Metalloenzymes represent an important target space for drug discovery. However, a major limitation to the development of metalloenzyme inhibitors has been the lack of established structure-activity relationships (SARs) for the metal ion cofactor(s) of a metalloenzyme. To address this shortcoming, a >350-component metal-binding pharmacophore (MBP) library was developed and used to both identify trends in metalloenzyme inhibition and to identify ideal fragment leads for fragment-based drug development (FBDD) campaigns. This library has proven a useful tool for FBDD, and library screens against over 25 therapeutically relevant metalloenzyme targets have routinely produced high hit rates, often in excess of 15% at 200 µM screening concentration.

Using influenza RNA polymerase PAN endonuclease as a representative metalloenzyme, we employed a bioinorganic perspective of metalloenzyme metal ion coordination to identify SARs unique to inhibition of this metalloenzyme. Identified trends highlighted the importance of ligand electronics (i.e., donor ability) of MBPs, in addition to MBP sterics, for achieving improved metalloenzyme inhibition and selectivity. By optimizing MBP preferences for PAN, we developed new classes of highly selective inhibitor fragments (MW <200) that display IC50 values <50 nM that also show good selectivity over other metalloenzymes. Based on these highly active MBPs for PAN inhibition, a FBDD campaign was pursued. Guided by principles of inorganic coordination chemistry and structure-based drug design (SBDD), MBP scaffolds were elaborated to further improve activity and selectivity. SARs were established and used to generate PAN inhibitors with unprecedented picomolar binding affinities. The activity of these inhibitors were analyzed and validated using a combination of FRET-based nuclease activity assays and differential scanning fluorometry (DSF), which was also shown to be a valuable tool for evaluating highly active inhibitors against this target. Lead compounds were found to be highly selective for PAN over several related dinuclear and mononuclear metalloenzymes. Several of these inhibitors were assayed against live virus in MDCK cells and were found to inhibit influenza viral infection. Combining principles of bioinorganic and medicinal chemistry has ultimately resulted in the development the most active in vitro influenza endonuclease inhibitors reported to date.