A Phenotypic‐based Drug Discovery Approach to Inhibit the Misfolding of Hyperphosphorylated Tau Isoform 1N4R

Abstract

The vast majority of Alzheimer’s disease (AD) drug development efforts have failed because of significant gap in the understanding of the pathophysiological processes and insufficient effort in identifying, optimizing, and validating small molecules inhibitors of aggregation. AD involves the misfolding of amyloid‐beta which forms Aβ plaques and, subsequently, activates different types of microglial cells leading to a pro‐inflammatory state. Microglia when activated have been known to increase tau phosphorylation and drive the trans‐synaptic spread of aggregated hyperphosphorylated tau (p‐tau). Most importantly, the spatiotemporal distribution of neurofibrillary tangles predicts cognitive impairment. Instead of the unmodified tau that shows minimal effects on cell viability, our research focuses on the control of p‐tau that possesses disease‐relevant molecular features, including hyperphosphorylation and cytotoxicity. Our drug discovery project is centered on the isoform 1N4R due to its prevalence (1N) and long‐distance trans‐synaptic propagation (4R). We identified compounds that inhibit the aggregation and cytotoxicity of p‐tau, which represent a novel approach for AD drug discovery and development. Due to lack of binding pocket(s) and fast‐switching conformation of p‐tau, we opted for a phenotypic‐based drug screening approach to identify and optimize hit candidates. In our current project, the anti‐aggregation activity and cellular protection of newly synthesized small molecules were evaluated with biophysical methods (thioflavin S fluorescence assays, photo‐induced cross‐linking of unmodified proteins, dynamic light scattering) and survival assays with human neuroblastoma SH‐SY5Y cells. We prepared and tested 3 different series of small molecules and challenged these molecules with our first‐tier screening assay, i.e. thioflavin S fluorescence assays. Ureas and sulfonylureas series failed to inhibit the aggregation of p‐tau 1N4R. The best prototypical representative of the sulfonamide series, compound JF‐19‐73, effectively antagonized the cytotoxicity of p‐tau in vitro at low micromolar concentrations, suggesting the potential for neuroprotection in vivo. Other representatives of the sulfonamide series exhibited an intermediate effect to antagonize the aggregation and cytotoxicity of p‐tau isoform 1N4R. This project opens doors in targeting undruggable prone‐to‐aggregate proteins such as p‐tau and provides a molecular scaffold for further optimization and pre‐clinical studies focused on AD drug development.