Protein stability effects in aggregate-based enzyme inhibition.

Small molecule aggregates are a leading cause of artifacts in early drug discovery, but little is known about their interactions with proteins, nor why some proteins are more susceptible to inhibition. A possible reason for this apparent selectivity is that aggregation-based inhibition, as a stoichiometric process, is sensitive to protein concentration, which varies across assays. Alternatively, local protein unfolding by aggregates may lead to selectivity, since stability varies among proteins. To deconvolute these effects, we used differentially stable point mutants of a single protein, TEM-1 β-lactamase. Broadly, destabilized mutants had higher affinities and were more inhibited to and by aggregates versus to more stable variants. Addition of the irreversible inhibitor moxalactam destabilized many mutants, and these typically bound with tighter to a colloidal particle, while mutant it stabilized bound weaker. These results suggest that less stable enzymes are more easily sequestered and inhibited by colloidal aggregates.