Screening in a spirit haunted world

Abstract

High-throughput screening (HTS) campaigns can be dominated by hits that ultimately turn out to be non-drug-like. These "nuisance" compounds often behave strangely, with steep dose-response curves, absence of clear structure-activity relationships, and high sensitivity to assay conditions. Several mechanisms contribute to these artifacts, including chemically reactive molecules, those that absorb light in assays and those that affect redox conditions. One of the most common mechanisms behind artifactual inhibition is discussed in this review: at micromolar concentrations organic molecules can aggregate to form particles in aqueous buffers, and these aggregates can sequester and thereby inhibit protein targets. Aggregation-based inhibition is baffling from a chemical perspective, but viewed biophysically such behavior is expected. The range of molecules that behave this way, their rapid detection in a screening environment and their possible biological implications will be considered here.

FIGURE 1

Aggregating promiscuous inhibitors have characteristic physical features and behaviors that distinguish them from classical inhibitors. Their distinct physical properties lend themselves to detection by direct biophysical measurement of particle size and shape; their presence is often indicated by steep dose-response curves. (a) Particle formation by the promiscuous inhibitor tetraiodophenolphthalein, as visualized by transmission electron microscopy (Bar = 100 nm) and (b) by dynamic light scattering. (c) The dose-response curve of Rottlerin, a promiscuous inhibitor at micromolar concentrations (circles) compared to that of BZB, a pure competitive inhibitor (squares), both acting against the enzyme β-lactamase. Reprinted, with permission, from Refs. [8] and [13].

FIGURE 2

Direct association between enzymes and aggregates. Several lines of evidence suggest that aggregates act by direct association with enzymes, including co-precipitation of enzyme by aggregates and the concentration of green fluorescent protein (GFP) fluorescence on the surface of aggregates. Co-precipitation can be measured by incubating the enzyme with an aggregating inhibitor followed by spinning the aggregate down in a microfuge and running the precipitant via an SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). Localization of GFP can be observed by the punctuate GFP fluorescence that develops in the presence of an aggregating inhibitor. (a) Co-precipitation of β-lactamase by tetraiodophenolphthalein, a promiscuous aggregator, followed by gel electrophoresis. SDS-PAGE and silver-stain analysis of supernatants and pellets from centrifugation of β-lactamase in the presence or absence of inhibitor, with and without Triton X-100. (b) Same as in (a), except with the promiscuous inhibitor 4-bromophenylazo-(4′)-phenol. (c) Fluorescence of GFP in the absence and (d) in the presence of the aggregator tetrai-odophenophthalein. (e) Same as in (c), but with addition of the detergent Triton-X 100 at 0.01% concentration. Scale bar = 5 μm. Adapted, with permission, from Ref. [16].

FIGURE 3

Many ‘drug-like’ molecules form promiscuous aggregates at micromolar concentrations. More than 1000 such compounds were evaluated for detergent-dependent inhibition and dynamic light-scattering, using 96-well plate based assays. Molecules were tested in different sets: two sets of predicted aggregators and two sets of predicted non-aggregators, derived using different computational models, were tested, as was a fifth category chosen at random from a ‘drug-like’ subset of the ChemDiv collection (see text). Reprinted, with permission, from Ref. [17].