TY - JOUR
T1 - Small-molecule inhibitors for the Prp8 intein as antifungal agents
AU - Li, Zhong
AU - Tharappel, Anil Mathew
AU - Xu, Jimin
AU - Lang, Yuekun
AU - Green, Cathleen M.
AU - Zhang, Jing
AU - Lin, Qishan
AU - Chaturvedi, Sudha
AU - Zhou, Jia
AU - Belfort, Marlene
AU - Li, Hongmin
N1 - Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
PY - 2021/1/12
Y1 - 2021/1/12
N2 - 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.
AB - 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.
KW - Antifungal
KW - Cryptococcus
KW - Protein splicing
KW - Prp8 intein
KW - Small-molecule inhibitor
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U2 - 10.1073/pnas.2008815118
DO - 10.1073/pnas.2008815118
M3 - Article
C2 - 33397721
AN - SCOPUS:85098997920
SN - 0027-8424
VL - 118
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 2
M1 - e2008815118
ER -