Current Drug Targets - Volume 7, Issue 11, 2006

Protein kinases play essential roles in cellular processes and are well recognized as important therapeutic targets in many human diseases. The serine/threonine protein kinase Glycogen synthase kinase-3 (GSK-3) is an extraordinary example in this regard. This enzyme, which was characterized originally as a glycogen synthase kinase, has emerged, unexpectedly, as a drug-discovery target in several pathological disorders, including diabetes, affective disorders, and Alzheimer's disease. The unique biochemical and cellular features of GSK-3, which distinguish it from other protein kinases, are most likely the key to understanding the sophisticated function of GSK-3 in biological systems. These features justify the efforts invested toward the development of selective GSK-3 inhibitors as a promising therapeutic treatment. In this issue of Current Drug Targets, articles are gathered from eight groups with different expertise areas that, together, uncover the multiple aspects of GSK-3. This gives the reader a unique, special opportunity to gain a panoramic view in the filed, and to uncover the challenges that still wait to be resolved.

Glycogen synthase kinase-3 (GSK-3) has attracted much scrutiny due to its plethora of cellular functions, novel mechanisms of regulation and its potential as a therapeutic target for several common diseases. In mammals, GSK-3 is encoded by two genes, termed GSK-3α and GSK-3β, that yield related but distinct protein-serine kinases. GSK-3 is unusual in that its protein kinase activity tends to be high in resting cells and cellular stimuli, such as hormones and growth factors, result in its catalytic inactivation. Further, many of the substrate proteins of GSK-3 are functionally inhibited by phosphorylation. Thus, signals that inhibit GSK-3 often cause activation of its diverse array of target proteins. Regulation of GSK-3 is important for normal development, regulation of metabolism, neuronal growth and differentiation and modulation of cell death. Dysregulation of GSK-3 activity has been implicated in human pathologies such as neurodegenerative diseases and type-2 diabetes. In this introductory chapter we provide a primer on the modes of GSK-3 regulation and a description of the various signaling pathways and cellular processes in which GSK-3 is an active participant.

Alzheimer's disease (AD) is a common neurodegenerative disorder that presents clinically as inexorable cognitive impairment and decline in performance of activities of daily living. AD is characterized pathologically by neuronal depopulation, extracellular amyloid plaques, and intraneuronal accumulation of neurofibrillary tangles (NFTs). Accumulation of these polypeptide aggregates is generally believed to be integral to the pathogenesis of AD. Recent evidence implicates the protein kinase glycogen synthase kinase 3 (GSK-3) in the regulation of both of these processes. GSK-3 has long been studied as one of several tau protein kinases, and has more recently been shown to be involved in the generation of Aβ peptides. GSK-3 activity may also promote cell death and conversely, inhibition of GSK-3 has been associated with increased cell survival under a variety of cytotoxic conditions. Thus drugs that target GSK-3 could attack AD pathogenesis on multiple fronts simultaneously. Here we will briefly review the molecular understanding of AD pathogenesis as it stands at this point, and then discuss the emerging role of GSK-3 in regulating these processes.

There exists an immediate need to develop novel medications for the treatment of mood disorders such as bipolar disorder and depression. Initial interest in glycogen synthase kinase-3 (GSK-3) as a target for the treatment of mood disorders arose from the finding that the mood stabilizing drug lithium directly inhibited the enzyme. More recent preclinical evidence implicates the modulation of GSK- 3 in either the direct or downstream mechanism of action of many other mood stabilizer and antidepressant medications currently in use. One of the cellular targets of GSK-3, which may mediate some of the effects of lithium and other drugs, is β-catenin, a transcription factor that is rapidly degraded when GSK-3 is active. Recent rodent behavioral data (both genetic and pharmacological) supports GSK-3 representing a therapeutically relevant target of lithium. This includes antidepressant-like behavior in the forced swim test and antimaniclike response to amphetamine following administration of the GSK-3 inhibitor AR-A014418, a findings that is concomitant with an increase in brain β-catenin. The evidence described in this review suggests that regulating GSK-3 may represent a target for novel medications to treat mood disorders.

GSK3 is a multifunctional protein kinase that is pivotal for the regulation of metabolism, the cytoskeleton, and gene expression. Multicellular eukaryotes utilize GSK3 as a molecular switch to specify distinct cell fates, but also to organize these cells spatially within the developing organism. We discuss the central role of GSK3 in control of the Wnt, Hedgehog, cAMP (in Dictyostelium), and other signaling pathways, but also focus on significant new evidence that GSK3 is required to establish cell polarity.

Glycogen synthase kinase-3 (GSK3) has recently been linked to mood disorders and schizophrenia, and the neurotransmitter systems and therapeutic treatments associated with these diseases. GSK3 is a widely influential enzyme that is capable of phosphorylating, and thereby regulating, over forty known substrates. Four mechanisms regulating GSK3 (phosphorylation, protein complexes, localization, and substrate phosphorylation) combine to provide substrate-specific regulation of the actions of GSK3. Several intracellular signaling cascades converge on GSK3 to modulate its activity, and several neurotransmitter systems also regulate GSK3, including serotonergic, dopaminergic, cholinergic, and glutamatergic systems. Because of changes in these neurotransmitter systems and the actions of therapeutic drugs, GSK3 has been linked to the mood disorders, bipolar disorder and depression, and to schizophrenia. Inhibition of GSK3 may be an important therapeutic target of mood stabilizers, and regulation of GSK3 may be involved in the therapeutic effects of other drugs used in psychiatry. Dysregulated GSK3 in bipolar disorder, depression, and schizophrenia could have multiple effects that could impair neural plasticity, such as modulation of neuronal architecture, neurogenesis, gene expression, and the ability of neurons to respond to stressful, potentially lethal, conditions. In part because of these key actions of GSK3 and its associations with mood disorders and schizophrenia, much research is currently being devoted to identifying new selective inhibitors of GSK3.

A reduced ability of insulin to activate glucose transport in skeletal muscle, termed insulin resistance, is a primary defect leading to the development of impaired glucose tolerance and type 2 diabetes. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase with important roles in the regulation of glycogen synthesis, protein synthesis, gene transcription, and cell differentiation in various cell types. An emerging body of evidence has implicated GSK-3 in the multifactorial etiology of skeletal muscle insulin resistance in obese animal models and in obese human type 2 diabetic subjects. Overexpression and overactivity of GSK-3 in skeletal muscle of rodent models of obesity and obese type 2 diabetic humans are associated with an impaired ability of insulin to activate glucose disposal and glycogen synthase. New insights into the importance of GSK-3 as a regulator of insulin action on glucose transport activity in muscle have come from studies utilizing selective and sensitive inhibitors of GSK-3. These studies have demonstrated that selective inhibition of GSK-3 in insulin-resistant skeletal muscle causes improvements in insulin-stimulated glucose transport activity that are likely caused by enhanced post-insulin receptor insulin signaling and GLUT-4 glucose transporter translocation. An additional important action of these GSK-3 inhibitors in the context of obese-associated type 2 diabetes is a reduction of hepatic glucose production, likely via downregulation of genes associated with gluconeogensis. It is clear from these studies that selectively targeting GSK-3 in skeletal muscle may be an important new strategy for the treatment of obesity-associated insulin-resistant states characterized by GSK-3 overactivity in insulinsensitive tissues.

Protein kinases represent a large family of enzymes involved in regulating complex molecular machineries that control many cellular functions, from survival and proliferation to apoptosis. Abnormal protein kinase activity has been involved in a variety of pathophysiologic states, including cancer, inflammatory and autoimmune disorders, and cardiac diseases, and protein kinases have become one of the major therapeutical targets of the past 10 years. The major problem associated with ATP-competitive kinase inhibition is target specificity, since many other enzymes, kinases and not-kinases alike, utilize ATP: less specific inhibitors would be expected to exhibit undesirable toxicities that would limit their potential utility as therapeutic agents. The purpose of this review is to offer the reader an idea of the evolution of the methodologies utilized in the quest for selective kinase inhibitors, from the more traditional, screening-based methods to the newer technology of chemogenomics, proteomics and chemical genetics.

Yeast cells carry four homologs of GSK-3β, RIM11, MCK1, MRK1 and YGK3. The significant homologs are RIM11 and MCK1 that presumably arose from a recent genome duplication followed by a rapid divergence. Accordingly, these homologs phosphorylate specific substrates. Rim11 is essential for entry into meiosis, whereas Mck1 is essential for growth at elevated and low temperatures. Both kinases transmit nutrient signals, but Mck1 transmits additional signals including stress signals such as, temperature, osmotic shock and Ca2+. Consequently, Mck1 plays a role in multiple functions, including cell wall integrity, meiosis and centromere function. The other two homologs, MRK1 and YGK3 that belong to the RIM11 and MCK1 phylogenetic trees, respectively, show no distinct phenotype. These paralogs posses redundant roles, though less important, with Rim11 and Mck1 functions. This review summarizes the cellular roles of these kinases, their mode of regulation, and the signals that they transmit.

Ageing is an issue, which inevitably affects all of us, and conditions that accompany ageing - such as cardiovascular disease, cancer, neurodegenerative disorders and diabetes - account for the majority of healthcare costs. This is a rapidly increasing problem, as, average life expectancy worldwide has increased by 20 years since 1950, to stand at 66 years now and the number of people over 65 is expected to double until 2025 in many industrialized countries in Europe and the US. It can be expected that the trend towards an increased lifespan - independent of the intake of specially designed treatment - has not come to an end yet. Drs. Oeppen and Vaupel (Cambridge University, United Kingdom and Max Planck Institute for Demographic Research, Germany) have observed that maximum life expectancy has steadily increased for the past 160 years. The increase in the record expectation of life should be slowing, when close to a natural maximum, an observation which, however, has not been made. While the magnitude of the consequences of these world-wide demographic changes, however, is not reflected by existing therapeutic options, there is an increasing interest and awareness in the scientific community for the treatment of age-related disorders, dysfunctions and diseases; furthermore, an increasing emphasis is placed on basic research witnessing unprecedented growth and acceptance. Although the fountain of youth certainly is out of reach, the recent past has seen exciting developments bringing us closer to the identification of causes of ageing and to the generation of agents increasing longevity. Exciting innovations have been described in biotechnology including conditions to regrow damaged or diseased tissues and organs, in stem cell technology to permit development of a supply source for human cells, tissues, and organs and genetic engineering advancements. The view dominated by evolutionary research considering ageing as a general deterioration, thus precluding monogenetic causes of longevity, has been changed dramatically within the last decade, which has seen spectacular increases of lifespan in model organisms as a result of single-gene mutations. These findings have fuelled the screening for specifically interfering agents and will prompt the search for further single-gene targets employing timely screening technologies such as high-throughput tests using RNAi. The latter is also being used to correct defective RNA splices that, when expressed, cause disease. Screening for potential treatments, however, requires appropriate models and feasible assays. Whereas lifespan studies are performed in simple organisms like yeast, worms (C. elegans) or flies (Drosophila) requiring several days or weeks, an evaluation in a mouse model may take several months or years. Recent genetic studies on these animals revealed that the lifespan of these species can be dramatically increased by the mutation of one or a few genes. The identification of these genes, however, suggests that ageing can be manipulated, because many genes that affect longevity in model organisms have human homologs. However, it still remains to be shown to which extent these or other gene activities represent drugable targets. With regard to low molecular weight agents, medicinal chemistry has developed efficient toolkits and e.g. Chemical Genetics, a phenotypedriven research targeting the lifespan of model systems and animal models, appears to be fruitful. Given the lack of knowledge on lifeextending mechanisms a random or biased screening approach is reasonable besides attempts to address distinct known targets for interference based on mechanistic considerations. For instance, a lifespan directed screening of agents with known pharmacological activities such as the testing of anticonvulsive drugs by Kerry Kornfeld and colleagues provides highly interesting data pointing to a previously unknown connection between neural function and longevity. Alternative underlying mechanisms are expected to include the preservation or the restoration of important functions in cellular control and repair mechanisms or of pathways affecting the expression and function of proteins controlling the levels of reactive oxygen species and improving stress resistance such as antioxidant enzymes like catalase or administration of the glutathione precursor cysteine, which might reduce age-associated degeneration. Drugs mimicking caloric restriction decrease weight, may decrease oxidative stress, increase insulin sensitivity, modulate the neuroendocrine system and prolong life in ways similar to caloric restriction....

Various molecular and cellular alterations to our tissues accumulate throughout life as intrinsic side-effects of metabolism. These alterations are initially harmless, but some, which we may term "damage", are pathogenic when sufficiently abundant. The slowness of their accumulation explains why decline of tissue and organismal function generally does not appear until the age of 40 or older. Aging is thus best viewed as a two-part process in which metabolism causes accumulating damage and sufficiently abundant damage causes pathology. Hence, a promising approach to avoiding age-related pathology is periodically to repair the various types of damage and so maintain them at a sub-pathogenic level. Some examples of such types of damage are intracellular and others extracellular. Several types of intracellular damage are highly challenging - sophisticated cellular and genetic therapies will be needed to combat them, which are surely at least 20 years away and maybe much more. Extracellular damage, by contrast, generally appears more amenable to pharmaceutical repair which may be feasible in a shorter timeframe. In this article, the major types of age-related extracellular damage and promising avenues for their repair are reviewed.

Recent research indicates that aging is affected by many genes and thus many biochemical pathways. This has led to a failure to find pharmaceuticals that significantly ameliorate the human aging process. Progress in evolutionary and genetic research, however, suggests the possibility of combining experimental evolution, genomic analysis, and mass screening of pharmaceuticals and botanicals to produce effective therapeutics for human aging. The starting point for this strategy is model systems that have outbred populations with substantially increased lifespan. These are easily produced by tuning the force of natural selection in the laboratory. Such biological material is then a good candidate for genomic analysis, leading to the identification of numerous biochemical pathways involved in increased lifespan, in the model system. These biochemical pathways would then be available for pharmaceutical development, first in fruit flies, then in rodents, and eventually in a clinical human population. We include a discussion of the pharmacological methods appropriate to this strategy of drug discovery.

A significant increase of the elderly in populations of developed countries is followed by increase morbidity and mortality from main age-related diseases - cardiovascular and neuro-degenerative, cancer, diabetes mellitus, declining in a resistance to infections. Obviously, the development of means of the prevention of the premature ageing and these diseases in humans are crucial at present. However, data on such type means rather scarce, contradictory and often not reliable from the points of view of the adequacy of the experiments to current scientific requirements, as well as the interpretation of the results and safety. Available data on the life span extension and adverse effects of chemical compounds and drugs suggested as geroprotectors are critically analyzed: antidiabetic drugs, growth and thyroid hormones, glucocorticoids, DHEA, sex steroids and contraceptives, melatonin and peptide preparations modulating the pineal gland, antioxidants, chelate agents and lathyrogens, adaptogens and herbs, neurotropic drugs, inhibitors of monoamine oxidase, immunomodulators and some other. Most of the results could not convincingly evidence the life span extension and safety of the suggested geroprotectors. We believe that it is necessary to establish an international program for the expert evaluation of the life span extension potential of pharmacological interventions for humans. The scope of the program should be to evaluate chemical, immunological, dietary and behavioural interventions that may lead to life span extension or retard premature ageing and the objective - preparation of critical reviews and evaluations on evidence of the life span extending properties of a wide range of potential geroprotectors and strategies by international groups of working experts. The program may assist national and international authorities in devising programs of health promotion and premature ageing prevention

The popular use of antioxidative vitamins illustrates the growing awareness of oxidative stress as an important hazard to our health and as an important factor in the ageing process. Superoxide radicals and superoxide-derived reactive oxygen species (ROS) are constantly formed in most cells and tissues. To ensure that ROS can function as biological signaling molecules without excessive tissue damage, ROS are typically scavenged by antioxidants such as glutathione and the vitamins A, C, and E. “Oxidative stress” occurs if the production of ROS is abnormally increased or antioxidant concentrations are decreased. Genetic studies in mice, Drosophila, and C.elegans suggested that ageing may be mechanistically linked to oxidative stress. Several manifestations of oxidative stress were shown to increase with age, whereas tissue levels of vitamin E, plasma concentrations of vitamin C, and intracellular glutathione concentrations decrease with age. In at least two independent studies, cysteine supplementation on top of the normal protein diet has shown significant beneficial effects on each of several different parameters relevant to ageing, including skeletal muscle functions. As the quality of life in old age is severely compromised by the loss of skeletal muscle function, and as muscle function can be measured with satisfactory precision, loss of muscle function is one of the most attractive surrogate parameters of ageing. The mechanisms by which a deficit in glutathione and its precursor cysteine contributes to various ageing-related degenerative processes appears to be related largely but not exclusively to the dysregulation of redox-regulated biological signaling cascades.

Selegiline inhibits the activity of monoamine oxidase B, enhances the release of dopamine, blocks the uptake of dopamine, acts as a calmodulin antagonist, and enhances the level of cyclic AMP, which in turn protects dopaminergic neurons. It possesses cognition- enhancing functions, rejuvenates serum insulin-like growth factor I in aged rats, and enhances life expectancy in rodents. Selegiline possesses neurotrophic-like actions, and rescues axotomized motorneurons independent of monoamine oxidase B inhibition. It enhances the synthesis of nerve growth factor, protects dopaminergic neurons from glutamate-mediated neurotoxicity, and protects dopaminergic neurons from toxic factors present in the spinal fluid of parkinsonian patients, and the said effect may be mediated via elaborating brain derived neurotrophic factor. Selegiline increases the striatal superoxide dismutase, protects against peroxynitrite- and nitric oxide-induced apoptosis, and guards dopaminergic neurons from toxicity induced by glutathione depletion. It stimulates the biosynthesis of interleukin 1-β and interleukin-6, is an immunoenhancing substance, possesses antiapoptotic actions, and is neuroprotectant in nature. Selegiline has been shown to be efficacious in Parkinson's disease, global ischemia, Gille de la Tourette syndrome, and narcolepsy. Its therapeutic efficacy in Alzheimer's disease remains uncertain. In Alzheimer's disease, short term studies of selegiline suggest a beneficial effect; whereas long term studies are less convincing.

While at present there is no scientific consensus on the reasons for cellular and organismal ageing - or indeed on a comprehensive definition for ageing - scientific efforts to unravel the complex biochemistry behind the ageing process have recently met with considerable success. Despite a still somewhat fragmented understanding of the phenomenon of ageing, a distinction has therefore become possible between those biochemical and physiological events that are causal to ageing, and those that merely accompany the process. Such a distinction is an important prerequisite for the selection of targets for pharmacological intervention, and for the design of “antiageing drugs” directed against these targets. This review looks from a chemical viewpoint at currently used model systems for the ageing process, at small molecules showing anti-ageing properties in these screens, and at their mechanisms of action.

Endocannabinoids are a new class of lipids, which include amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Δ9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. AEA is released “on demand” from membrane lipids, and its activity at the receptors is limited by cellular uptake followed by intracellular hydrolysis. Together with AEA and congeners, the proteins which bind, synthesize, transport and hydrolyze AEA form the “endocannabinoid system”. Endogenous cannabinoids are present in the central nervous system and in peripheral tissues, suggesting a physiological role as broad spectrum modulators. This review summarizes the main features of the endocannabinoid system, and the latest advances on its involvement in ageing of central and peripheral cells. In addition, the therapeutic potential of recently developed drugs able to modulate the endocannabinoid tone for the treatment of ageing and age-related human pathologies will be reviewed.

Histone deacetylases (HDACs) are enzymes that are able to deacetylate lysine side chains in histones and certain non-histone proteins which leads to altered states of conformation and activity for the proteins in question. Three classes of histone deacetylases have been recognized in humans. Class I and II are zinc-dependent amidohydrolases and eleven subtypes have been discovered (HDAC1-11). Class III enzymes depend in their catalysis on NAD+ and subsequently, O-acetyl ADP ribose and nicotinamide are formed as a consequence of the acetyl transfer. Due to the homology to the yeast histone deacetylase Sir2p the NAD+-dependent deacetylases are also termed sirtuins and seven members (Sirt1-7) are known in humans. Sirtuins are found from bacteria to eukaryotes and altogether about 60 isoforms have been characterized in different organisms. Sirtuins have been implicated in the regulation of molecular mechanisms of aging. The overexpression of sirtuin enzymatic activity leads to an increase of lifespan in Saccharomyces cerevisiae and Caenorhabditis elegans that can also be reached by calorie restriction. Sirtuins have been proposed to act as sensors for glucose uptake that respond to the levels of NAD+ but more complex ways of action have been suggested as well. This article will present the members of the human sirtuin family with their respective functions and review the existing druglike inhibitors and activators of sirtuin activity.