title: Quantitative analyses of aggregation, autofluorescence, and reactivity artifacts in a screen for inhibitors of a thiol protease.
abstract: The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of nonspecific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds, and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.5% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.
authors.affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892-3370, USA.
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: Rafaela S
authors.initials: RS
authors.lastname: Ferreira
authors.forename: Carleen
authors.initials: C
authors.lastname: Klumpp
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Mott
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Thomas
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.forename: Brian K
authors.initials: BK
authors.lastname: Shoichet
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
mesh_terms: D016477:Artifacts; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors; D013329:Structure-Activity Relationship; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
chemical_list: D001426:Bacterial Proteins; D015853:Cysteine Proteinase Inhibitors; D003902:Detergents; D015800:Protozoan Proteins; D065093:beta-Lactamase Inhibitors; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi; C048795:AmpC beta-lactamases; D001618:beta-Lactamases