Lo, Chih Hung2021-10-132021-10-132019-08https://hdl.handle.net/11299/225017University of Minnesota Ph.D. dissertation. August 2019. Major: Biomedical Engineering. Advisors: Jonathan Sachs, David Thomas. 1 computer file (PDF); xvii, 319 pages.Protein–protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Here, we investigated two classes of protein, including a transmembrane protein (tumor necrosis factor receptor 1 (TNFR1)) and intrinsically disordered proteins (tau and huntingtin (HTT)), which are implicated in autoimmune diseases and neurodegenerative diseases respectively. Receptor-specific inhibition of TNFR1 signaling is a highly sought after strategy for treatment of inflammatory diseases such as rheumatoid arthritis. In this study, we investigated the structure-function relationship of TNFR1 by engineering a TNFR1 fluorescence resonance energy transfer (FRET) biosensor to monitor the structural and conformational changes of the receptor. We have also shown using small-molecule tool compounds, that the disruption of receptor-receptor interactions (competitive inhibition) and perturbation of the receptor conformational dynamics (allosteric inhibition) are both feasible approaches to inhibit TNFR1 signaling. We have also made a major discovery showing that long-range structural couplings, between TNFR1 membrane distal and proximal domains, mediated through the ligand-binding loop, determine the conformational states of the receptor that act as a molecular switch in receptor function. In addition to deepening the understanding of a novel mechanism in TNF receptor activation, we have optimized a lead compound through medicinal chemistry by improving its potency by more than sixty-fold to the nanomolar range, thereby advancing therapeutic developments in these clinically important targets. The heterogeneity of tau and HTT pathology is one of the major challenges that plagues current clinical trials, hence impeding the discovery of a cure for Alzheimer’s disease (AD) and Huntington’s disease (HD). We have engineered novel FRET biosensors of these proteins to target the ensemble of heterogeneous protein oligomers or aggregates in cells. The biosensors are not only capable of monitoring oligomer conformations, but can also be used as a high-throughput screening platform. Using these technologies, we have discovered small-molecule inhibitors of tau oligomerization or HTT aggregation that rescue cell cytotoxicity with nanomolar potency.enFREToligomerizationprotein-protein interactionsSARtauTNFR1Thesis or Dissertation