Project Summary Protein phosphorylation plays a major role in perturbation-induced signal transduction. The Library of Integrated Network-based Signatures (LINCS) P100 project has generated targeted (using 96 carefully chosen phosphosites) and comprehensive (using DIA, data independent acquisition) mass spectrometry (MS)-based phosphoproteomic datasets characterizing cell states perturbed using a collection of common bioactive therapeutic (“known”) compounds. The analysis of phosphoproteomics data acquired using DIA is challenging, and as a result the comprehensive LINCS DIA data has not yet been leveraged to its full potential. The Molecular Transducers of Physical Activity (MoTrPAC) consortium has also used phosphoproteomics (along with other omics data) to quantify the molecular effects of exercise. Using methods like post-translational modification site- specific enrichment analysis (PTM-SEA, Krug et al. MCP, 2019), perturbation-induced phosphorylation signatures can be correlated with exercise-induced phosphorylation changes to identify known compounds that can mimic the effects of physical activity, providing an exciting opportunity to enhance the combined utility and impact of the LINCS and MoTrPAC Common Fund datasets. The goal of our proposal is to test the hypothesis that there are known compounds that can mimic the effects of physical activity. To accomplish this goal, the PTM signatures database (PTMsigDB) will be significantly expanded using the LINCS DIA data.These signatures will then be correlated with phosphoproteomic changes induced by physical activity provided by MoTrPAC to nominate exercise-mimetic drugs. With these goals in mind we will 1) Develop and apply an automated, cloud-based pipeline to greatly expand phospho-perturbation profiles from existing LINCS data from 100 to several thousand distinct phosphosites; 2) Derive perturbation-specific phosphoproteomic signatures from the greatly expanded phospho-profiling data; and 3) Identify exercise mimetic drugs using PTM signature enrichment in MoTrPAC phosphoproteomic data from acute and long-term exercise. Successful application of the proposed strategy will nominate a list of exercise-mimetic drugs with the potential to initiate entirely new avenues of research focused on finding new therapeutic approaches to combat aging or muscle wasting (cachexia) in cancer patients.