Development of small-molecule P21-activated kinase (PAK1) activator: management of heart failure and cardiac hypertrophy

P21-activated kinase (PAK1), a serine/threonine protein kinase, has significant homeostatic functions in the myocardium. PAK1’s activation of protective signaling in cardiac pathways has been reported to modulate cardiac hypertrophy and associated ventricular arrhythmias. Clinical significance of PAK1 has been emphasized by the growing evidence on the protective role of PAK in the myocardium, which offers important implications for therapeutic targeting. Based on this evidence, we designed the PAK1-activating peptide (PAP) to establish whether disrupting the auto-inhibitory domain (AID) of PAK1 would stimulate PAK1’s activation through preventing the inhibitory mechanism. The results showed that PAP both considerably stimulated PAK1 activation and had an anti-hypertrophic effect in cardiomyocytes, proving to be a viable method of regulating PAK1 activation with important therapeutic benefits.
For clinical translation, the PAK1-activating peptide was successfully redesigned into a small- molecule PAK1 activator that can directly stimulate PAK1 activity. Computational in silico methods were used to perform virtual ligand screening in the AID of PAK1. Compounds with high negative energy scores were synthesized, and their efficacy in PAK1 activation was tested using in vitro kinase assays. To acquire an in-depth understanding of the active site, we used the structure-based drug discovery for drug optimization. Such detailed understanding of the active site would be necessary to comprehensively understand the protein-ligand interactions and to design novel ligands with improved selectivity, efficacy, and physiochemical profiles. The lead compounds showed a 3-fold stimulatory effect on PAK1 with significantly enhanced physiochemical properties.
The interaction between protein-ligands was also biophysically examined using photoaffinity labelling coupled with mass spectrometry (PAL-MS). Based on the results, we identified that the compounds were binding at the interface between the kinase inhibitory segment of AID and the kinase domain, i.e. the domain where the interaction is crucial for kinase activity regulation. Such mechanisms of kinase activation have not been demonstrated in previous research. Furthermore, along with discovering small molecules with significant stimulatory effects on the kinase activity, the present study is the first to demonstrate that enzymatic regulation can be achieved through hindering the inhibitory mechanism of enzymes. In future research, these mechanisms of action can be applied to other drug discoveries, which can then be used as a footprint to design other kinase activators. In addition, previous therapeutic targets regulating beneficiary signaling pathways can now be targeted for drug discovery and activated to confer clinical elevation in various pathological conditions.
In order to gain further insight into the physiological effect of PAK1 activation, selected lead compounds were tested in functional studies. First, the results of immunohistochemical staining in isolated cardiomyocytes of PAK1 activator revealed a significant increase in fluorescence, indicating an increased PAK1 activation (phospho-PAK1). Second, a rapid perfusion of JB compound on isolated ventricular myocytes was found to have an antagonizing effect on isoprenaline-induced hypertrophy and arrhythmia. Third, the results of examining JB79’s cardio-protective effect against programmed electrical stimulation (PES)-induced arrhythmia in an ex vivo whole heart model, with the specific focus on PVC and VF, showed that a sufficient activation of PAK1 signaling at the concentration of 10mg/kg considerably increased the voltage tolerance for PES-induced VF and PVC, thereby reducing the incidence of cardiac arrhythmia. These results were further supported by our findings on PAK1 activators’ effect on the catecholaminergic polymorphic ventricular tachycardia (CPVT). After 25-minute PAK1 activator treatment, the damaged heart from isoprenaline-induced fibrillation and arrhythmia exhibited a significant overall improvement in contractility. Finally, the results of our functional study on the TAC-induced cardiac hypertrophy model demonstrated that prolonged treatment of PAK1 activator also provides significant protection against pressured overload cardiac myopathy. Taken together, the results of the present study demonstrate that PAK1 activators are a potential novel therapeutic strategy for both prophylactic and therapeutic measures involving cardiac hypertrophy and arrhythmia.

Keywords: drug discovery
kinase activator
pharmacology