Objective

To validate ROCK inhibition as a therapeutic strategy for Huntington’s disease (HD), we have developed a potent, selective and brain penetrant ROCK inhibitor suitable for in vivo proof-of-concept (PoC) studies in HD animal models. KiNativ™ technology was used to confirm dose- and time-dependent target engagement and kinase selectivity in mice brain tissue. Whilst KiNativ™ data confirms in vivo target engagement, we were unaware of how this translated to a relevant ROCK inhibition-mediated biological response. To address this, our efforts were targeted towards development of a pharmacodynamic (PD) ROCK biomarker assay to better inform dose regimen for a PoC study.

Using in vitro cell lines, we identified several ROCK biomarker candidates that represented downstream protein phosphorylation events. One of these candidates, phospho-MYPT1 (T850), translated to an in vivo context and therefore represented a proximal ROCK biomarker event. In order to capture this biomarker event, we showed that tissue fixation using microwave irradiation in combination with harsh lysis buffer conditions were essential to capture and extract soluble phospho-MYPT1 (T850). Based on these findings we developed and validated a Meso Scale Discovery (MSD) assay that can measure phosphorylated-(T850) and total-MYPT1 proteins from brain tissue.More importantly, in vivo dosing of our candidate compound to mice showed dose- and time-dependent inhibition of MYPT1 phosphorylation that closely correlated with the KiNativ™ target engagement data. Collectively, the PK-PD and KiNativ™ results will enable us to make a data-driven decision when designing the dose regimen for a PoC study.