Abstract
Huntington’s disease is a fatal neurodegenerative disorder caused by a polyglutamine (poly-Q) expansion (≥35 repeats) of the huntingtin protein (httex1), leading to fibril accumulation in neuronal inclusion bodies. Recent studies have dissected httex1 aggregation using solution NMR spectroscopy. [1,2] By analyzing both non-pathogenic httex1Q7 and pathogenic httex1Q35, distinct stages of the aggregation process corresponding to different kinetic regimes were characterized. Pre-nucleation events corresponding to the formation of short-lived oligomeric species (dimers, tetramers) on the microsecond timescale were studied in the httex1Q7 construct via concentration-dependent chemical shift (δex) changes, 15N R1ρ measurements and 15N CPMG relaxation dispersion experiments. In contrast, nucleation and fibril formation in httex1Q35 were monitored using time-resolved SOFASTHMQC allowing observation of time-dependent chemical shift changes and variations in NMR crosspeak volume/intensity. These observables were globally fitted to a kinetic model incorporating tetramerization, conversion to nuclei, elongation, and secondary nucleation. Building on this framework, we extended the model including the action of small-molecule inhibitors at different stages of either pre-nucleation, nucleation or aggregation. The extended model considers smallmolecule interactions with monomeric httex1 species (m) and nuclei (P), whose lifetimes are compatible with the timescale of small-molecule binding (milliseconds to seconds). While direct binding of inhibitors to short-lived oligomers is excluded from the time-dependent kinetic model due to timescale incompatibility, relaxation-based NMR experiments with httex1Q7 can still reveal inhibitor-induced modulation of early oligomerization equilibria and exchange kinetics. This theoretical framework advances classical amyloid aggregation models by incorporating reversible binding equilibria of small-molecule inhibitors across multiple aggregation stages. In this context, flavan-3-ols represent a promising molecular class for probing the mechanisms that govern early nucleation events.3 Ultimately, our goal is to provide a conceptual and methodological platform to guide the rational design of inhibitors targeting nucleating species in amyloid-related disorders