses reveal that isoxazole 1 is an uncompetitive inhibitor, binding uniquely within the CoA tunnel, disrupting its formation by inducing an atypical conformation of the enzyme's C-terminal domain. Despite the high sequence and structural conservation among ACS enzymes, isoxazole 1 exhibits remarkable selectivity for CnAcs1 and does not inhibit homologous enzymes in Candida spp. or humans. Molecular dynamics and structure-activity relationship studies identify critical features of isoxazole 1, including the cyclopropyl-isoxazole and amide carbonyl moieties, as determinants of its potency and specificity.These findings establish isoxazole 1 as a lead compound for antifungal drug development and highlight the potential of targeting ACS for therapeutic interventions in fungal diseases.