Synthetic Protein Mimetic Modulators of Tau Aggregation

Abstract

Alzheimer's disease is a major neurodegenerative disorder characterized by progressive cognitive decline and extensive neuropathological changes. A central pathological hallmark of Alzheimer's disease and related Tauopathies is the abnormal aggregation of the microtubule-associated protein Tau. However, Tau is an intrinsically disordered protein that lacks a stable three-dimensional conformation, making it a challenging therapeutic target for conventional drug discovery approaches. The objective of this study was to identify synthetic oligopyridyl dimeric protein mimetics capable of modulating the aggregation of the K18 Tau fragment. To accomplish this objective, a systematic biochemical screening workflow was developed to evaluate a structurally diverse library of candidate compounds. The experimental workflow began with high-throughput screening of a library of 330 oligopyridyl dimeric protein mimetics using the ClearTau aggregation platform in combination with a Thioflavin T (ThT) fluorescence assay. Nineteen compounds exhibiting reduced endpoint ThT fluorescence relative to untreated controls were selected for secondary validation under standardized assay conditions. The three highest-performing compounds identified during secondary screening were subsequently subjected to kinetic aggregation analysis to evaluate their effects on the temporal progression of K18 Tau aggregation. Of the 330 compounds evaluated, nineteen advanced to secondary screening, from which three lead compounds were identified based on their reproducible aggregation profiles. Among these, Compound 2.14 consistently demonstrated the greatest reduction in ThT fluorescence and exhibited the most pronounced alteration of aggregation kinetics throughout the 120-hour assay. These findings indicate that structural variation within the oligopyridyl dimeric scaffold influences aggregation behavior under the experimental conditions employed in this study. The screening workflow established in this study successfully identified oligopyridyl dimeric protein mimetics capable of modulating K18 Tau aggregation and provides a reproducible platform for the systematic evaluation of large synthetic libraries. More broadly, this work demonstrates that combining rational protein mimetic design with sequential biochemical screening represents a practical strategy for discovering modulators of intrinsically disordered proteins and establishes a foundation for future mechanistic, structure-activity relationship, and biological investigations targeting Tau aggregation.