Summary/Abstract Fungal pathogens have an enormous impact on human health worldwide. In the U.S. alone, bloodstream infections have increased by over 200% in recent decades, associated with an increasing number of people with compromised immune function due to treatment for cancer, organ transplantation, and HIV. Poor clinical outcome for most invasive fungal infections is attributable to the very limited number of effective antifungals available and the emergence of clinical resistance to each of the three main modes of action they target. Protein kinases have emerged as richly rewarding targets in the development of drugs for diverse diseases, ranging from cancer to metabolic disorders, but kinases as a class have remained completely untapped in the quest for new antifungals. To begin to fill this void, we tested a panel of well-characterized, structurally diverse kinase inhibitors for activity against a drug-resistant isolate of Candida albicans, the most common human fungal pathogen. This screen identified several compounds which were active against C. albicans and the emerging pathogen, Candida auris. Using chemical genomic approaches, we established the primary target of our most active compounds as Yck2, a fungal member of the widely expressed casein kinase 1 (CK1) family. Using genetic techniques, we confirmed that Yck2 is required for growth in culture under host-relevant conditions, is required to maintain echinocandin-resistance in culture, and enables the virulence of echinocandin-resistant C. albicans in both immune-competent and immune-compromised mice. Now, we will exploit selectivity handles revealed by co-crystal structures of the Yck2 kinase domain in complex with our lead and several other inhibitors to optimize potency, fungal selectivity, and pharmacological properties. Pursuing two scaffolds in parallel as a de-risking strategy, our goal is to deliver one or more advanced leads for future development of a clinical drug candidate. To achieve this goal, our multidisciplinary team will use its expertise in chemistry, structural biology, pharmacology, and fungal biology to pursue the following aims: AIM 1: Structure-enabled synthesis of Yck2 inhibitors with improved antifungal activity AIM 2: Optimize cellular and whole animal pharmacology of fungal Yck2 inhibitors AIM 3: Evaluate tolerability and efficacy in mouse models of systemic fungal infection by drug-resistant clinical isolates with and without concurrent sub-therapeutic echinocandin treatment The Yck2 inhibitors we develop in achieving these aims are expected to possess single agent activity in vivo as well as reverse/prevent resistance to echinocandins. The development of these compounds will be invaluable not only from the perspective of establishing a new target space for discovery and development of mechanistically distinct single-agent antifungals but also in pioneering a resistance-aversive combination approach to antifungal therapy that has proven essential in controlling other infectious diseases.

Project Narrative Invasive fungal infections pose an escalating threat to human health and have enormous economic consequences. This project will optimize compounds that inhibit a newly discovered antifungal target to deliver one or more advanced leads for future development of a clinical drug candidate. In doing so, it will pioneer protein kinases as a greatly needed new target class for highly effective single-agent antifungals which also possess the potential for use in resistance- aversive combination regimens, a treatment strategy that has been missing for antifungals but has proven key to controlling other life-threatening infectious diseases.