A detergent-based assay for the detection of promiscuous inhibitors

Abstract

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.

Figure 1

Transmission electron micrograph of aggregates of tetraiodophenolphthalein (dark edged circles) associated with β-galactosidase. This association inhibits the enzyme by sequestering it from substrate, although whether these are adsorption or absorption effects remains uncertain at this time. Bar, 200 nm. Reproduced from ref.