Mechanisms of Specific versus Nonspecific Interactions of Aggregation-Prone Inhibitors and Attenuators
- Stephen BoultonStephen BoultonDepartment of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4L8, CanadaMore by Stephen Boulton,
- Rajeevan SelvaratnamRajeevan SelvaratnamDepartment of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4L8, CanadaDepartment of Laboratory Medicine, University Health Network, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 2C4, CanadaMore by Rajeevan Selvaratnam,
- Rashik AhmedRashik AhmedDepartment of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4L8, CanadaMore by Rashik Ahmed,
- Katherine VanKatherine VanDepartment of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4L8, CanadaMore by Katherine Van,
- Xiaodong ChengXiaodong ChengDepartment of Integrative Biology and Pharmacology and Texas Therapeutics Institute, McGovern Medical School, University of Texas Health Science Center, Houston, Texas 77030, United StatesMore by Xiaodong Cheng, and
- Giuseppe Melacini*Giuseppe Melacini*E-mail: [email protected]Department of Biochemistry and Biomedical Sciences, Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario L8S 4L8, CanadaMore by Giuseppe Melacini
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 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.
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