A macrocyclic peptide platform for the discovery of functional tau epitopes

PROJECT ABSTRACT The accumulation of tau protein deposits is characteristic of several neurodegenerative diseases that manifest with symptoms of dementia, cognitive decline, and movement disorders. The conformational plasticity of tau allows it to play a role in many important cellular processes; however, its misfolding and self-assembly into pathological filaments results in both loss of normal function and gain of toxic function. Disease-associated filamentous tau exhibits cross-b architecture, wherein protein monomers adopt b-arch folds that stack in register. These filaments can seed the misfolding of physiological tau and propagate across neurons in a prion-like manner. Recent cryo-EM structural data demonstrate that the conformations of tau protomers within pathological protofilaments can vary by disease, even when they are comprised of the same isoform and sequence. This raises the intriguing possibility of a link between conformational fold, seeding capacity, and disease progression. Current models of tau propagation based on co-factor-induced aggregation fail to capture the structural diversity of pathological tau folds, thus limiting their relevance. Given the scarcity and variability of seed-competent patient-derived extracts, efforts to recapitulate pathological tau folds are urgently needed. The current project will address this need through the structure-based design of peptidomimetics that mimic the form and function of tauopathic filaments. At the core of our approach is the diversity-oriented synthesis of conformationally constrained peptide macrocycles that adopt stable b-arch folds. These molecules, termed "mini-taus", will serve as broadly useful chemical biology tools to probe tau seeding and to generate conformation-specific antibodies. Our overarching hypothesis is that peptide stapling will potentiate the structure and function of minimal tau epitopes and will enable the development of anti-tau therapies targeting pathological folds. In Aim 1, we will carry out the diversity-oriented synthesis of a macrocyclic b-arch peptide library based on the high-resolution structures of 4R tauopathic filaments. These macrocycles will be tested for their ability to seed endogenous inclusions of full-length tau in engineered cells and primary neurons in a macrocycle- and sequence-dependent manner. In Aim 2, we will confirm that seed-competent b-arch macrocycles self-assemble into pathological cross- b folds using a combination of CD, X-ray fiber diffraction, and high-resolution cryo-EM. In Aim 3, we will establish in vivo seeding by a lead mini-tau and generate a single-domain antibody with potent and selective immunoreactivity with patient-derived 4R tauopathic filaments. The potentiation of b-arch form and function through peptide stapling has the potential to afford unique insights into the misfolding and propagation of tau as well as other amyloids implicated in disease.