Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells.

Huntington's disease (HD) is the most common inherited neurodegenerative disorder and one of the nine polyglutamine (polyQ) diseases. HD is characterized by the pathological aggregation of the misfolded huntingtin exon 1 protein (Httex1) with abnormally long polyQ expansion due to genetic mutation. While there is currently no effective treatment for HD, inhibition of aggregate formation represents a direct approach in mediating the toxicity associated with Httex1 misfolding. To exploit this therapeutic window, we engineered two fluorescence resonance energy transfer (FRET) based biosensors that monitor the aggregation of Httex1 with different expanded Q-lengths (Q39 and Q72) in living cells. These FRET biosensors, together with a high-precision fluorescence lifetime detection platform, enable high-throughput screening of small molecules that target Httex1 aggregation. We found six small molecules that decreased the FRET of the biosensors and reduced Httex1-Q72-induced neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced SPR and EPR techniques, we confirmed that the compounds directly bind to Httex1 fibrils and inhibit aggregate formation. This strategy in targeting the Httex1 aggregates can be applicable to other proteins involved in polyQ related diseases.

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies.

OBJECTIVE: Understanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. BACKGROUND: Tauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. NEW/UPDATED HYPOTHESIS: We hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. MAJOR CHALLENGES FOR THE HYPOTHESIS: Our approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. LINKAGE TO OTHER MAJOR THEORIES: Our biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease-associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.

Noncompetitive inhibitors of TNFR1 probe conformational activation states.

Tumor necrosis factor receptor 1 (TNFR1) is a central mediator of the inflammatory pathway and is associated with several autoimmune diseases such as rheumatoid arthritis. A revision to the canonical model of TNFR1 activation suggests that activation involves conformational rearrangements of preassembled receptor dimers. Here, we identified small-molecule allosteric inhibitors of TNFR1 activation and probed receptor dimerization and function. Specifically, we used a fluorescence lifetime-based high-throughput screen and biochemical, biophysical, and cellular assays to identify small molecules that noncompetitively inhibited the receptor without reducing ligand affinity or disrupting receptor dimerization. We also found that residues in the ligand-binding loop that are critical to the dynamic coupling between the extracellular and the transmembrane domains played a key gatekeeper role in the conformational dynamics associated with signal propagation. Last, using a simple structure-activity relationship analysis, we demonstrated that these newly found molecules could be further optimized for improved potency and specificity. Together, these data solidify and deepen the new model for TNFR1 activation.