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 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.

Keywords: Cryptococcus; Prp8 intein; antifungal; protein splicing; small-molecule inhibitor.

Fig. 1.

Validation of the split-GFP–Prp8 intein-splicing assay. (A) The Prp8 intein-splicing assay based on split-GFP. GFP (200 nM) and GFP–Prp8i (200 nM) were used with DMF or cisplatin (40 µM) for fluorescence detection. n = 8. ***P < 0.001. (B) Dose–response fitting of inhibition of splicing of the GFP–Prp8i by cisplatin. GFP–Prp8i (200 nM) was used. Cisplatin was in twofold serial dilutions with concentrations ranging from 100 µM (30 µg/mL) to 0.78 µM (0.23 µg/mL). n = 3.

Fig. 2.

Pilot screening reveals a strong intein inhibitor. (A) Chemical structures of hit compounds. (B) Inhibition of split-GFP–Prp8 splicing by hit compounds at 20-µM concentration. (C) Inhibition of the split RLuc–Prp8 intein splicing by hit compounds at 20 µM concentration. (D and E) Dose-dependent inhibition of the split-GFP–Prp8i (200 nM) and RLuc–Prp8 (2 nM) intein splicing by 6G-318S, which was in twofold serial dilutions with concentrations ranging from 100 µM to 0.78 µM. n = 3. (F) Dose-dependent inhibition of MIG–Prp8 splicing by 6G-318S. (Left) SDS/PAGE analysis of each sample; (Right) relative percentage reduction of the MIG precursor upon treatment, compared to the starting material (T0). %P decrease is calculated as (%P_T0 − %P_Sample)/(%P_T0 − %P_DMF) × 100, where %P indicates percent of the MIG precursor at time point 0 (P_T0) and/or after ∼18 h of treatment with DMF (P_DMF) or 6G-318S at different concentrations (P_Sample). n = 3.

Fig. 3.

Analogs of 6G-318S have lower inhibitory activity but are intein-specific. (A) Chemical structure of 6G-318S analogs. (B) Inhibition of the split-GFP–Prp8i splicing by the 6G-318S analogs at 20 µM. (C) Dose-dependent inhibition of the GFP–Prp8i intein splicing by 6G-319S, which was in twofold serial dilutions with concentrations ranging from 100 µM to 0.78 µM. n = 3. (D) No inhibition of the trypsin and papain protease activities by intein inhibitors. Compound concentration for cisplatin, 6G-318S, and 6G-319S was set at 400 µM. Control inhibitor AP-2HCl was at 3.5 µM. n = 3.

Fig. 4.

Improved cytotoxicity profile with 6G-319S. Cell viability assay with 6G-318S (A) and 6G-319S (B). A549 cells were incubated with various concentrations of the drugs and then assayed for viability at 48 h postincubation. Experimental data were fitted using a sigmoidal function. Cell viability with DMSO control was set as 100% viable. Medium alone with DMSO was set as 0% viability. n = 3.

Fig. 5.

Direct binding of 6G-318S and 6G-319S to the Prp8 intein. (A) PTSA for binding of compounds to the Prp8 intein. ∆T_m was defined as T_m-drug − T_m-DMSO. (B) Deconvolution of MS spectra of the recombinant WT Prp8 intein (pXI version) and its complex with 6G-318S and 6G-319S. (C) Proposed mechanism of inhibition of the Prp8 intein splicing by compounds 6G-318S and 6G-319S. Chemical reaction as illustrated. (D) Deconvolution of MS spectra of the recombinant C1A Prp8 intein mutant (pXI version) and its complex with 6G-318S and 6G-319S. (E) Kinetic binding data. SPR sensograms are shown with different colors for the binding of 6G-318S at different concentrations to recombinant Cga Prp8 or mutant inteins (pXI version), which were coupled to a ProteOn GLH sensor chip. Global fitting of data to a 1:1 binding model is shown in black.

Fig. 6.

6G-318S inhibits Prp8 intein splicing in vivo. (A) The antibody serum is specific to the Prp8 intein-containing fungi C. gattii and C. neoformans. Western blot analysis of cell lysates of C. gattii, C. neoformans, and C. albicans using a polyclonal anti-Prp8 intein serum. (B) Dose-dependent inhibition of Prp8 intein splicing in C. neoformans H99 by 6G-318S. (C) Time-course of inhibition of Prp8 intein splicing in C. neoformans H99 by 6G-318S. Western blot analysis of cell lysates of C. neoformans H99 treated with DMF or 6G-318S (0.16 μg/mL), using the polyclonal anti-Prp8 intein serum. (D) Dose-dependent accumulation of high molecular band, likely corresponding to Prp8 precursor in 6G-318S-treated C. neoformans H99 cells. (Left) Western blot analysis of cell lysates of C. neoformans H99 treated with DMF or 6G-318S at indicated concentrations, using the polyclonal anti-Prp8 intein serum; (Right) relative ratio between unspliced Prp8 protein precursor (P8P) and spliced intein (Int). n = 3. (E) MS/MS spectra obtained from the fragmentation of Prp8 precursor, including both Prp8 intein and extein peptides. Fragment ions corresponding to y- and b-ions were observed (red lines).