Abstract 2755: Limited proteolysis coupled to mass spectrometry (LiP-MS), a novel drug target deconvolution strategy

Background High attrition rates in target-centric drug development approaches, as well as a limited number of targets, have shifted the focus of drug development back towards phenotypic screening. In parallel, novel proteomics-based target deconvolution approaches to drug target identification have gained popularity. Limited proteolysis coupled with mass spectrometry (LiP-MS) is a new target deconvolution technique that exploits protein structural alterations driven by drug binding. A major advantage of LiP-MS is its unique focus on detection of peptides that report on ligand binding induced structural changes that are generated by a limited digestion and identified by proteomic analysis. Here we demonstrate the performance of LiP-MS using the protein phosphatase inhibitor Calyculin A, as well as two well known kinase inhibitors, selumetinib (SE) and staurosporine (ST), in HeLa cell lysate. Materials and Methods Mechanically sheared HeLa cell lysate was incubated with compound at multiple concentrations. Next, a limited digest was performed using proteinase K. Finally, the limited digests were processed to peptides with trypsin for mass spectrometry analysis. A project-specific spectral library was generated using data-dependent acquisition (DDA) mass spectrometry and for quantitative analysis data-independent acquisition (DIA) data were recorded and analyzed using Spectronaut Pulsar X. Results LiP-MS identifies several peptides for the phosphatase inhibitor Calyculin A, with IC50 values of 52 nM and 17 nM for PP1A and PP2A respectively. Additionally, a previously unknown target PP1B was also identified among the same family, although with a higher IC50 (74 nM). Through structural inspection of ligand-sensitive peptides we were able to map the drug’s binding site within the phosphatases and predict distal conformational changes, demonstrating that LiP-MS can be used to provide structural insights to ligand-protein binding. Similar dose response relationships were observed for both specific (SE) and broad (ST) kinase inhibitors. Amongst the top 200 identified target candidate peptides ranked by LiP score, GO enrichment analysis confirmed a highly significant 3-fold enrichment for kinase targets (p < 0.00002) in ST-treated lysate, while no such enrichment was observed for SE. However, in SE-treated lysate robust identification of multiple MEK1 peptides, one of the compound’s main targets, was observed. In the case of both kinase inhibitors LiP peptides could