Sun, C.; Nagarajan, B.; Rijal, A.; Lyons, C. E.; Hamza, A.; Wang, W.; Fang, X.; Cen, Y.; Liu, Q.

Fungal infections remain a major cause of morbidity and mortality worldwide; however, therapeutic options are limited and are increasingly compromised by antifungal resistance. Heat Shock Protein 110 kDa (Hsp110) is an essential molecular chaperone required for protein homeostasis (proteostasis) and fungal viability and has recently emerged as a promising antifungal target. We previously identified 2H, a first-in-class inhibitor of fungal Hsp110 proteins that exhibits broad-spectrum antifungal activity through disruption of proteostasis. Heat Shock Transcription Factor 1 (HSF1), the master regulator of cellular stress responses and chaperone expression, has also been proposed as a potential antifungal target, although direct pharmacological evidence supporting this strategy remains limited. Here, we investigated the effects of combining the HSF1 inhibitor DTHIB with the fungal Hsp110 inhibitor 2H in Cryptococcus neoformans and Candida albicans. Although DTHIB alone exhibited minimal antifungal activity, it significantly enhanced the growth-inhibitory and fungicidal effects of 2H. Notably, this potentiating effect was specific to 2H and was not observed with fluconazole. Consistent with the roles of Hsp110 and HSF1 in proteostasis, 2H induced extensive accumulation of detergent-insoluble protein aggregates in C. neoformans, whereas HSF1 inhibition further increased both the abundance and diversity of aggregated proteins. Quantitative proteomic analyses demonstrated that 2H induces proteome-wide protein aggregation, impacting proteins involved in a broad range of essential cellular processes, with high-molecular-weight proteins exhibiting particular susceptibility. Together, these findings provide pharmacological evidence supporting HSF1 as a promising antifungal sensitization target and demonstrate the utility of 2H as a chemical probe for studying Hsp110-dependent proteostasis. Simultaneous disruption of both HSF1-mediated stress responses and Hsp110-dependent proteostasis promotes extensive proteome instability and enhances antifungal activity, highlighting a potential new strategy for antifungal drug development.