Small-molecule inhibitors for the Prp8 intein as antifungal agents

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus 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 Cne 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 Cne Prp8 intein in vivo in Escherichia coli and in C. neoformans. Moreover, the compounds repressed growth of WT C. neoformans and C. gattii. In contrast, the inhibitors were less potent at inhibiting growth of the inteinless Candida albicans. Drug resistance was observed when the Prp8 intein was overexpressed in C. neoformans, 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.

Many microbial pathogens contain self-splicing elements called inteins, which are internal proteins that self-excise from their intein-hosting proteins and catalyze ligation of the flanking sequences (exteins) with a natural peptide bond (1–4). Overall, more than 1,700 inteins have been identified (5). Among intein-containing deadly human pathogens is Mycobacterium tuberculosis, which has inteins in three critical genes, involved in replication (dnaB), iron-sulfur cluster assembly (sufB), and recombination (recA). M. tuberculosis infections cause 2 million annual tuberculosis-related deaths worldwide (6). Pathogenic fungi, such as Crypotococcus neoformans (Cne), Crypotococcus gattii (Cga), and Aspergillus fumigatus also encode inteins, in the pre-mRNA processing factor 8 (Prp8) gene (7, 8). Globally, over 300 million people are affected by invasive fungal infections, with estimated deaths of over 1 million people every year (9–12). Moreover, the emergence of severely drug-resistant strains of M. tuberculosis and pathogenic fungi, plus the deadly synergistic association with HIV/AIDS, represent significant public health challenges (13–18).Since inteins consistently interrupt highly conserved sites of intein-hosting proteins, splicing inhibition can cause a disruption of functions that are essential for the pathogen’s survival. Inteins are therefore attractive targets for drug development (7, 8, 19–21). Additionally, inteins do not occur in multicellular organisms including humans nor in unicellular organisms including bacteria normally associated with the human gut flora, making intein-inhibiting drugs highly selective for intein-containing pathogens, such as M. tuberculosis, C. neoformans, C. gattii, and A. fumigatus.Previously we found that cisplatin, a Food and Drug Administration (FDA)-approved chemotherapeutic drug, inhibited fungal Prp8 intein splicing in vitro and reduced fungal burden in vivo (8). The action of cisplatin is by the platinum ion being coordinated by the catalytic Cys1 of the inetin, thereby inhibiting the first step of splicing and subsequent branched intermediate formation and extein ligation. However, the high cytotoxicity of cisplatin and its derivatives (22–24) may limit their use in immunocompromised patients. In the present study, we performed a pilot screening of a small-molecule library and found a compound and its derivative that impede fungal Prp8 intein splicing in a dose-dependent manner. In addition, these molecules inhibited the Prp8 intein-containing fungi C. neoformans and C. gattii, but not the yeast Candida albicans, a major human pathogen that does not encode a Prp8 intein. Furthermore, C. neoformans treated with the small molecules led to accumulation of unspliced Prp8 precursor. The potency of these inhibitors is better than or comparable to the current frontline antifungal drugs. Mechanistic studies indicated that the small molecules inhibited Prp8 intein splicing by covalently binding to the Prp8 intein active-site residue Cys1, the nucleophile that initiates the protein splicing reaction.