PKC Delta and Epsilon in Drug Targeting and ...

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Recent Patents on DNA & Gene Sequences 2009, 3, 96-101

PKC Delta and Epsilon in Drug Targeting and Therapeutics Tomo Yonezawa, Riho Kurata, Minoru Kimura and Hidetoshi Inoko Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Ishehara, Kanagawa 259-1193, Japan Received: January 15, 2009; Accepted: March 18, 2009; Revised: March 30, 2009

Abstract: Protein kinase C (PKC) belongs to the serine and threonine kinase family. At least ten PKC isoforms have been identified and subdivided into three groups: classical (alpha, beta I, beta II and gamma), novel (delta, epsilon, theta and eta), and atypical (zeta and iota/lambda). Two calcium-insensitive isoforms of novel PKC, PKC delta and epsilon, have received particular attention as promising targets for new drugs. PKCs play a multifaceted role in cellular responses in a range of tissues. Professor Mochly-Rosen’s group and KAI Pharmaceuticals Inc. have developed drugs targeted against PKC delta (KAI-9803) and epsilon (KAI-1678). These drugs ameliorate pathological conditions in acute myocardial infarction and reduce pain via specific modulation of membrane-translocation of PKC delta or epsilon. Another research group has recently used the KinAceTM approach to produce PKC epsilon-abrogating peptides (KCe-12 and KCe-16) that are based on the catalytic domain of PKC. These peptides specifically inhibit PKC epsilon and ameliorate pathological conditions in a rodent insulin resistance model. This review describes the development of these therapeutic drugs targeting PKC delta and epsilon by two independent groups in the light of recent patents.

Key words: Protein kinase C (PKC), selective peptide modulator, KAI-9803, KAI-1678, KCe-12, KCe-16. INTRODUCTION PROTEIN KINASE C FAMILY Protein kinase C (PKC) was first identified and characterized by Nishizuka and co-workers in 1977 [1]. PKC was initially shown to have a histone kinase activity [1] that is activated by phosphatidylserine (PS), diacylglycerol (DAG) in a Ca2+-dependent manner, or 4-phorbol 12-myristate 13-acetate (PMA) [2]. The discovery of PKC represented an outstanding breakthrough in our understanding of signal transduction in cells [2]. PKC belongs to the AGC (cAMP-dependent, cGMP-dependent and PKC) family of serine and threonine kinases; PKCs form a multi-gene family with at least 10 isoforms that differ in their tissue expression, subcellular localization, activator and cofactor requirements, and substrate specificity [3]. These PKC isoforms have a highly conserved catalytic domain at their carboxy terminus: this domain consists of a motif required for ATP and substrate binding and for catalysis. The isoforms also have a variable regulatory domain containing an autoinhibitory pseudosubstrate domain and several membrane binding modules, such as C1 and C2, at their amino terminus [4] Fig. (1). The ten known PKC isoforms fall into three groups: classical PKC (cPKC), including alpha, beta I, beta II (beta I and beta II are alternative spliced isoforms of the same gene), and gamma; novel PKC (nPKC), including delta, epsilon, theta and eta; and atypical PKC (aPKC), including zeta and iota/lambda (the mouse homologue of PKC iota has been named PKC lambda) [4]. The regulatory *Address correspondence to this author at the Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Bohseidai, Ishehara, Kanagawa 259-1193, Japan; Tel: +81-463-94-1121; Fax: +81-463-94-8884; E-mail: [email protected]; [email protected] 1872-2156/09 $100.00+.00

domain of cPKC contains twin C1 domains, termed C1A and C1B, consisting of about 50 residues; each domain has 6 cysteines and 2 histidines which act as zinc-finger motifs that function in DAG/PMA binding. The cPKC regulatory domain also contains a C2 domain that binds anionic phospholipids in a calcium-dependent manner. nPKC possesses twin C1 domains and a C2 domain although the order of nPKC C1 and C2 domains are different to those of cPKC. Importantly, the nPKC C2 domain lacks the critical calciumcoordinating acidic residues that are the determinants for calcium binding. aPKC lacks a calcium-sensitive C2 domain and contains an atypical C1 domain that consists of only one cysteine-rich membrane targeting structure for binding PIP3 or ceraminide. cPKCs are activated by calcium, DAG and PS; nPKCs are activated by DAG and PS but not calcium. aPKCs require neither DAG nor calcium for activation [4] Fig. (1). In the steady state, the pseudo-substrate region of the regulatory domain binds with the catalytic domain by intramolecular interaction, maintaining PKC in the inactive form [4] Fig. (1). A basic outline of the regulation of PKC activation and function has been established over the last decade. The initial step is an increase in cytosolic calcium concentration and/or in lipid second messengers by activation of some cell surface receptors, such as G-protein coupled cell-surface receptor or receptor tyrosine kinase; activation of these receptors results in the switching of PKC from the inactive to the active form. The second step is the translocation of active PKC into the cellular membrane fraction, including golgi, endoplasmic reticulum, mitochondria and nucleus. Finally, the translocated active enzyme phosphorylates specific substrates. PKC activation has been shown to be involved in pathological states such as cancer [5], pain [6], diabetes [7, 8] cardiac attack [9, 10], stroke [11, 12], Alzheimer’s disease [13], heart failure [14], angiogenesis [15] and regulation of the © 2009 Bentham Science Publishers Ltd.

Selective Inhibitors of PKC

Recent Patents on DNA & Gene Sequences 2009, Vol. 3, No. 2

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Fig. (1). Overview of PKC isoforms. The PKC family consists of at least ten isoforms, which are subdivided into three classes that differ in structure and activator requirement. The region in the regulatory domain labeled PS indicates the pseudosubstrate domain. In the list of activators, PS indicates phosphatidylserine. DAG and PIP3 indicate diacylglycerol and phosphatidylinositol (3,4,5)- trisphosphate, respectively.

immune response [16]. Several pharmaceutical companies have sought to develop selective modulators for each PKC isoform. Various small molecule modulators against PKC targeting to ATP biding site, such as UCN01 (a.k.a. 7hydroxystaurosporine) and enzastaurin (a.k.a.LY317615) which is an oral administered inhibitor of PKCbeta/ Phosphoinositide 3-kinase (PI3K)/Akt and can pass through the blood-brain barrier, or phospholipids binding site, such as bryostatin (a.k.a.LY333531) which is an oral administered activator and can pass through the blood-brain barrier, has been established [17]. Another unique approach for generation of inhibitor is to design an anti-sense oligonucleotide against PKC. Aprinocarsen (a.k.a. LY900003) targeting 3´UTR of PKC alpha mRNA inhibits mRNA and protein expression and has the anti-tumor activity, which led to Phase III clinical trail in combination with cisplatin and gemcitabine or carboplatin and paclitaxel in patients with advanced non-small lung cancer [17, 18]. This trial has revealed that aprinocarsen did not add significantly to the chemotherapy regimens and enhanced toxicities such as thrombocytopenia, epistaxis and thrombosis/embolism [17, 18]. Development of PKC isoform specific modulators has proven to be a big challenge. In this review, we shed light on two alternative approaches for generation of selective-PKC modulator using short-peptide and its patents. RECEPTOR FOR ACTIVATED C KINASE (RACK) Multiple PKCs are present in the same cell and are activated by the same hormones, growth factors and neurotransmitters [3]. Biochemical assays have also shown that each PKC has a similar substrate selectivity [3]. However, the translocation pattern of each PKC is distinct before and after activation [19, 20], suggesting that subcellular localization of PKCs is finely regulated by the binding of an isoform-specific anchoring protein. Mochly-Rosen and coworkers proposed the concept of “RACKs” (receptor for activated C-kinase) and identified two RACK proteins [21, 22]. In the presence of an activator, PKC beta II specifically

binds with RACK1 via a region within the C2 domain [21, 23]. RACK1 consists of seven repeats of an approximately 40 amino acid segment, called the tryptophan-aspartic acid (WD40) motif, which was first identified in the beta subunit of the heterotrimeric G protein [24]. RACK2 also contains seven WD40 repeats and specifically binds with active PKC epsilon [22]. RACK2 was first identified as the coatomer protein complex, subunit beta 2 (COPB2 a.k.a. beta prime cop), a component of nonclathrin-coated vesicles in golgi [25, 26]. The WD40 repeats allow the protein to bind simultaneously with different proteins [27, 28]. RACKs act as a scaffold for a multiple protein complex that includes activated PKC. Other PKC isoforms can bind multiple proteins via their RACKs during activation. Binding of activated PKC to their RACKs directs PKC isoform functions to a specific subset of substrates. GENE-TARGETING STRATEGIES Recent gene-targeting studies on PKC have provided important insights into the physiological functions of PKC isoforms in mammals. PKC gamma is highly expressed in the brain, particularly in the hippocampus [29, 30]. PKC gamma-null mice are viable, develop normally, and have synaptic transmission that is indistinguishable from wildtype mice. However, the hippocampuses of these mice show greatly reduced synaptic plasticity, long-term potentiation (LTP), but not long-term depression (LTD) and paired-pulse facilitation (PPF) [29, 30]. Thus, PKC gamma is important for brain functions involved in learning and memory [29, 30]. Mice homozygous for a targeted disruption of the gene encoding PKC beta I and II are immunodeficient due to impairment of humoral immune responses and suppression of B cell responses, a phenotype similar to that of X-linked immunodeficient mice [31]. Further analysis has shown that PKC beta plays a critical role in B cell receptor-mediated survival signaling for NF-kappaB activation [32]. Thus, PKC beta I and II are important for B cell activation and are

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functionally linked to NF-kappaB activation in antigen receptor-mediated signal transduction. Investigation of PKC epsilon-null mice has shown that this isoform is involved in the regulation of the GABAA receptor in the brain [33] and in lipopolysaccharide-mediated signaling in macrophages [34]. In addition, PKC epsilon-null mice were recently shown to have enhanced production and secretion of insulin in the pancreas, which increased uptake of blood glucose in peripheral tissues and ameliorated pathological changes associated with diet-induced insulin resistance [35]. In PKC theta-null mice, although T cells mature normally, T cell receptor (TCR)-mediated NF-kappaB activation is impaired [36]. Thus, PKC theta is essential for TCR-mediated T-cell activation, but not TCR-dependent thymocyte maturation. In PKC delta-null mice, smooth muscle cells are resistant to apoptosis induced by angiotensin II and endothelin [37]. Activation of antigen-specific degranulation is observed in mast cells [38]. In B cell, B cells tolerance is suppressed by enhancement of maturation and differentiation [39, 40]. From a study of PKC zeta-null mice embryonic fibroblasts, it became clear that PKC zeta has an important role in the regulation of NF-kappaB transcriptional activity [41]. Therefore, lack of PKC zeta induces impairment of B cell receptor signaling, and inhibition of cell proliferation and survival [42]. In mice, deficiency of PKC lambda results in embryonic lethality around embryonic day 9 [43]. Tissue-specific PKC lambda-deficiency can produce an insulin resistance phenotype [44, 45]. PKC eta-null mice exhibit increased susceptibility to tumor formation and impairment of epithelial regeneration in wound healing [46]. PKC alpha-deficient mice display cardiac hypercontractility [47], and enhancement of insulin sensitivity in muscle and white adipocytes [48]. PATENT Peptide Modulator for Translocation of PKC [49, 50] Phospholipase C, Phospholipase D and synaptic vesicle specific protein, synaptotagmin also translocates cytosolic into membrane following activation as well as PKC [51, 52]. These proteins have a domain of approximately 150 residues that is homologous with the C2 domain of PKCs. Although the C2 domain was initially characterized as binding lipids, Mochly-Rosen and co-workers hypothesized that the domain might also be critical for translocation and binding of RACKs in PKCs, and were able to demonstrate that synaptotagmin C2 does indeed bind RACKs [53]. A sequence homology analysis of PKC beta II C2 and synaptotagmin C2 revealed several putative RACK binding sites consisting of 8-13 residues. Short peptides derived from these sites inhibited phorbol ester-induced PKC beta II translocation in cardiomyocytes [54]. Using a similar strategy, they generated short peptides from the C2 domain of PKC delta [55-58] and PKC epsilon [59-62] and found that these peptides also inhibited translocation of activated

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PKC. These peptides mimic regions of the C2 domain and compete with PKC for binding to RACKs; consequently, they abrogate kinase-dependent signaling Fig (2A). Professor Daria Mochly-Rosen is the founder of KAI Pharmaceuticals Inc., a company that plans to develop PKC modulators, including the above mentioned peptide inhibitor, for use in therapeutics. KAI Pharmaceuticals Inc. operates drug discovery and development programs with multiple, clinical-stage trials for new therapies for cardiovascular disease and pain. KAI has two promising lead products: KAI-9803 (SEQ ID NO.2) [49] and KAI-1678 (SEQ ID NO.1) [50]. KAI-9803 (a.k.a. deltaV1-1 [53-56]) is a PKC delta-specific translocation inhibitor derived from the C2 domain and has been put through a Phase IIb clinical trial in patients with acute myocardial infarction. This trial is funded by the Bristol-Myers Squibb Company under a global development and commercialization collaboration. Animal studies showed that the peptide is safe in a wide range of concentrations. They also demonstrated that treatment with the peptide after myocardial infarction or stroke reduced infarct size by about 70% and reduced functional impairment of heart and brain via inhibition of apoptosis [9, 56]. This therapeutic effect is consistent with the phenotype of PKC delta-null mice smooth muscle cells [37]. Ikeno et al. patent claims four peptide sequences including KAI-9803 (SEQ ID NO.2-4) and their administration methods of vasodilator alone or in combination with bradykinin, adenosine, prostacyclin, iloprost, cicaprost; nicotinic acid, niacin, a beta adrenergic blocking drug in patients with acute myocardial infarction. [49]. The second candidate, KAI-1678 [50] (a.k.a. epsilonV1-2 [59-62]), is a PKC epsilon-specific translocation inhibitor derived from the C2 domain. It too has just completed a Phase IIa clinical trial in patients with moderate to severe postoperative pain. PKC epsilon localizes and functions in peripheral neurons such as nociceptive neurons [63]. Transient receptor potential vanilloid-1 (TRPV1) is critical for sensing pain [64]; pro-inflammatory signals, including ATP and bradykinin, enhance TRPV1 activity in a PKC-dependent manner [65, 66]. Additionally, PKC epsilon directly phosphorylates TRPV1 [67]. In rodent neuropathic pain models, KAI-1678 attenuates the response of nociceptive neurons [62, 68]. It is possible that current treatments for pain, such as opioids and non-steroidal anti-inflammatory drugs (NSAIDs), might be substituted in future by use of KAI-1678. Mochly-Daria et al. patent claims short peptide sequence of KAI-1678 (SEQ ID NO.1) and their administration methods using cell-permeable carrier peptides derived from Drosophila antennapedia (SEQ ID NO. 4) and Human immunodeficiency virus transactivating regulatory protein (Tat) (SEQ ID NO.5) in patients with allograft transplantation [50]. “KinAceTM” Approach for Generation of PKC Inhibitors [69, 70] Professor Eleazar Shafrir’s group and Keryx Biopharmaceuticals Inc. recently produced PKC epsilon abrogating peptides, named KCe-12 and KCe-16 [69, 71]. These peptides were copied from the catalytic domain using the KinAceTM approach [70, 71]. KinAceTM is based on short peptides, derived from specific regions in the catalytic domain of a kinase, that are involved in kinase-substrate



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Fig. (2). Two unique functional strategies for identifying peptide inhibitors that target PKC. (A) KAI pharmaceuticals sought to identify competitive inhibitors of the interactions between the specific anchoring protein, RACK, and PKC among short peptides derived from the PKC C2 domain. (B) Keryx Biopharmaceuticals examined inhibition of the interactions between PKC and a substrate by competitive short peptides derived from the kinase region of PKC. KD indicates the kinase domain of PKC.

interactions [70, 72] Fig. (2B). From analysis of protein sequence, three-dimensional structure and mutational protein-protein interaction in many kinases, the targeting domains, such as the HJ-alphaG or alphaD regions, are substrate binding sites in the catalytic domain of the kinase and are conserved structural patterns in all kinases [70, 72]. These peptides mimic regions of the kinase and compete with the kinase for binding to a substrate; consequently, they abrogate kinase-dependent signaling Fig. (2). Niv et al. have demonstrated that short myristoylated peptides from the target regions of c-Kit and Lyn belonging to tyrosine kinases and 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt belonging to serine/threonine kinases selectively inhibit the signaling of the kinase from which it is derived and cancer cell proliferation in the micromolar range [70, 72]. Thus, the advantage of KinAceTM is to generate selective-kinase inhibitor without known function or solved crystal structure. An alternative approach to KinAceTM has been developed for screening many candidate peptides derived from the substrates, pseudosubstrate, regulator interacting with kinase, and the non-catalytic domains of the substrate binding domain. This novel technology is unique and effective for screening for drugs targeted to kinases and has been used to isolate myristoylated-peptides, KCe-12 (SEQ ID NO.6) and KCe-16 (SEQ ID NO.7) [69, 71], derived from the alphaD region of PKC epsilon (SEQ ID NO.2). Intraperitoneal injections of these peptides prevent nutritional diabetes and protect muscle IRS-1 from PKC epsilon-induced serine phosphorylation, and abrogate insulin resistance in the desert gerbil model for type 2 diabetes [71]. The claims of Shafrir et al. patent encompass not only the isolated two peptides sequences, but also two domain sequences of alphaD and HJ-alphaG region in PKC epsilon (SEQ ID NO.5) regarding the use as medicament for treatment of type 2 diabetes, insulin resistance, hyperglycemia, diabetic complications and metabolic disorders. Shafrir et al. also claimed analogs

modulated from these peptides (Formula I-III, SEQ ID NO. 3, 4 and 8,). CURRENT & FUTURE DEVELOPMENTS Two patents have been obtained for the short peptides described in this review. These novel peptides are unique and powerful therapeutic reagents for targeting PKCs. From rodent study by daily intraperitoneal injection of Tat- or Tatconjugated PKC modulators (20 nM) for 2 weeks, short peptides including Tat-conjugated KAI-9803 and KAI-1678 have no apparent toxicity, activation of compensatory pathways, enhancement of immune response and tolerance to the positive effect of these peptides [73]. KCe-12 and KCe16 may also have no toxicity effects. Additionally, these selective-PKC inhibitory peptides may have potential as new therapies for other diseases. The identification of the direct substrates of PKCs during development of inhibitory peptides has also led to the identification of down-stream effector molecules that are involved in therapeutic effects. These down-stream molecules have some potential as new drug targets. Professor Mochly-Rosen and colleagues used a proteomics approach to show that the direct substrate of PKC epsilon is mitochondrial aldehyde dehydrogenase 2 (ALDH2) in ischemic heart tissue, and generated a small molecule modulator of ALDH2 activity, Alda-1 [74, 75]. In addition to KAI-9803, pretreatment by Alda-1 reduces infarct size by 60% in an ischemic rat model. This effect may be mediated by inhibition of the formation of cytotoxic aldehydes. With respect to delivery of therapeutic peptides to the brain, the inhibitory peptides will need to be modified to enable them to cross the blood-brain barrier in order that they can be used in treatment of stroke, Alzheimer’s disease and pain. Nevertheless, these short inhibitory peptides and have great therapeutic potential, and the technologies used to produce them will undoubtedly drive advances in medical research and for developing drugs.

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ACKNOWLEDGEMENTS This work was supported by a Grant-in-Aid for Young Scientists (Start-up) from Japan Society for Promotion of Science (JSPS-20810031).

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