A Hit Prioritization Strategy for Compound Library Screening Using LiP-MS and Molecular Dynamics Simulations Applied...

An important step in screening small-molecule libraries for drug discovery is hit prioritization and validation in order to rule out false positives. This is usually performed using biochemical and biophysical assays, so the development of orthogonal assays that are highly sensitive and can accelerate the hit-to-lead process would be valuable. Limited proteolysis combined with mass spectrometry (LiP-MS) is a technique used to study changes in protein structure upon ligand binding. In LiP-MS, proteins are exposed to low concentrations of proteases under native conditions. The resulting proteolytic patterns are sensitive to the protein structure near the cleavage site, which can change upon ligand binding. We have characterized the interaction of small-molecule inhibitors of the KRas G12D mutant oncoprotein by LiP-MS combined with molecular dynamics (MD). Intact mass spectrometry and top-down analysis were used to detect and identify KRas G12D cleavage products in the presence and absence of inhibitors, thereby locating the cleavage sites within the protein. Cleavage sites that were found to be protected upon compound binding correlated well with the switch II binding site. The degree of cleavage depended on the binding affinity and the presence of specific functional groups in the inhibitor's structure. A comparison of MD simulations for the ligand-free and ligand-bound proteins revealed the atomistic mechanisms by which the cleavage sites, located in flexible and disordered regions, were stabilized upon compound binding. We suggest that LiP-MS combined with MD (LiP-MS-MD) could be valuable in small-molecule screening campaigns and can be added to the current list of methods for high-quality hit selection in early stage drug discovery.