Incorporating Hotspot Mapping and Allostery in Structure Based Drug Design.

Hotspots are defined as regions on the protein surface that disproportionately contribute to binding free energy. Mixed-solvent molecular dynamics (MixMD) is a hotspot mapping technique that relies on molecular dynamics simulations of binary solvent mixtures. Previous work in the group on MixMD has established the technique’s effectiveness in capturing binding sites of small organic compounds. The MixMD approach embraces full protein flexibility while allowing for competition between probes and water. Sites preferentially mapped by probe molecules are more likely to be hotspots. First, we establish a rigorous protocol for the identification of hotspots on the binding surface. There are two important requirements: 1) the high-ranking hotspots must be mapped at very high signal-to-noise ratio and 2) the hotspots must be mapped by multiple probes. We have focused our probe molecule repertoire to include acetonitrile, isopropanol, and pyrimidine as these probes allowed us to capture a range of interaction types that include hydrophilic, hydrophobic, hydrogen-bonding and aromatic interactions. Second, we use MixMD to identify both competitive and allosteric sites on proteins. The test cases include Abl Kinase, Androgen Receptor, Chk1 Kinase, Glucokinase, Pdk1 Kinase, Protein-Tyrosine Phosphatase 1B, and Farnesyl Pyrophosphate Synthase. The success of the technique is demonstrated by the fact that the top four sites map the competitive and allosteric sites. We then present methodological developments for characterizing the free energies and entropies of binding sites identified by MixMD. Finally, the significance of these findings is strengthened by a successful prospective application of MixMD on Heat Shock Protein 27. Taken together, these studies demonstrate the powerful utility of MixMD in structure based drug design.