Anti-Fungals Targeting Prp8
The Wadsworth Center discovered small molecule intein splicing inhibitors targeting Prp8, an essential protein for Cryptococcus cellular function. This opens prospects for a mechanistically novel class of antifungals. Microbial inteins are attractive drug targets because humans do not have inteins, and therefore drugs targeting inteins will not disrupt normal human functions. Because Cryptococcus Prp8 inteins share high sequence identity (78-83%) and are absolutely conserved (i.e., unchanging) among the Prp8 inteins, it is likely that Prp8 intein splicing inhibitors would be effective against all, or most, Cryptococcus fungal species. The molecules also showed synergistic effects with FDA-approved antifungals.
Cryptococcus neoformans is an opportunistic human pathogen, causing cryptococcal meningitis in immunocompromised individuals, with a mortality rate of more than 50%. We previously found that the Prp8 intein was a viable drug target for C. neoformans. In an effort to find new therapies, we developed screening assays and identified a small molecule and its fluoride derivative as potent Prp8 intein-splicing inhibitors. These inhibitors bind covalently to an intein active-site residue and inhibit protein splicing, leading to nonfunctional Prp8, which is essential for fungal growth. These molecules inhibited growth of C. neoformans and showed synergistic or additive effects with Food and Drug Administration-approved antimycotics. Overall, the identification of these intein-splicing inhibitors opens up prospects for a new class of antifungals.
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.
- Treatment for infections from Cryptococcus fungal species.
- Synergistic companion treatment with FDA-approved antifungals.
These molecules inhibited growth of C. neoformans and showed synergistic or additive effects with Food and Drug Administration-approved antimycotics.
Targets highly conserved region existing across all or most Cryptococcus fungal species.
No off-target activity has been observed.
Synergistic with FDA approved antifungal amphotericin B – reduced MICs more than fourfold.
Reduced MICs twofold for 5-FC, IZ and voriconazole.
Attractive drug targets because humans do not have inteins, and therefore drugs targeting inteins will not disrupt normal human functions.
A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in 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 wild-type C. neoformans and C. gattii.
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.
Patents and Licensing:
U.S. Application No.: 18/001,316
Small-molecule Inhibitors for the Prp8 Intein as Antifungal Agents. Li, Z., Tharappel, A. M., Xu, J., Lang, Y., Green, C. M., Zhang, J., et al. (2021). Proc. Natl. Acad. Sci. USA 118, e2008815118. doi:10.1073/pnas.2008815118
Tharappel Anil Mathew, Li Zhong, Li Hongmin Frontiers in Molecular Biosciences Vol.9 2022 DOI=10.3389/fmolb.2022.821146
Director Intellectual Property & Licensing