Towards new antifungal drugs: A FRET-based HTS assay to identify selective C. albicans HSP90 and HSP90 - Sba1 interaction inhibitors

Increase in drug resistance of pathogenic fungi as well as a growing immunocompromised patient population in risk of fungal infections create a need for novel anti-infective drugs [1].

HSP90 forms homodimers and is an essential chaperone involved in a plethora of protein-protein-interactions (PPI) [2]. The HSP90 conformational cycle is driven by the binding and hydrolysis of ATP by HSP90. Whereas HSP90 is highly conserved across species, co-chaperones of HSP90 like Sba1 are less conserved, providing a potential target structure for the development of selective antifungal drugs [3]. A prerequisite of HSP90 – Sba1 interaction is the prior binding of ATP to HSP90.

In this study, a high-throughput capable in vitro Förster Resonance Energy Transfer (FRET) based assay for the identification of HSP90 - Sba1 interaction inhibitors was developed. The assay is also suitable to identify ATP-competitive HSP90 inhibitors. HSP90-mNeonGreen (donor) and Sba1-mScarlet-I (acceptor) fusion protein constructs showed specific interaction in the assay. Known ATP-competitive HSP90 inhibitors such as geldanamycin can be reliably identified and characterized with the assay. This demonstrates the applicability of the assay to identify and characterize the activity of small molecule inhibitors. Competition experiments with Sba1 for Sba1-mScarlet-I binding to HSP90-mNeonGreen provide evidence for the suitability as a screening assay to also identify direct PPI inhibitors. Analogous setups for the homologous human HSP90 – p23 interaction enable the determination of selectivity for C. albicans PPI inhibition. A small nucleoside-mimetic library of 320 compounds was screened for inhibition of the HSP90 – Sba1 interaction. Overall, the developed assay showed low data variability and a robust separation, resulting in a Z-factor of consistently > 0.5 [4].

References
1. Aldholmi, M.; Marchand, P.; Ourliac-Garnier, I.; Le Pape, P.; Ganesan, A. A Decade of Antifungal Leads from Natural Products: 2010-2019. Pharmaceuticals (Basel) 2019, 12, doi:10.3390/ph12040182.
2. Schopf, F.H.; Biebl, M.M.; Buchner, J. The HSP90 chaperone machinery. Nat. Rev. Mol. Cell Biol. 2017, 18, 345–360, doi:10.1038/nrm.2017.20.
3. Gu, X.; Xue, W.; Yin, Y.; Liu, H.; Li, S.; Sun, X. The Hsp90 Co-chaperones Sti1, Aha1, and P23 Regulate Adaptive Responses to Antifungal Azoles. Front. Microbiol. 2016, 7, 1571, doi:10.3389/fmicb.2016.01571.
4. Ji-Hu Zhang, Thomas D. Y. Chung, and Kevin R. Oldenburg. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.