Chemoproteomic Screening of Covalent Ligands Reveals UBA5 As a Novel Pancreatic Cancer Target Allison M. Roberts David K. Miyamoto Tucker R. Huffman Leslie A. Bateman Ashley N. Ives David Akopian Martin J. Heslin Carlo M. Contreras Michael Rape Christine F. Skibola Daniel K. Nomura 10.1021/acschembio.7b00020.s003 https://acs.figshare.com/articles/dataset/Chemoproteomic_Screening_of_Covalent_Ligands_Reveals_UBA5_As_a_Novel_Pancreatic_Cancer_Target/4654627 Chemical genetic screening of small-molecule libraries has been a promising strategy for discovering unique and novel therapeutic compounds. However, identifying the targets of lead molecules that arise from these screens has remained a major bottleneck in understanding the mechanism of action of these compounds. Here, we have coupled the screening of a cysteine-reactive fragment-based covalent ligand library with an isotopic tandem orthogonal proteolysis-enabled activity-based protein profiling (isoTOP-ABPP) chemoproteomic platform to rapidly couple the discovery of lead small molecules that impair pancreatic cancer pathogenicity with the identification of druggable hotspots for potential cancer therapy. Through this coupled approach, we have discovered a covalent ligand DKM 2–93 that impairs pancreatic cancer cell survival and <i>in vivo</i> tumor growth through covalently modifying the catalytic cysteine of the ubiquitin-like modifier activating enzyme 5 (UBA5), thereby inhibiting its activity as a protein that activates the ubiquitin-like protein UFM1 to UFMylate proteins. We show that UBA5 is a novel pancreatic cancer therapeutic target and show DKM 2–93 as a relatively selective lead inhibitor of UBA5. Our results underscore the utility of coupling the screening of covalent ligand libraries with isoTOP-ABPP platforms for mining the proteome for druggable hotspots for cancer therapy. 2017-02-10 00:00:00 DKM platform screening covalent ligand libraries compound pancreatic cancer pathogenicity Novel Pancreatic Cancer Target Chemical cysteine-reactive fragment-based covalent ligand library isoTOP-ABPP UBA 5 impairs pancreatic cancer cell survival druggable hotspots tandem orthogonal proteolysis-enabled activity-based protein vivo tumor growth ubiquitin-like protein UFM 1 molecule novel pancreatic cancer cancer therapy