A detergent-based assay for the detection of promiscuous inhibitors.
At micromolar concentrations, many small molecules self-associate into colloidal aggregates that non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor inhibition of beta-lactamase in the absence and presence of detergent. Inhibition that is attenuated in the presence of detergent is characteristic of an aggregate-based mechanism. In the 96-well-format assay described here, about 200 molecules can be tested, in duplicate, per hour for detergent-dependent sensitivity. Furthermore, we also describe simple experiments that can offer additional confirmation of aggregate-based inhibition.
Feng
Brian Y
BY
Department of Pharmaceutical Chemistry, and Graduate Group in Chemistry and Chemical Biology, University of California-San Francisco, 1700 4th Street, California 94158-2330, USA.
Shoichet
Brian K
BK
D003902:Detergents / Q000737:chemistry* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D065093:beta-Lactamase Inhibitors
D003902:Detergents; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors