CaMKII: Therapeutic target in vascular diseases warranted for Clinical and Translational Discovery?

There has been a rich history of studies on the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) in the brain, heart, and other tissues. CaMKII consists of four genes encoded by CaMKIIα, CaMKIIβ, CaMKIIδ and CaMKIIγ. CaMKIIα and CaMKIIβ are enriched in the neural tissues, while CaMKIIδ and CaMKIIγ are ubiquitously expressed. CaMKII isoforms consist of catalytic, regulatory and association domains. To date, five amino acid sites in the regulatory domain have been identified to regulate the kinase activity, including the autophosphorylation sites Thr287 and Thr306/307, oxidation site Met281/282, O-GlcNAcylation site Ser280 and S-nitrosylation site Cys290 (Figure 1). Sustained and excessive activation of CaMKII has been linked to arrythmia, hypertrophy, heart failure, hypertension, ischemic heart diseases and diabetes. Pharmacological inhibitors and genetic ablation or suppression of CaMKII activity have elevated CaMKII as a key target in heart failure, arrythmia and other forms of heart disease. However, whether CaMKII is a druggable target in vascular diseases is uncertain. As an important part of the cardiovascular system, blood vessels are the major targets of vasoconstriction or vasodilation for the treatment of cardiocerebrovascular diseases, particularly hypertension and atherosclerosis. The contraction or relaxation of blood vessels are governed by the outer layer vascular smooth muscle cells (VSMCs) and the inner layer endothelial cells (ECs). Recently, a growing body of evidence suggests a crucial involvement of calmodulin-dependent protein kinase II (CaMKII) in the regulation of vascular contractility and vascular remodeling. CaMKIIδ-6 has been identified as the predominant isoform in ECs. Differentiated VSMCs primarily express CaMKIIγ which regulates contractile activity and calcium haemostasis, whereas the expression level of CaMKIIδ is higher in proliferative VSMCs, which controls gene transcription, proliferation, and migration in vitro, and regulates vascular hypertrophic and hyperplastic remodeling in vivo. Although there are controversial conclusions of CaMKII activation and vasorelaxation in mesenteric arteries,1, 2 studies in large conductance blood vessels show that CaMKII activity is increased in response to vasoconstrictions. CaMKII regulates the activation of myosin light chain kinase (MLCK) by phosphorylation and controls the interaction between α-SMA and myosin to affect the VSMC elasticity, while reducing calcium level inhibits the activity of MLCK to promote vascular smooth muscle relaxation.3 Several studies showed that CaMKII inhibition decreased angiotensin II-induced blood pressure, intravascular injuries, and altered the expression of muscle contractile genes in VSMC.4, 5 The antisense oligonucleotides suppressing CaMKIIγ isoform inhibits ferret aorta contraction ex vivo. The activation of CaMKII has been found to promote the phosphorylation of eNOS and increase the release of NO in ECs, thus regulates vasoconstriction in VSMCs. Despite endothelial CaMKII inhibition having no significant effect on NO-dependent vasodilation or blood pressure in vivo under baseline conditions,6 CaMKII plays an important role in transient bradykinin-driven eNOS activation in vitro. Therefore, CaMKII may provide a promising nodal point for intervention that may allow simultaneous prevention of cardiac stress and vascular diseases, however to date, there is no drug targeting CaMKII for clinical therapy of vascular diseases. There are mainly two types of well-established CaMKII inhibitors, including the peptide substrates (AIP, AC3I, CaMKIIN, CAMKIINtide, CN21) and small molecular compounds (KN-93, KN-62). Identification of the autoinhibitory regulatory domain of CaMKII led to development of the substrate competitive inhibitors of CaMKII, autocamitide-2 related inhibitory peptide (AIP) and AC3I (Figure 1). CaMKIIN is an endogenous protein that inhibits CaMKII with high affinity. Identification of the core inhibitory domain of CaMKIIN generated a 28 amino acid peptide inhibitor termed CaMKIINtide. CN21 is derived from CaMKIIN amino acid 43–63 showing full potency of CaMKII inhibition. The peptide substrate inhibitors are mainly used in the academic field with unknown off-target effects. Importantly, they may block the interaction of CaMKII with some anchoring proteins, and the peptide nature limits their membrane permeability and bioavailability. Compared with other type of inhibitors, small-molecule compounds have many merits, such as avoidance of immune responses, bioavailability, and cost effectiveness. KN-62 and KN-93 are competitive with Ca2+/CaM but not competitive with ATP. Thus, they are allosteric inhibitors for the initial step of CaMKII activation, which are ineffective against activated CaMKII. Additionally, KN-62 and KN-93 have many off-targets, for example, CaMKI, CaMKIV, the potassium channels and the L-type calcium channels, thus limiting its application. Recently, four ATP-competitive CaMKII inhibitors, GS-680, AS105, RA608 and RA306, have been identified with the first three to inhibit arrhythmogenesis and RA306 to reverse cardiac dysfunction in dilated cardiomyopathy.7-10 Using a high-throughput screening system for the kinase activity of CaMKII-δ9, we recently identified Hesperadin as a specific ATP-competitive inhibitor of CaMKII-δ with dual functions of cardioprotective and anti-tumour effects. Hesperadin directly binds to CaMKII-δ to inhibit its autonomous activity and avoid the off-target effects in CaMKII-α in the brain or the potassium channels in the hearts.11 However, their effects and application in vasodilation remain to be elucidated. Recently, Lu et al.12 reported a brand new druggable site on CaMKII that can be targeted by a pre-existing traditional Chinese medicine with demonstrated benefits in alleviating cardiovascular symptoms among hospitalised atherosclerotic patients. Through iTRAQ1-based differential proteomic analysis and subsequent verification, CaMKII was identified as a potential target. Furthermore, the group identified that ligustilide, one of the active ingredients in this Chinese medicine, and its metabolite (E-lig) are irreversible inhibitors for CaMKII at cysteine 116, which is located within the catalytic domain (Figure 1). This inhibitory action induced potent vasodilation in isolated rat thoracic aorta and improved cardiac output in atherosclerotic mice. All of these findings suggest that inhibiting the catalytic domain of CaMKII can be employed as a feasible therapeutic strategy in relieving vascular dysfunction. While it is exciting to identify a new drug entity, there are two major limitations regarding the potential application of ligustilide in treating vascular disease. First, the effective doses for both ligustilide and E-lig do not appear to be optimal, as both are in micromolar range. Furthermore, at the comparable dosing range, the effects of ligustilide and E-lig are not superior to that of KN-93. Thus, further lead optimization is warranted to increase drug potency. Second, a higher risk of side effects is expected with the mode of action since cysteine 116 is universal for all CaMKII isoforms. Together with a higher effective dosage, we expect to observe multiple levels of side effects in different organ systems. Therefore, the feasibility of targeting this amino acid as a therapeutic strategy should be carefully examined pre-clinically. In summary, identifying a new mode of CaMKII inhibition in this study is definitely an exciting advance in the field. It is foreseeable that further compound development will explore on this target and address the shortcomings in drug potency. Furthermore, this discovery also opens a new research avenue to examine which mode of inhibition suits the timing of CaMKII regulation in the disease context. Lastly, this study is an exemplary success in the attempt to improve our understanding of traditional Chinese medicine by identifying the active ingredients and the underlying molecular targets. We look forward to many more similar studies to bridge traditional Chinese medicine and modern medicine in the future.