Protein Aggregation Small Molecule Inhibitor Discovery and Mechanisms

Epidemiological and clinical studies showed significant association between type 2 diabetes (T2D) and the risk for Alzheimer's disease. Pancreatic hormone amylin is a highly amyloidogenic protein and amylin amyloid deposition in the pancreas are hallmark features of T2D. Recent clinical studies showed that amylin plaques were deposited in the brain of diabetic patients, but not in healthy controls. We performed cell‐based studies, demonstrating that amylin amyloid is highly toxic to pancreatic β‐cells INS1 and neuronal cells SH‐SY5Y and Neuro2A. From a collection of natural compounds used in alternative medicine, we identified multiple potent inhibitors, including rosmarinic acid (RA) and baicalein (200 nM and 1 μM respectively in apparent IC 50 ). These lead compounds disaggregate amylin fibrils from transmission electron microscopic observations and significantly reduce amylin‐induced cytotoxicity. Dissecting the functional groups of these compounds, we demonstrated, for the first time to our knowledge, that the vicinal hydroxyl groups of the catechol groups played key functional roles in amyloid inhibition in more than a dozen catechol‐containing compounds, including physiological neurotransmitters epinephrine, norepinephrine, and dopamine. Compounds with multiple catechol groups, such as rosmarinic acid, exhibited additive/synergistic effects. We provided further mass spectrometric evidence that incubating several of these catechol‐containing inhibitors with amylin leads to covalent adducts consistent with Schiff base conjugation as a mechanism for blocking toxic amyloid formation. The inhibition effects by these compounds were also demonstrated in molecular simulation analyses, providing additional non‐covalent inhibition mechanisms. To expand protein aggregation inhibitor discovery, we applied the concept of drug repurposing. We developed a 384‐well plate based screening platform and screened a NIH Clinical Collection small molecule library that are currently in phased trials. We were able to rapidly identify 10–30 each of “hits” against four different amyloidogenic proteins: amylin, Aβ, tau, and α‐synuclein. Excitingly, we were able to identify compounds that can inhibit all four amyloids but also “protein‐specific” inhibitors. Mechanisms for “general” protein aggregation inhibitors and for “protein‐specific” aggregation inhibitors will be elucidated. Support or Funding Information This work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, Diabetes Action Research and Education Foundation (DAREF), and Virginia Tech Center for Drug Discovery (VTCDD).