| Autor: |
Huth SW; Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.; Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States., Oakley JV; Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.; Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States., Seath CP; Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.; Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States., Geri JB; Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.; Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States., Trowbridge AD; Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.; Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States., Parker DL Jr; Discovery Chemistry, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States., Rodriguez-Rivera FP; Discovery Chemistry, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States., Schwaid AG; Discovery Chemistry, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States., Ramil C; Discovery Chemistry, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States., Ryu KA; Merck Exploratory Science Center, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States., White CH; Merck Exploratory Science Center, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States., Fadeyi OO; Merck Exploratory Science Center, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States., Oslund RC; Merck Exploratory Science Center, Merck & Co., Inc., Cambridge, Massachusetts 02141, United States., MacMillan DWC; Merck Center for Catalysis at Princeton University, Princeton, New Jersey 08544, United States.; Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States. |
| Abstrakt: |
The characterization of ligand binding modes is a crucial step in the drug discovery process and is especially important in campaigns arising from phenotypic screening, where the protein target and binding mode are unknown at the outset. Elucidation of target binding regions is typically achieved by X-ray crystallography or photoaffinity labeling (PAL) approaches; yet, these methods present significant challenges. X-ray crystallography is a mainstay technique that has revolutionized drug discovery, but in many cases structural characterization is challenging or impossible. PAL has also enabled binding site mapping with peptide- and amino-acid-level resolution; however, the stoichiometric activation mode can lead to poor signal and coverage of the resident binding pocket. Additionally, each PAL probe can have its own fragmentation pattern, complicating the analysis by mass spectrometry. Here, we establish a robust and general photocatalytic approach toward the mapping of protein binding sites, which we define as identification of residues proximal to the ligand binding pocket. By utilizing a catalytic mode of activation, we obtain sets of labeled amino acids in the proximity of the target protein binding site. We use this methodology to map, in vitro, the binding sites of six protein targets, including several kinases and molecular glue targets, and furthermore to investigate the binding site of the STAT3 inhibitor MM-206, a ligand with no known crystal structure. Finally, we demonstrate the successful mapping of drug binding sites in live cells. These results establish μMap as a powerful method for the generation of amino-acid- and peptide-level target engagement data. |
