Abstract Huntington’s disease is caused by polyglutamine expansions in the huntingtin protein. These polyQ expansions make the huntingtin protein, and its naturally occurring exon1 fragment (Httex1), more aggregation prone. Deposition of fibrillar Httex1 aggregates in the brain is a hallmark of the disease in patients and animal models. We have shown that Httex1 aggregation is a step-wise process wherein Httex1 gives rise to different misfolded species prior to formation of fibrils. Early species in the misfolding pathway are of particular interest as they can cause the formation of seeds, which cause further misfolding of monomeric Httex1. This process not only enhances toxicity in a given cell, but it can also cause spreading of misfolding throughout the brain. In addition, earlier misfolding intermediates could also directly contribute to disease, as their toxicity has been observed in cell culture experiments. Despite their importance, early misfolding intermediates cannot easily be detected in biological tissues and it has not been possible to interfere with their seeding ability or toxicity. In this project, we aim to address these fundamental problems by developing genetically encoded ligands (peptides) that bind early misfolding intermediates and by testing their potential biomarker or therapeutic utility. To accomplish these goals, we have assembled a team of three PIs with expertise in peptide ligand discovery (Roberts), huntingtin protein structure and function (Langen), and cell-based and animal-based disease models (Chen). The Langen lab has laid the biochemical foundation for the proposal by identifying and characterizing different forms of Httex1 aggregates. Working together, the Roberts lab has used directed evolution and mRNA display to generate Httex1 directed (HD) peptide ligands against protofibrils. The Langen and Chen lab have demonstrated that HD peptides inhibit Httex1 misfolding in vitro and in cultured cells. Importantly, HD peptides also protect from Httex1 toxicity in cultured cells. In Aim 1, we propose to extend this work by characterizing the interactions of HD peptides with protofibrils using biophysical methods. Specifically, we will determine the HD peptide’s affinity, specificity, molecular mechanism of interaction with protofibrils and we will evaluate their ability to inhibit misfolding. Moreover, we will use peptide multimerization and other optimizations to achieve ultra-high affinity binding. Aim 2 then uses these well characterized binders in animal and cell models to evaluate their utility as biomarkers and therapeutics in cell cultures and animal models. In aim 3, we will generate binders for the earliest misfolding intermediate, the a-helical oligomers, and test our prediction that binders to these species block the formation of seeds and protect from Httex1 misfolding and toxicity.