Kinase Inhibitors. Fig. 1. Intracellular signaling cascades involved in the long-term stabilization of mood by lithium (Li) and valproic acid (VPA). Activation of receptors coupled to PI hydrolysis results in the breakdown of phosphoinositide 4,5-biphosphate (PIP2) into two second messengers: IP3 and diacylglycerol (DAG), which is an endogenous activator of PKC. Lithium is an uncompetitive inhibitor of inositol monophosphatases, whereas both lithium and VPA, upon chronic administration, decrease myo-inositol uptake. These perturbations by mood stabilizers likely contribute to the reduction in PKC activity and the reduced levels of PKC-a, PKC-e, and myristoylated alanine-rich C kinase substrate (MARCKS), a major PKC substrate in the CNS (MARCKS proteins play important roles in multiple cell functions, including cell architecture, cell cycle, and exocytosis, and are regulated by Ca2+/calmodulin and PKC.) In the Wnt signaling pathway, binding of the Wnt signal to the Wnt receptor (WntR) activates an intermediary protein, Disheveled, which regulates GSK-3p. GSK-3P regulates cytoskeletal proteins, and also has an important role in determining cell survival and cell death. Lithium (and possibly VPA) directly inhibits GSK-3P, which may underlie, at least in part, the increases in b-catenin that occur after chronic treatment with these agents. The ERK MAP kinase cascade regulates several important transcription factors, most notably CREB and activator protein-1 (AP-1) (AP-1 is a transcription factor that regulates gene expression in response to diverse stimuli.) Recent studies have demonstrated that both lithium and VPA activate the ERK MAP kinase cascade, which may contribute to the long-term changes in synaptic plasticity and morphology that follow chronic treatment. Together, regulation of these signaling pathways through selective kinase inhibition might bring about an enhancement of synaptic connectivity, potentially necessary for long-term stabilization of mood. (Reprinted from Manji C (2002) Nat Med 8:557-558 With permission.)
Preclinical and Clinical Evidence of the Role of PKC Inhibition in the Pathophysiology and Treatment of Bipolar Disorder
Evidence from numerous studies using widely varying methodologies implicates PKC in the pathophysiology and treatment of ► bipolar disorder. These include peripheral blood cell studies that have showed altered PKC isozyme levels and activity in bipolar subjects, and postmortem studies similarly demonstrate altered PKC levels and activity in subjects with bipolar disorder. Importantly, lithium and ► valproate - two efficacious medications with mood-stabilizer properties, widely utilized in the treatment of bipolar disorder - decrease PKC levels in an isozyme-specific manner and decrease PKC activity. Lithium interacts with the PI/PKC pathway via the inhibition of inositol mono-phosphatase (IMPase), resulting in decreased free myo-inositol and the subsequent production of DAG, with the downstream effect of decreasing PKC levels and activity. Valproate - structurally dissimilar from lithium - also appears to inhibit PKC, suggesting that this inhibition is important to the common therapeutic effects of both medications.
Based on the clinical observation that ► psychostimulants (amphetamine and cocaine) worsen manic symptoms in hypomanic patients and induce manic relapses in subjects with a personal or family history of bipolar disorder, stimulant-induced behavioral alterations in rodents are utilized as experimental models of mania. Lithium treatment prevents these forms of psychostimulant-induced behaviors, which provides additional validation to these models. Notably, the behavioral alterations induced by psychostimulants in rodent are associated with changes in PKC, and furthermore, pharmacological inhibitors of this kinase-attenuate amphetamine-induced hyperlocomotion (Fig. 2a, b). In fact, excessive activation of PKC dramatically impairs cognitive functions of the prefrontal cortex, while PKC inhibition protects it, suggesting that PKC might play a role in some of the cognitive features of mania.
The first study in humans with a fairly selective PKC inhibitor was a small, open-label trial, in which treatment with tamoxifen produced a greater than 50% decrease in manic symptoms in five of seven subjects. Noticeably, this effect was recently confirmed in a ► double-blind, placebo-controlled, 3-week study of 16 manic patients (Zarate et al. 2007), effects found as early as day 5 of treatment with the PKC inhibitor (Fig. 2c). The potential role of the PKC signaling pathway in the pathophysiology of bipolar disorder has been further strengthened by the identification of a bipolar susceptibility gene, which is an upstream regulator of this kinase. Diacylglycerol kinase eta
(DGKH), a major regulator of DAG that activates all known classical and novel isoforms of PKC, was identified as a risk gene for bipolar disorder in two recent independent genome-wide association studies (Baum et al. 2008; Wellcome Trust Case Control Consortium 2007).
A large number of structurally distinct PKC inhibitors have been identified or developed, mostly for use in cancer treatment, and Tamoxifen has been investigated and found efficacious in three independent clinical trials for bipolar disorder. PKC inhibitors that are used in treatment or have undergone clinical trials for cancer include the already-mentioned tamoxifen (treatment for estrogen receptor-positive breast cancer); the lipid analogs safingol (Phase-I trial with doxorubicin for solid tumors) and miltefosine (Phase-II trial of topical treatment for cutaneous breast cancer metastases); the indolocarbazoles UCN-01 (Phase-II trials for several cancers) and PKC412 (Phase-II trials for solid tumors); the bisindolylmalemide enzastaurin (Phase-II trials for glioblastoma and B-cell lymphoma); and the PKCa-specific antisense oligonucleotide ISIS3521 (several Phase-II and Phase-III trials) (Serova et al. 2006). The PKC inhibitors that have been investigated thus far are relatively nonspecific, with the exception of the ► antisense oligonucleotides, that unmodified, however, very rarely cross the brain-blood barrier and will probably require forms of central delivery or adaptations to increase its lipophilicity. The potential of isoform-, cell type-, and substrate-selective PKC inhibitors has therefore not been fully explored.
The development of highly selective PKC inhibitors is critical for the possibility of novel therapies with good efficacy, tolerability, and safety profiles. As mentioned, strategies such as the use of non-ATP-competitive inhibitors, or inhibitors that target binding proteins or substrates that are less promiscuous than the target kinase, may prove useful in the development of anti-PKC agents. A substrate-competitive inhibitor has been designed for PKCa, and peptides corresponding to PKC-anchoring proteins like RACK (receptors for activated C kinases) selectively inhibit specific PKC isozyme activity. A related strategy is to target PKC in specific subcellular locations. Because PKC isoforms are translocated upon activation, targeting location-specific anchoring proteins may produce isoform- and/or substrate-specific inhibition. Finally, combination strategies may prove beneficial for increasing specificity and sustained therapeutic effectiveness and reducing resistance. Examples of target combinations might include kinase-targeted agents with (a) binding or scaffolding proteins that are location- and/or
Kinase Inhibitors. Fig. 2. Preclinical and clinical behavioral evidence of the effect of protein kinase inhibition: (a) Effects of two injections of tamoxifen, a PKC inhibitor, (1 mg/kg) on amphetamine-induced hyperactivity in rats, measured in a large open field after acute or (b) chronic (7 daily injections) amphetamine treatment. The y-axis represents total distance traveled during the 45-min test session. Both acute and chronic amphetamine increased total distance traveled, and this increase was attenuated by tamoxifen pretreatment without affecting nonamphetamine-treated rats. Sal = Saline; Con = vehicle; Amp = amphetamine; Tam = tamoxifen; Sens = sensitized. * p <0.05. (c) Clinical study: change in scores on the Young Mania Rating Scale (YMRS) over three weeks (n = 16). Subjects on tamoxifen showed significant improvement in mania when compared with placebo as early as 5 days, an effect that remained significant throughout the 3-week trial. * p< 0.05. (d) Immobility time in the forced swim test (in seconds) in Rats; n =14 per group; bar graphs represent mean ± SE. Animals treated with AR-A014418 (a selective GSK-3 inhibitor) demonstrated significantly reduced immobility time (suggesting an antidepressant-like action) when compared with vehicle-treated animals. * p<0.05. DMSO = Vehicle. (Modified and reproduced from Einat, Yuan, Szabo, Dogra, Manji (2007) Neuropsychobiology 55:123-131 (a and b); Zarate et al. 2007 (c); and Gould, Einat, Bhat, Manji (2004) Int J of Neuropsychopharmacol 7:387-390 (d). With permission.)
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