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

Abstract

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 aggregationbased 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.

The Epac1 Protein: Pharmacological Modulators, Cardiac Signalosome and Pathophysiology

“…Some Epac small-molecule inhibitors, including ESI-09, are prone to aggregation-based inhibition leading to false positives arising from nonspecific binding. The mechanisms by which additives such as Triton X-100 and serum albumin can attenuate this adverse effect have been studied in detail by combination of biophysical techniques [36]. Biochemical characterization and SAR studies indeed suggested that ESI-09 inhibited activity of both Epac1 and Epac2 at concentrations well below those that induce protein denaturation [37].…”

Recent Advances in EPAC-Targeted Therapies: A Biophysical Perspective

“…This property may limit the maximum CE3F4R concentration used in therapeutic applications and increases susceptibility to non-specific effects such as aggregation-based inhibition. This led Boulton et al to investigate CE3F4R’s non-specific interactions with EPAC1 using diverse techniques with varying degrees of resolution [47]. The formation of CE3F4R aggregates in vitro was studied using two commonly-used techniques, namely dynamic light scattering (DLS) and transmission electron microscopy (TEM) [34,35,42,47].…”

“…This led Boulton et al to investigate CE3F4R’s non-specific interactions with EPAC1 using diverse techniques with varying degrees of resolution [47]. The formation of CE3F4R aggregates in vitro was studied using two commonly-used techniques, namely dynamic light scattering (DLS) and transmission electron microscopy (TEM) [34,35,42,47]. With both techniques, sub-micrometer aggregates were observed with sizes between 60 to 900 nm [47].…”

“…The formation of CE3F4R aggregates in vitro was studied using two commonly-used techniques, namely dynamic light scattering (DLS) and transmission electron microscopy (TEM) [34,35,42,47]. With both techniques, sub-micrometer aggregates were observed with sizes between 60 to 900 nm [47]. According to TEM, however, unlike most aggregation-prone inhibitors, CE3F4R aggregates display a more varied, irregular morphology [34,35].…”

“…The objective of this review is to summarize both the specific interactions underlying EPAC inhibition by two promising EPAC-specific inhibitors—CE3F4R [45] and ESI-09 [46]—as well as the non-specific interactions that result from their colloidal aggregate formation and subsequent aggregation-based EPAC inhibition in aqueous systems [47]. In this manner, we hope to provide key biophysical insight to the EPAC community in evaluating the inhibitory potential of current commercially available aggregation-prone EPAC inhibitors, in optimizing existing lead compounds, and in informing the future development of EPAC-specific inhibitors.…”