Small-molecule inhibitors for the Prp8 intein as antifungal agents

Zhong Li, Anil Mathew Tharappel, Jimin Xu, Yuekun Lang, Cathleen M. Green, Jing Zhang, Qishan Lin, Sudha Chaturvedi, Jia Zhou, Marlene Belfort, Hongmin Li

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article numbere2008815118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number2
DOIs
StatePublished - Jan 12 2021

Keywords

  • Antifungal
  • Cryptococcus
  • Protein splicing
  • Prp8 intein
  • Small-molecule inhibitor

ASJC Scopus subject areas

  • General

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