Advancing Fragment-Based Methods for Dinuclear Metalloenzyme Inhibitors

PROJECT SUMMARY/ABSTRACT This research program is focused on new approaches for the discovery of metalloenzyme inhibitors (MEIs). Metalloenzymes are involved in the propagation of numerous diseases, including bacterial/viral infections, hypertension, cancer, and others. The PI (Cohen) has developed a robust workflow and platform for MEI discovery by uniting the principles of bioinorganic and medicinal chemistry. This program has become internationally recognized as one of the few efforts focused on the specific challenges faced when addressing metalloenzyme inhibition. Our approach to developing MEIs centers on the advancement and utilization of a metal-binding pharmacophore (MBP) fragment library for use in fragment-based drug discovery (FBDD) against metalloenzymes. MBPs are fragments that can engage the active site metal ion(s) in metalloenzymes and provide a starting scaffold for FBDD-based discovery of MEIs. In addition to MBPs, this effort has employed the concept of isostere replacement to create metal-binding isosteres (MBIs) that can improve the drug-like properties of MEIs. In this next phase of our project, we will turn our attention to dinuclear metalloenzymes, which possess two metal ions in the metalloenzyme active site and present unique challenges for MEI development. In this effort, we will develop new sublibraries of MBIs to specifically engage dinuclear metalloenzymes and will apply these new fragments to identify inhibitors of the influenza N-terminal endonuclease domain of the polymerase acidic protein (PAN), the Dengue virus non-structural protein 5 (NS5) RNA-dependent RNA polymerase, the Verona integron-encoded metallo-β-lactamases (VIMs), and the phosphotriesterase-related enzyme (PTER). In addition, we will perform studies to validate the on- target selectivity of MEIs in biochemical and cellular assays. Specifically, enzyme counter screens, thermal shift assays, and pull-down assays with MBPs, MBIs, and MEIs will be pursued. These experiments will also be complimented by targeted protein degradation (TPD) studies to confirm on-target selectivity, as well as advance our program into this exciting therapeutic area. The previous project period generated many collaborations, numerous conference presentations, and >20 publications. In addition, a startup company (Blacksmith Medicines) is advancing our discoveries into clinical realities. We will continue our internal and collaborative efforts to discover best- and first-in-class MEIs that can address infectious disease and other human ailments.