Research rationale: Despite significant progress in comprehending the genetic and metabolic underpinnings of cardiomyocyte dysfunction, there is still a pressing need for targeted treatments to address hypertrophy and progressive remodelling (e.g., fibrosis), seen in HCM [1]. PAK1, a regulator of ion channels and myofilaments in cardiomyocytes, has shown promise in curbing pathological hypertrophy [2]. Our hypothesis centres on the potential therapeutic benefits of pharmacologically activating PAK1 in managing hypertrophy and adverse remodelling in HCM.

Methods: Molecular docking and high-throughput kinase assays using RapidFire-mass spectrometry were employed for virtual and physical screening to develop PAK1 activators. The effect of these activators on cellular hypertrophy was assessed using hypertrophic neonatal rat cardiomyocytes (n=200-300) induced by isoprenaline (ISO, 40 μM). Subsequently, the small molecule PAK1 activator JB2020A was evaluated in a well-characterized transgenic mouse model expressing the hypertrophic cardiomyopathy-causing mutation Actc1^E99K, known for its rapid disease progression [3]. A six-week oral treatment regime (10 mg/kg/day of JB2020A, Vehicle, and WT, N=5 in each group) was initiated at 4 weeks of age. The therapeutic efficacy of these pharmacological interventions and the endpoints were assessed using echocardiography, histology including Sirius red staining and H&E staining, and biomarker assessment using western blot. Data were analysed as mean ± SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns, not significantly different according to one-way ANOVA with Tukey’s posthoc test.

Results: We have identified potent and effective small molecule PAK1 activators that significantly increased PAK1 activity by 3 to 5-fold, with an EC₅₀ range between 0.5 and 2.5 μΜ. Through the evaluation of PAK1 activators on cellular hypertrophy, JB2020A not only prevented ISO-induced hypertrophy but also reversed pre-existing cellular hypertrophy induced by ISO 24 hours earlier. Moreover, after 6 weeks of JB2020A treatment, we observed a significant reduction in cardiomyocyte hypertrophy and cardiac fibrosis, accompanied by preserved cardiac function in Actc1^E99K HCM mice compared to vehicle treatment. These cardio-protective effects were associated with increases in phosphorylated PAK1 observed after activator treatment. Furthermore, JB2020A treatment resulted in a reduction in pro-apoptotic CHOP expression and an upregulation of protective endoplasmic reticulum (ER) response molecules (such as ATF4 and Xbp1), suggesting an amelioration of ER stress in HCM.

Conclusions: Collectively, these findings underscore the therapeutic potential of small molecule PAK1 activators as a novel approach for addressing hypertrophy and progressive remodelling in HCM.