Small-molecule inhibitors for the Prp8 intein as antifungal agents.

Abstract

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats <i>Cryptococcus neoformans</i> (<i>Cne</i>) and <i>Cryptococcus gattii</i> (<i>Cga</i>), the causative agents of cryptococcosis. Inhibition of protein splicing in <i>Cryptococcus</i> sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the <i>Cne</i> Prp8 intein using a split-GFP splicing assay. This revealed the compound 6G-318S, with IC₅₀ values in the low micromolar range in the split-GFP assay and in a complementary split-luciferase system. A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in the inhibition of splicing. 6G-318S and its derivative inhibited splicing of the <i>Cne</i> Prp8 intein in vivo in <i>Escherichia coli</i> and in <i>C. neoformans</i> Moreover, the compounds repressed growth of WT <i>C. neoformans</i> and <i>C. gattii</i> In contrast, the inhibitors were less potent at inhibiting growth of the inteinless <i>Candida albicans</i> Drug resistance was observed when the Prp8 intein was overexpressed in <i>C. neoformans</i>, indicating specificity of this molecule toward the target. No off-target activity was observed, such as inhibition of serine/cysteine proteases. The inhibitors bound covalently to the Prp8 intein and binding was reduced when the active-site residue Cys1 was mutated. 6G-318S showed a synergistic effect with amphotericin B and additive to indifferent effects with a few other clinically used antimycotics. Overall, the identification of these small-molecule intein-splicing inhibitors opens up prospects for a new class of antifungals.