Mechanisms of Specific versus Nonspecific Interactions of Aggregation-Prone Inhibitors and Attenuators

A common source of
false positives in drug discovery is ligand
self-association into large colloidal assemblies that nonspecifically
inhibit target proteins. However, the mechanisms of aggregation-based
inhibition (ABI) and ABI-attenuation by additives, such as Triton
X-100 (TX) and human serum albumin (HSA), are not fully understood.
Here, we investigate the molecular basis of ABI and ABI-attenuation
through the lens of NMR and coupled thermodynamic cycles. We unexpectedly
discover a new class of aggregating ligands that exhibit negligible
interactions with proteins but act as competitive sinks for the free
inhibitor, resulting in bell-shaped dose–response curves. TX
attenuates ABI by converting inhibitory, protein-binding aggregates
into nonbinding coaggregates, whereas HSA minimizes nonspecific ligand
interactions by functioning as a reservoir for free inhibitor and
preventing self-association. Hence, both TX and HSA are useful tools
to minimize false positives arising from nonspecific binding but at
the cost of potentially introducing false negatives due to suppression
of specific interactions.