Discovery and optimization of antifungal acetyl CoA synthetase inhibitors

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PROJECT SUMMARY Recently, we discovered that a small molecule inhibitor of acetyl CoA synthetase (ACS), AR-12, has broad spectrum fungicidal activity in vitro and promising activity in vivo. Consistent with this broad spectrum of activity, genetic studies indicate that ACS is essential for viability in multiple fungi (C. albicans, Fusarium, S. cerevisiae). In contrast, ACS is not essential in mammals. This is likely because, in mammals and plants, the vast majority of acetyl CoA is derived from ATP-citrate lyase (ACL) and not ACS. The most important exception to this rule is the cancer cell where ACS is the predominant source of acetyl CoA. Consequently, ACS has emerged as an anti-cancer target. Although the development of AR-12 stalled, we propose that its target, ACS, remains worthy of further exploration as the basis for a new class of antifungal drugs.To identify novel inhibitors of fungal ACSs, we have developed a multi-disciplinary approach based on: 1) two complementary small molecule screening strategies; 2) the structural characterization ACS-inhibitor complexes from multiple pathogenic fungi: 3) whole cell assays of ACS function and inhibition, and 4) medicinal chemistry strategies that have already yielded micromolar inhibitors of ACS. An STD-NMR screen with C. neoformans Acs1 and identified 492 ACS interacting molecular fragments, of which the vast majority also interacted with multiple fungal ACS enzymes. In Aim 1, we will further characterize these hits. As a parallel strategy, we adapted our ACS activity assay for high throughput screening (HTS) with the goal of directly identifying small molecule ACS inhibitors. Our chemistry plan (Aim 2) is guided, in part, by the hypothesis that molecules mimicking the acetyl adenosine-monophosphate ester (AcAMP) intermediate are likely to be effective inhibitors. In Aim 2A, we will characterize the acetyl-PO3 binding pocket by a structure-activity study of AcAMP mimics derived from molecules already crystallized in the active site of fungal ACSs. Biochemically stable, potent acetyl-PO3 isosteres emerging from this analysis will then be linked with putative ATP/AMP-binding pocket-targeted fragments to assemble candidate non-nucleoside, bi- substrate ACS inhibitors. To complement this hypothesis-based strategy, candidate inhibitors will also be assembled from other strongly interacting fragments and we will optimize inhibitors directly identified in the ACS activity-based HTS screen (Aims 2B&C). New molecules will be evaluated (Aim 3) with a testing funnel that includes biochemical characterization of ACS inhibition, antifungal activity against a range of pathogenic fungi, whole cell assays of on-target activity against ACS, and initial in vitro toxicity/ADME characterization. Our goal is to identify a lead ACS inhibitor scaffold along with a back-up series for further pre-clinical development as broad-spectrum antifungal drug candidates.