Calcimycin Inhibits and by Targeting the Prp8 Intein Splicing.

Drug resistance is a significant concern in the treatment of diseases, including cryptococcosis caused by () and (). Alternative drug targets are necessary to overcome drug resistance before it attains a critical stage. Splicing of inteins from pro-protein precursors is crucial for activities of essential proteins hosting intein elements in many organisms, including human pathogens such as and . Through a high-throughput screening, we identified calcimycin (CMN) as a potent Prp8 intein splicing inhibitor with a minimum inhibitory concentration (MIC) of 1.5 μg/mL against the wild-type H99 (-WT or ). In contrast, CMN inhibited the intein-less mutant strain (-Mut) with a 16-fold higher MIC. Interestingly, and a few species were resistant to CMN. Further studies indicated that CMN reduced virulence factors such as urease activity, melanin production, and biofilm formation in . CMN also inhibited intracellular infection in macrophages. In a target-specific split nanoluciferase assay, the IC of CMN was 4.6 μg/mL. Binding of CMN to recombinant Prp8 intein was demonstrated by thermal shift assay and microscale thermophoresis. Treating cells with CMN reduced intein splicing. CMN was fungistatic and showed a synergistic effect with the known antifungal drug amphotericin B. Finally, CMN treatment at 20 mg/kg body weight led to 60% reduction in lung fungal load in a cryptococcal pulmonary infection mouse model. Overall, CMN represents a potent antifungal with a novel mechanism of action to treat and possibly infections.