title: Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2.
abstract: Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe's target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (Ki= 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP.
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Sang Joon
authors.initials: SJ
authors.lastname: Won
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Eschweiler
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Jaimeen D
authors.initials: JD
authors.lastname: Majmudar
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Fei San
authors.initials: FS
authors.lastname: Chong
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Sin Ye
authors.initials: SY
authors.lastname: Hwang
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Brandon T
authors.initials: BT
authors.lastname: Ruotolo
authors.affiliation: Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
authors.forename: Brent R
authors.initials: BR
authors.lastname: Martin
keywords: Activity-based protein profiling; chemical proteomics; inhibitor selectivity; native mass spectrometry