Parameter Choice Matters: Validating Probe Parameters for Use in Mixed-Solvent Simulations

Probe mapping is a common approach for identifying potential binding sites in structure-based drug design; however, it typically relies on energy minimizations of probes in the gas phase and a static protein structure. The mixed-solvent molecular dynamics (MixMD) approach was recently developed to account for full protein flexibility and solvation effects in hot-spot mapping. Our first study used only acetonitrile as a probe, and here, we have augmented the set of functional group probes through careful testing and parameter validation. A diverse range of probes are needed in order to map complex binding interactions. A small variation in probe parameters can adversely effect mixed-solvent behavior, which we highlight with isopropanol. We tested 11 solvents to identify six with appropriate behavior in TIP3P water to use as organic probes in the MixMD method. In addition to acetonitrile and isopropanol, we have identified acetone, <i>N</i>-methylacetamide, imidazole, and pyrimidine. These probe solvents will enable MixMD studies to recover hydrogen-bonding sites, hydrophobic pockets, protein–protein interactions, and aromatic hotspots. Also, we show that ternary-solvent systems can be incorporated within a single simulation. Importantly, these binary and ternary solvents do not require artificial repulsion terms like other methods. Within merely 5 ns, layered solvent boxes become evenly mixed for soluble probes. We used radial distribution functions to evaluate solvent behavior, determine adequate mixing, and confirm the absence of phase separation. We recommend that radial distribution functions should be used to assess adequate sampling in all mixed-solvent techniques rather than the current practice of examining the solvent ratios at the edges of the solvent box