Structure-based design of antifungal inhibitors targeting the glycosylphosphatidylinositol (GPI) ethanolamine phosphate transferase, MCD4 - the center for disease control (CDC) and world health…

Structure-based design of antifungal inhibitors targeting the glycosylphosphatidylinositol (GPI) ethanolamine phosphate transferase, MCD4 - the center for disease control (CDC) and world health organization (who) have both recently emphasized a critical need for the development of new antifungal agents with novel mechanism of action(S) (MOA) to address expanding clinical resistance to standard of care (SOC) antifungal therapeutics, emerging fungal pathogens, and the unacceptably high rates of morbidity and mortality associated with invasive mycoses despite current treatment options. Candida albicans is the principal clinically relevant invasive fungal pathogen in the U.S. And a leading cause of bloodstream infections (CANDIDEMIA). The recent emergence of C. Auris is also alarming as IT is often multidrug resistant to most if not all antifungal drug classes. Aspergillus fumigatus is the principal mold pathogen and is the cause of invasive aspergillosis, where mortality rates can exceed 50% despite SOC treatment. Only three major drug classes exist to treat life-threatening infections: amphotericin B (and various formulations), azoles, and echinocandins. Each drug class, however, possesses its own limitations (spectrum, administration, resistance, drug-drug interactions, and/or toxicity). Also concerning, few mechanistically novel antifungals have advanced beyond phase 1 clinical development. One exception is fosmanogepix (FMGX), a potent and selective inhibitor of glycosylphosphatidylinositol (GPI) biosynthesis. GPI biosynthesis serves as an attractive antifungal drug target as enzymes involved in this pathway are highly conserved and essential for fungal growth. We have identified the natural product, M743, which also inhibits GPI biosynthesis but by inhibiting a distinct (MCD4-MEDIATED) step in the pathway. M743 offers significant antifungal drug development advantages, including 1) potent and broad spectrum in vitro antifungal activity against multiple who-designated critical priority fungal pathogens, 2) a novel MOA to overcome existing clinical drug resistance mechanisms, 3) preliminary in vivo efficacy, 4) synergistic activity in combination with manogepix (the active form of FMGX), and 5) the future opportunity to benefit from FMGX clinical development learnings. To assist its development, we propose to perform cryo-EM studies to identify key molecular contacts between M743 and its fungal target versus the human ortholog, PIG-N. These studies will guide hypothesis-based MED chem synthesis of new analogs to explore the opportunity of rationally improving fungal potency and selectivity of the series. Specific aims: aim 1. Expression, purification, and high-resolution cryo-EM structural elucidation of C. Albicans (CA) MCD4 as well as its human ortholog, PIG-N in APO and M720-bound form. Milestone 1. Obtain high-resolution structures (< 2.80 Å) of CAMCD4 and PIG-N in both APO and M720-bound form. Aim 2. Hypothesis-driven MED chem synthesis of up to 30 new semi-synthetic analogs based on aim 1 structural insights to identify new analogs displaying improved antifungal selectivity while maintaining or improving existing potency of M743. Milestone 2. Identify (N > 1) new analogs with reduced cytotoxicity against HEPG2, HEK293, and caco-2 cell lines (TI> 200) while maintaining or improving candida and aspergillus whole-cell activity and CAMCD4 IC50 versus M743.