Discovery and development of novel antifungal agents for the treatment of Candida auris infections

Fungal infections are one of the leading causes of death in humans, causing infections that range from mild superficial infections to severe, and life-threatening invasive infections that affect the bloodstream and vital organs. Invasive fungal infections have a high mortality rate, leading to approximately 1.5 million deaths annually. The most common pathogens responsible for these infections are Candida, Cryptococcus, and Aspergillus. Currently, treatment options for invasive fungal infections are limited to three main classes of antifungal drugs: azoles, polyenes, and echinocandins. The emergence of new fungal species, such as Candida auris, which displays high resistance and mortality rates (30-60%), has further complicated treatment efforts. Thus, there is a critical need for new therapeutic strategies to combat these life-threatening pathogens. C. auris isolates have demonstrated significant resistance, especially to azoles (fluconazole) and polyenes (amphotericin B, AmB). To address this, we screened approximately 2,600 FDA-approved drugs and clinical compounds to identify agents capable of inhibiting C. auris growth and enhancing or restoring the antifungal activity of existing antifungals. This screening revealed that HIV protease inhibitors, such as lopinavir, atazanavir, saquinavir and ritonavir, significantly enhanced the antifungal activity of azoles (fluconazole, voriconazole, itraconazole, and posaconazole) and polyene (AmB). Mechanistic studies showed that the HIV protease inhibitors inhibited the fungal efflux pump, and interfered with glucose utilization, leading to reduced ATP levels in C. auris. Moreover, HIV protease inhibitors, in combination with AmB, were able to inhibit the virulence factors of Candida species. Furthermore, HIV protease inhibitors, in combination with itraconazole or posaconazole, resulted in a significant reduction of the C. auris burden in mice kidneys. Two additional drugs, darapladib and rilapladib, phospholipase A2 inhibitors, were identified as potent inhibitors of C. auris. Darapladib and rilapladib demonstrated superior killing kinetics compared to itraconazole. Interestingly, C. auris did not develop any detectable resistance to both drugs at sub-inhibitory concentration over 16-passages. Mechanistic studies revealed that darapladib increased the plasma membrane permeability and caused DNA leakage, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. We further validated the broad-spectrum, fungicidal, and potent activity of darapladib in combination with AmB, showing significant synergy against multidrug-resistant fungal pathogens, including C. auris, Aspergillus fumigatus and mucormycosis-associated species. Additionally, darapladib demonstrated a superior safety profile compared to AmB, exhibiting lower affinity for cholesterol, and no toxicity in combination with AmB to kidney cells. Finally, rilapladib (at 2× MIC) inhibited the C. auris burden by 1.5 Log10, whereas darapladib (at 2× MIC) achieved complete eradication of the C. auris burden in an in vivo C. elegans model.