title: A high-throughput, multiplexed assay for superfamily-wide profiling of enzyme activity.
abstract: The selectivity of an enzyme inhibitor is a key determinant of its usefulness as a tool compound or its safety as a drug. Yet selectivity is never assessed comprehensively in the early stages of the drug discovery process, and only rarely in the later stages, because technical limitations prohibit doing otherwise. Here, we report EnPlex, an efficient, high-throughput method for simultaneously assessing inhibitor potency and specificity, and pilot its application to 96 serine hydrolases. EnPlex analysis of widely used serine hydrolase inhibitors revealed numerous previously unrecognized off-target interactions, some of which may help to explain previously confounding adverse effects. In addition, EnPlex screening of a hydrolase-directed library of boronic acid- and nitrile-containing compounds provided structure-activity relationships in both potency and selectivity dimensions from which lead candidates could be more effectively prioritized. Follow-up of a series of dipeptidyl peptidase 4 inhibitors showed that EnPlex indeed predicted efficacy and safety in animal models. These results demonstrate the feasibility and value of high-throughput, superfamily-wide selectivity profiling and suggest that such profiling can be incorporated into the earliest stages of drug discovery.
authors.affiliation: The Eli and Edythe L. Broad Institute, Cambridge, Massachusetts, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Bachovchin
authors.affiliation: The Eli and Edythe L. Broad Institute, Cambridge, Massachusetts, USA.
authors.forename: Luke W
authors.initials: LW
authors.lastname: Koblan
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Wengen
authors.initials: W
authors.lastname: Wu
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Youhua
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Iwona
authors.initials: I
authors.lastname: Woznica
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Shu
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Jack H
authors.initials: JH
authors.lastname: Lai
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Poplawski
authors.affiliation: Arisaph Pharmaceuticals, Boston, Massachusetts, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Kiritsy
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Healey
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: DiMare
authors.affiliation: Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA.
authors.forename: David G
authors.initials: DG
authors.lastname: Sanford
authors.affiliation: Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Munford
authors.affiliation: 1] Department of Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA. [2] Arisaph Pharmaceuticals, Boston, Massachusetts, USA.
authors.forename: William W
authors.initials: WW
authors.lastname: Bachovchin
authors.affiliation: 1] The Eli and Edythe L. Broad Institute, Cambridge, Massachusetts, USA. [2] Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [3] Harvard Medical School, Boston, Massachusetts, USA. [4] Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Todd R
authors.initials: TR
authors.lastname: Golub
mesh_terms: D000818:Animals; D001897:Boronic Acids / Q000737:chemistry / Q000494:pharmacology; D002219:Carbamates / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors; D054873:Dipeptidyl-Peptidase IV Inhibitors / Q000494:pharmacology*; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005951:Glucose Tolerance Test; D005971:Glutamates / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008252:Macaca fascicularis; D008297:Male; D008810:Mice, Inbred C57BL; D009570:Nitriles / Q000737:chemistry; D009842:Oligopeptides / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D057057:Serine Proteases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology
chemical_list: C000592053:ARI-2408; D001897:Boronic Acids; D002219:Carbamates; D054873:Dipeptidyl-Peptidase IV Inhibitors; D004791:Enzyme Inhibitors; D005971:Glutamates; C000592052:JZL195; D008070:Lipopolysaccharides; D009570:Nitriles; D009842:Oligopeptides; D010879:Piperazines; D015842:Serine Proteinase Inhibitors; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide; D011392:Proline; D002265:Carboxylic Ester Hydrolases; C048964:acyloxyacyl hydrolase; D057057:Serine Proteases