E001 Photoaffinity probe-mediated target identification for HTT PET ligand

<h3>Background</h3> The PET (positron emission tomography) radioligand [11C]CHDI-180R was designed to target mutant huntingtin (mHTT) aggregates in Huntington9s disease (HD) brains. It has been validated in preclinical HD models and has demonstrated promising kinetic properties in a first-in-human study. <h3>Aim</h3> In this investigation, focus was to identify mHTT proteoforms that CHDI-180 binds to, which is crucial for understanding its pharmacology and developing second generation tracers. <h3>Methods</h3> Probes with photoreactive chloroalkane tag were designed to selectively isolate mHTT target(s). UV-induced covalent photo-cross-linkage is applied to secure the binding between probe and mHTT. Targeted mHTT proteoform(s) cross-linked to ligand is enriched through covalent capture onto beads. After capture, target-ligand complex is selectively released from beads for characterization by using mass spectrometry, mass photometry (MP), gel electrophoresis (AGERA) and negative stain electron microscopy (nsEM). MP and nsEM have been optimized for analysis of various mHTT proteoforms. <h3>Results</h3> We show optimization of key steps, including coupling of ligands to beads and linker cleavage. Our results indicate that all 6 PAL ligands can be successfully coupled and cleaved. The extent of coupling and cleavage varied based on applied conditions and ligand investigated. Results from a time course of recombinantly derived aggregated mHTT analyzed by nsEM and MP will be presented. <h3>Conclusions</h3> Our studies revealed that nsEM and MP are valuable tools to characterize mHTT aggregates. Optimized methods will be applied to characterize mHTT proteoforms to which CHDI-180 binds and to compare CHDI-180 and other HTT PET tracer target(s) in animals and human post-mortem brain samples.