The most widely used inhibitor for study of cellular and in vivo functions of CaMKII has been KN-93 (Sumi et al., 1991; Figure 2A). It is one of a remarkable number of tool inhibitors developed by Hidaka and his colleagues for PKA, protein kinase C (PKC), CaMKII, and MLCK, many of which became commercially available and widely used. However, these were not on the path to therapeutic use and are therefore not optimized for potency, selectivity, or pharmacokinetics. KN-93 supplanted KN-62 which shares structural elements and mechanism of action (Tokumitsu et al., 1990). Inhibition by both is competitive with Ca²⁺/CaM and not competitive with ATP. KN-62 binds to the holoenzyme and interferes with the ability of Ca²⁺/CaM to activate it, but does not directly bind to CaM, i.e., it is not a CaM antagonist at effective inhibitory concentrations. It is worth noting, however, that a “predecessor” of KN-93 with a very similar structure, HMN-709, is a CaM antagonist (Yokokura et al., 1996; Figure 2A). Neither KN-62 nor KN-93 inhibits the activity of autophosphorylated CaMKII, consistent with a block of activation but not of catalysis (Tokumitsu et al., 1990; Sumi et al., 1991). Such inhibition can be classified as “ATP non-competitive” or “allosteric” as binding likely occurs outside the active site. Based on an ischemic stroke model, it has been suggested previously that blocking catalytic activity is the more effective approach because autonomous activity is resistant to allosteric inhibition by KN-93 (Vest et al., 2010). The observed differing efficacies, however, may have been due to differences in inhibitor concentrations at the site of action.

KN-93 (and KN-62) likely blocks the ability of Ca²⁺/CaM to wrap around the CaM-binding segment and free it from the catalytic domain. The displacement of the autoinhibitory regulatory segment can be monitored by appropriate FRET pairs and indeed KN-93 blocks the change in FRET signal induced by Ca²⁺/CaM and the change promoted by either autophosphorylation, oxidation, or GlcNAcylation (Erickson et al., 2011, 2013). KN-93 blocks Ca²⁺/CaM from displacing and “presenting” Thr287 to an active neighboring subunit for phosphorylation (Rich and Schulman, 1998). KN-93 similarly blocks presentation of Ser280 for GlcNAcylation (Erickson et al., 2013). Once activated and autophosphorylated, however, KN-93 does not inhibit the kinase, nor is it likely to inhibit kinase made autonomously active by oxidation or GlcNAcylation.

The initial characterization of KN-93 and KN-62 showed them to be selective for CaMKII relative to PKA, PKC and MLCK (Tokumitsu et al., 1990; Sumi et al., 1991), but they were later shown to inhibit CaMKI and CaMKIV equally well (Mochizuki et al., 1993; Enslen et al., 1994). KN-93 is not very potent, inhibiting CaMKII with an IC50 ~1–4 μM depending on level of CaM and other assay conditions (Sumi et al., 1991; Anderson et al., 1998; Rezazadeh et al., 2006). A recent screen against 234 protein kinases shows that KN-93 is indeed very selective, but its targets now include Fyn, Haspin, Hck, Lck, MLCK, Tec, and TrkA (Gao et al., 2013).

Tool inhibitors are rarely optimized for potency or off-target effects and indeed KN-62 and KN-93, while inhibiting only a few protein kinases, do inhibit many of the ion channels that have been tested. KN-62 and KN-93 block modulation of the L-type Ca²⁺ channel by CaMKII but also have direct effects on the channel (Li et al., 1992; Anderson et al., 1998) so it is important to use their kinase-inactive controls (KN-04 and KN-92). KN-62 and KN-93 block macroscopic voltage-dependent K⁺ current (K_v) in smooth muscle cells at concentrations (0.3–3 μM) used to inhibit CaMKII (Ledoux et al., 1999). KN-92 similarly blocks the channel and is therefore useful in excluding K⁺ channel effects. A detailed analysis demonstrated that KN-93, but not KN-92, blocked members of the Kv1, Kv2, Kv3, Kv4, and Kv7 (hERG) voltage-gated potassium channel families at concentrations that also block CaMKII (Rezazadeh et al., 2006). To be broadly useful as a control, it would be ideal if KN-93 and KN-92 had the same pharmacology other than for CaMKII, but this is unfortunately not the case. For example, KN-92 is not as potent in direct inhibition of L-type Ca²⁺ channels as is KN-93 (Anderson et al., 1998) or as potent in direct inhibition of IP₃-induced Ca²⁺ release via IP₃R-1 (Smyth et al., 2002) and thus not every effect blocked by KN-93 and not by KN-92 can be ascribed to CaMKII. Despite these caveats, these inhibitors have been extremely useful as initial evidence for CaMKII action, as illustrated by several examples in Table 1.

It is a bit surprising, given the channel blocking effects of KN-93, that it has not been as problematic to use it for in situ inhibition of CaMKII, when compared to peptide inhibitors below. The interplay between its effect on K⁺ channels and L-type Ca²⁺ channels and inhibition of CaMKII are not well understood and complicate arrhythmia studies. Clearly, modification of K currents, such as in genetic mutations, can lead to arrhythmia, whereas ablation or peptide inhibition of CaMKII are anti-arrhythmic. KN-93 may therefore be anti-arrhythmic despite its K⁺ channel blockade because concurrent inhibition of CaMKII serves as an arrhythmia shield that blocks the pro-arrhythmic consequence of the K⁺ channel inhibition. Alternatively, a significant component of the anti-arrhythmic effect of KN-93 could be to reduce CaMKII activation by inhibiting the L-type Ca²⁺ channel and lowering free Ca²⁺ levels. Ultimately, inhibition of CaMKII with small molecule allosteric or ATP competitive inhibitors can be achieved without a significant K⁺ channel component, something that is harder to achieve with channel blockers used as anti-arrhythmic agents.

Identification of the autoinhibitory regulatory segment of CaMKIIα led to development of long inhibitory peptides lacking the CaM binding sequence (residues 273–302) that could be injected into cells, e.g., implicating CaMKII in functions such as long-term potentiation (Payne et al., 1988; Malinow et al., 1989). The N-terminal end of this peptide contains the autophosphorylation site that was the basis for peptides substrates such as autocamtide-2 and autocamtide-3 (Hanson et al., 1989) and substitution of the phosphorylatable Thr to an Ala generated the peptide inhibitors AIP (Ishida et al., 1995) and AC3-I (Braun and Schulman, 1995). They inhibit CaMKII with >100-fold selectivity relative to PKC, PKA and CaMKIV, although their selectivity has not been broadly profiled, e.g., green fluorescent protein (GFP)-AC3-I was found to inhibit cellular actions of protein kinase D1 (PKD1) as well as those of CaMKII (Backs et al., 2009). As exemplified in Table 1, peptide inhibitors with internalization sequences for cellular studies or the transgenic expression of the peptides in mice have been critical first steps in delineating cardiovascular functions of CaMKII. Transgenic expression of inhibitor/control pairs can also be used to delineate molecular pathways of gene expression (Singh et al., 2009) and phosphorylation initiated by CaMKII activation (Scholten et al., 2013).

Some caution is warranted in the use of peptide (or small molecule) inhibitors that are often optimistically described as “highly specific inhibitors” when experience or data should suggest otherwise. Useful first generation tool inhibitors are typically developed by academic labs with limited resources, so selectivity is based on a handful of kinases available to the lab rather than on the 50–300 kinases that should be tested. As a minimum, the off-target effects of AC3-I should be checked by use of a control peptide (AC3-C; Patel et al., 1999; Wu et al., 2009). Altered selectivity can also occur when peptides are fused to GFP in order to increase expression and metabolic stability, or modified by lipids or internalization sequences for cell permeation. For example, addition of Ant (Antennapedia) to another peptide inhibitor generated a direct CaM antagonistic sequence (Buard et al., 2010) and myristoylated AIP and AC3-C were shown to have some effects unrelated to CaMKII inhibition (Wu et al., 2009).

CaMKIIN or CaM-KIIN designates small endogenous proteins that inhibit CaMKII with high affinity that can be applied pharmacologically or genetically. CaMKIIN was discovered by use of a yeast two-hybrid screen whereby the catalytic domain of CaMKIIβ served as bait to clone interacting proteins from a rat neuronal library (Chang et al., 1998, 2001). Two small proteins sharing high homology were identified and termed CaM-KIINβ (79 amino acids) and CaM-KIINα(78 amino acids) to designate them as inhibitors, and often spelled CaMKIIN. The α and β in their names are unrelated to the CaMKII isoform, as either of these inhibits all CaMKII isoforms with IC50 of 50 nM (Chang et al., 2001). The protein has not been detected in heart although a related mRNA was detected (Zhang et al., 2001).

Identification of the core inhibitory domain of CaMKIIN led to generation of a 28 amino acid peptide inhibitor termed CaMKIINtide (Chang et al., 1998) that was subsequently shortened and modified to improve potency (Vest et al., 2007; Coultrap and Bayer, 2011; Gomez-Monterrey et al., 2013). CaMKIIN and CaMKIINtide only bind to the activated conformation of CaMKII, suggesting that they dock to the kinase surface exposed and shaped only after displacement of the autoinhibitory domain during activation. They should therefore inhibit CaMKII activated by bound Ca²⁺/CaM or autonomously active due to autophosphorylation, methionine oxidation, or GlcNAcylation. A 21-residue segment of CaMKIINtide was co-crystallized with CaMKII and shown to dock at the B/C sites using hydrophobic and basic residues at its N-terminal region to support potency and specificity and to extend to the A site where it precludes substrate from binding in a manner similar to protein kinase inhibitor (PKI) binding to PKA (Chao et al., 2010; Figure 1).

CaMIINtide has been modified to increase potency (Coultrap and Bayer, 2011; Gomez-Monterrey et al., 2013). In one series of optimizations, a shorter sequence of 21 amino acids (CN21a) was found to retain the potency of CaMKIINtide (Vest et al., 2007). A 19 amino acid sequence was then subjected to Ala scanning substitutions to identify critical residues and subsequent modifications produced a more potent and selective CaMKII inhibitor termed CN19o (Coultrap and Bayer, 2011). CN19o inhibited CaMKIIα with IC50 < 0.4 nM and improved selectivity for tested kinases, showing minimal or no inhibition at 5 μM against CaMKI, CaMKIV, DAPK1, AMPK, PKC, and PKA. A similar study generated a smaller optimized 17 amino acid peptide, CN17β, with IC50 of 30 nM and little inhibition of CaMKI or CaMKIV (Gomez-Monterrey et al., 2013).

CaMKIIN and CaMKIINtides are excellent experimental tools being adopted by the field but their use can result in additional effects by blocking interaction of CaMKII with some anchoring proteins and substrates that share the B/C docking sites (Figure 1). Translocation and docking to anchoring proteins aids in fidelity of signal transduction that would be disrupted by CaMKIIN and may generate secondary effects because anchoring proteins often cluster several signaling proteins that might be affected (Colbran, 2004; Schulman, 2004; Tsui et al., 2005). Mutations at the B/C sites block both binding of CN21a and CaMKII translocation/docking to the glutamate NR2B receptor (Leonard et al., 1999; Strack et al., 2000; Bayer and Schulman, 2001; Bayer et al., 2006; Vest et al., 2007) suggesting overlapping binding sites. Application of TatCN21 on neurons inhibits the kinase but also reduces the level of kinase at synaptic sites (Sanhueza et al., 2011), decreases clustering in dendrites, and produces aggregates with polyribosomes (Tao-Cheng et al., 2013). CaMKIINtides also block interaction with densin (Jiao et al., 2011) and Cav2.1 calcium channels (Magupalli et al., 2013) and possibly with β_IV-spectrin and other proteins (Hund et al., 2010). Finally, the biological function of CaMKIIN is not fully understood and it may directly affect cellular pathways unrelated to CaMKII inhibition or interference with its translocation.

The cardiovascular field has appropriately advanced from the AC3-I/AIP- to CaMKIIN-based inhibitors to delineate in situ and in vivo functions (Table 1). The inhibitor can be directly introduced in a regionally selective manner via locally applied adenoviral constructs, as a transgene targeted to selective tissues, and even directed to distinct intracellular sites with appropriate targeting sequences.

The inhibitory agents and approaches described above have been essential in identifying key roles of CaMKII in health and disease and make a compelling case for targeting CaMKII for several cardiovascular indications, so as one of us asked previously, “where are the drugs?” (Anderson et al., 2006). While it is possible that a CaMKIIN-based inhibitor could be developed as a therapeutic, e.g., for local expression at the sinoatrial node (SAN), new small molecule inhibitors will be needed for treating cardiovascular disease, and these will, in turn, provide better tools for advancing CaMKII research. The period covered since the review above has seen a large increase in the number of protein kinases targeted in oncology, with over 100 hundred in clinical development and many in clinical practice (Cohen and Alessi, 2013). The cardiovascular field, largely because of the greater safety requirements but also because of decades-long investments in ion channel blockers, has been slower in turning to this target class. The message emerging from data published by the CaMKII research field has been heard and now there are several programs targeting CaMKII for cardiovascular indications.

One of the early programs was initiated at Scios, expanded following its acquisition by Johnson and Johnson, and discontinued along with more advanced programs for strategic reasons when Scios was closed. The program did provide some potent ATP competitive inhibitors, along with structure – activity relationships that enables an understanding of how to inhibit CaMKII (Lu et al., 2008; Mavunkel et al., 2008). One of the lead compounds, a pyrimidine (Scios 15b) with IC50 of 9 nM in vitro and 320 nM in situ, is shown in Figure 2B. Bosutinib (and sunitinib) were developed as ATP competitive inhibitors of protein tyrosine kinases but have surprising potency at inhibiting CaMKII and a co-crystal of CaMKII with bosutinib has been published (Chao et al., 2011). Dainippon Sumitomo Pharma has had the most advanced CaMKII program and developed Rimacalib (SMP-114) for treatment of rheumatoid arthritis. It passed the Phase I safety trial, but appears not to have shown efficacy in a 24-week Phase II trial (Tagashira and Fukushima, 2008; Westra et al., 2010). The other compounds are all potent (2–60 nM) inhibitors generated during CaMKII inhibition programs at Sanofi (Beauverger et al., 2012) and Dainippon (Asano et al., 2010; Komiya et al., 2012). Allosteros Therapeutics is developing both ATP-competitive and allosteric or ATP non-competitive inhibitors for cardiovascular and other indications and a CaMKII program at Myogen (with Novartis) was part of an acquisition by Gilead with no publications of structures so far. We are aware of several other early stage CaMKII inhibitor programs and anticipate that both tool inhibitors for academic research as well as new chemical entities for treating cardiovascular disease will arise from several of these programs.

The biochemical properties of some the best characterized CaMKII inhibitors are summarized in Table 2.