1. Academic Validation
  2. Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2

Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2

  • ACS Med Chem Lett. 2016 Dec 9;8(2):215-220. doi: 10.1021/acsmedchemlett.6b00441.
Sang Joon Won 1 Joseph D Eschweiler 1 Jaimeen D Majmudar 1 Fei San Chong 1 Sin Ye Hwang 1 Brandon T Ruotolo 1 Brent R Martin 1
Affiliations

Affiliation

  • 1 Program in Chemical Biology and Department of Chemistry, University of Michigan , 930 North University Avenue, Ann Arbor, Michigan 48109, United States.
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.

Keywords

Activity-based protein profiling; chemical proteomics; inhibitor selectivity; native mass spectrometry.

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