BS8 RAF ‘paradox breaker’ PLX8394 activates ERK1/2 VIA craf, with no effect on angii-induced cardiac remodelling in mice

IntroductionThe ERK1/2 cascade, a key pathway involved in cardiac remodelling, is regulated by RAF kinases. Small molecule inhibitors of RAF have been developed due to activating oncogenic mutations, however paradoxical activity has been seen in early generations of inhibitors. Therefore, ‘paradox breaker’ inhibitors (e.g. PLX8394) have been developed and are undergoing clinical trials. Here, we investigated the effects of PLX8394 on vascular ERK1/2 signalling in vitro and on hypertensive cardiac remodelling in vivo.MethodsMurine endothelial cells (ECs) or human cardiac fibroblasts (HCFs) were incubated with PLX8394 and effects on RAF-ERK1/2 pathway activity determined by western blotting, with effects on cell migration and proliferation assessed via wound healing and BrdU assays. For in vivo characterisation, PLX8394 (5mg/kg/d) was infused with/without angiotensin-II (AngII; 0.8mg/kg/d) for 7 days by osmotic minipumps in male wildtype C57Bl/6J mice (n=8–11/group). Cardiac function/dimensions were assessed using echocardiography; effects on cardiac morphology were assessed by histological staining. mRNA expression was assessed by qPCR. Statistical tests used 1-way ANOVA with Holm-Sidak’s post-test.ResultsPLX8394 (5 min; 1uM) activated ERK1/2 (n=3; p=0.018) pathway via CRAF (n=3; p=0.047) in ECs with no change seen in BRAF activity. This was accompanied by increased BrdU incorporation (n=6; p=0.0002; p=0.0009) but significantly inhibited migration (n=6; p