Current Medicinal Chemistry (v.19, #9)

Glutamate is a highly abundant excitatory neurotransmitter in the central nervous system residing in vesicles within chemical synapses. Once a nerve impulse triggers the release of glutamate from the pre-synaptic cell, it binds to and activates both ionotropic (ion channel- forming) and metabotropic (G protein-activating) glutamate receptors on the opposing post-synaptic cell. Subsequently glutamate transporters, localized in neuronal and glial membranes, rapidly and efficiently remove glutamate from the extracellular space thus inactivating the signaling. Glutamate excitatory neurotransmisison is involved in most aspects of normal brain function including cognition, memory and learning. However in several neurological and psychiatric diseases excess glutamate accumulates outside the cells resulting in hyperactivation of post synaptic glutamate receptors and causing massive calcium influx, damage to mitochondria and activation of proapoptotic genes. For this reason pharmacological blockade of this so-called “excitotoxic” effect via antagonism of post synaptic glutamate receptors has been of significant therapeutic interest. Unfortunately, although post synaptic receptor blockers have shown significant promise in preclinical animal models, they have been fraught with untoward side effects in the clinic thus limiting their therapeutic utility [1, 2]. Given this limitation, alternative strategies have been sought targeting processes upstream of the excitotoxic insult, i.e. blocking glutamate release from presynaptic terminals. This approach rationalizes that diminishing the amount of glutamate in the synaptic cleft will attenuate several diverse downstream pathologic processes simultaneously. Lamotrigine and riluzole are two examples of such strategy. Both drugs are presynaptic sodium channel blockers which limit glutamate release and are used in clinical practice to alleviate symptoms of epilepsy and amyotrophic lateral sclerosis, respectively [3, 4]. Recent data from multiple laboratories has identified an alternative approach to attenuate excess glutamate transmission. This approach is based upon inhibiting the hydrolysis of N-Acetyl-aspartyl-glutamate (NAAG), the most abundant mammalian peptidic neurotransmitter. NAAG is hydrolyzed to N-acetyl-aspartyl and glutamate by Glutamate Carboxypeptidase II (GCPII), a glially localized membrane-bound binuclear zinc metallopeptidase [5]. Inhibiting GCPII has been shown to dampen excessive glutamate transmission by both decreasing extracellular NAAG-derived glutamate as well as increasing NAAG. Increased NAAG results in activation of presynaptic mGluR3 [6] and the release of the neuroprotective trophic factor TGFβ [7]. Many families of structurally distinct small molecule inhibitors of GCPII have been synthesized and shown to attenuate neurotoxicity in several animal models of disease whereby enhanced glutamate transmission is presumed pathogenic. These models include inflammatory and neuropathic pain, brain ischemia, motoneuron disease, spinal cord and traumatic brain injury, peripheral neuropathy, epilepsy, and drug abuse (for review see [8-10]). This new approach to modulate glutamate levels via GCPII inhibition is therapeutically exciting, since it does not appear to affect basal glutamate function but rather selectively inhibits excessive glutamate neurotransmission [11]. From a therapeutic standpoint, this is ideal. If this holds true in the clinic, GCPII inhibition could limit excess glutamate release and provide neuroprotection without the untoward side effects observed with potent glutamate receptor antagonists. Data from the first clinical evaluation of a small molecule GCPII inhibitor supports this possibility [12]. The current knowledge of the role of glutamate under normal and pathologic conditions as well as the utility and development of inhibitors of GCPII able to target glutamate synthetic machinery will be reviewed in the following chapters. REFERENCES [1] Low, S. J.; Roland, C. L., Review of NMDA antagonist-induced neurotoxicity and implications for clinical development. Int J Clin Pharmacol Ther 2004, 42 (1), 1-14. [2] Javitt, D. C.; Schoepp, D.; Kalivas, P. W.; Volkow, N. D.; Zarate, C.; Merchant, K.; Bear, M. F.; Umbricht, D.; Hajos, M.; Potter, W. Z.; Lee, C. M., Translating glutamate: from pathophysiology to treatment. Sci Transl Med 3 (102), 102mr2. [3] Gordon, P. H., Amyotrophic lateral sclerosis: pathophysiology, diagnosis and management. CNS Drugs 25 (1), 1-15. [4] Syed, T. U.; Sajatovic, M., Extended-release lamotrigine in the treatment of patients with epilepsy. Expert Opin Pharmacother 11 (9), 1579-85. [5] Slusher, B. S.; Robinson, M. B.; Tsai, G.; Simmons, M. L.; Richards, S. S.; Coyle, J. T., Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization. J Biol Chem 1990, 265 (34), 21297-301. [6] Wroblewska, B.; Wroblewski, J. T.; Pshenichkin, S.; Surin, A.; Sullivan, S. E.; Neale, J. H., N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells. J Neurochem 1997, 69 (1), 174-81. [7] Thomas, A. G.; Liu, W.; Olkowski, J. L.; Tang, Z.; Lin, Q.; Lu, X. C.; Slusher, B. S., Neuroprotection mediated by glutamate carboxypeptidase II (NAALADase) inhibition requires TGF-beta. Eur J Pharmacol 2001, 430 (1), 33-40. [8] Tsukamoto, T.; Wozniak, K. M.; Slusher, B. S., Progress in the discovery and development of glutamate carboxypeptidase II inhibitors. Drug Discov Today 2007, 12 (17- 18), 767-76. [9] Zhou, J.; Neale, J. H.; Pomper, M. G.; Kozikowski, A. P., NAAG peptidase inhibitors and their potential for diagnosis and therapy. Nat Rev Drug Discov 2005, 4 (12), 1015-26. [10] Barinka, C.; Rojas, C.; Slusher, B.; Pomper, M., Glutamate Carboxypeptidase II in Diagnosis and Treatment of Neurologic Disorders and Prostate Cancer. Curr Med Chem. 2012, Jan 2 [Epub ahead of publication] [11] Slusher, B. S.; Vornov, J. J.; Thomas, A. G.; Hurn, P. D.; Harukuni, I.; Bhardwaj, A.; Traystman, R. J.; Robinson, M. B.; Britton, P.; Lu, X. C.; Tortella, F. C.; Wozniak, K. M.; Yudkoff, M.; Potter, B. M.; Jackson, P. F., Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury. Nat Med 1999, 5 (12), 1396-402. [12] van der Post, J. P.; de Visser, S. J.; de Kam, M. L.; Woelfler, M.; Hilt, D. C.; Vornov, J.; Burak, E. S.; Bortey, E.; Slusher, B. S.; Limsakun, T.; Cohen, A. F.; van Gerven, J. M., The central nervous system effects, pharmacokinetics and safety of the NAALADase-inhibitor GPI 5693. Br J Clin Pharmacol 2005, 60 (2), 128-36.

Glutamate has been implicated in the pathogenesis of several diseases on the central nervous system, but recent studies have also suggested that it can be involved also in the onset and course of peripheral neuropathies. Given the increasing evidence of this possibility, several attempts have been performed in order to modulate its activity. Among them, glutamate carboxypeptidase II (GCP II) inhibition demonstrated promising results in different models of peripheral nerve damage, including diabetic and toxic neuropathies

Glutamate is one of the major neurotrasmitters in mammalian brain and changes in its concentration have been associated with a number of neurological disorders, including neurodegenerative, cerebrovascular diseases and epilepsy. Moreover, recently a possible role for glutamatergic system dysfunction has been suggested also in the peripheral nervous system. This chapter will revise the current knowledge in the distribution of glutamate and of its receptors and transporters in the central nervous system.

Glutamate carboxypeptidase II, also known as prostate specific membrane antigen or folate hydrolase I, is a type II transmembrane 750 amino acid membrane-bound glycoprotein, with a molecular weight in the human form of approximately 100 kDa and a demonstrated metallopeptidase activity. At the synaptic level it hydrolyzes N-acetylaspartylglutamate to N-acetyl-aspartate and glutamate. Its localization in the animal and human nervous system has only recently been clearly established, since many of the older studies gave conflicting results, likely due to the use of poorly characterized antibodies lacking epitope mapping and proper controls (i.e. immunohistochemistry complemented by western blot analysis and enzyme activity determination). In this chapter, we will review the available literature describing the animal and human distribution of glutamate carboxypeptidase in the central and peripheral nervous system.

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a zinc metallopeptidase that hydrolyzes N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate in the nervous system. Inhibition of GCPII has the potential to reduce extracellular glutamate and represents an opportune target for treating neurological disorders in which excess glutamate is considered pathogenic. Furthermore, GCPII was found to be identical to a tumor marker, prostate-specific membrane antigen (PSMA), and has drawn significant interest as a diagnostic and/or therapeutic target in oncology. Over the past 15 years, tremendous efforts have been made in the discovery of potent GCPII inhibitors, particularly those with phosphorus-, urea- and thiol-based zinc binding groups. In addition, significant progress has been made in understanding the three-dimensional structural characteristics of GCPII in complex with various ligands. The purpose of this review article is to analyze the structure-activity relationships (SAR) of GCPII inhibitors reported to date, which are classified on the basis of their zinc-binding group. SAR and crystallographic data are evaluated in detail for each of these series to highlight the future challenges and opportunities to identify clinically viable GCPII inhibitors.

Glutamate and Multiple Sclerosis by M. Frigo (1295-1299).
Multiple sclerosis (MS) has been considered for a long time a typical inflammatory demyelinating disease of the central nervous system due to autoimmunity targeting oligodendrocytes with sparing of axons until advanced stages of the disease. For this reason, most of the earliest experimental studies focused on the role of cytokines and chemokines at the site of oligodendrocytes loss and on the importance in MS pathogenesis of classical inflammatory mechanisms. As a result, several attempts to treat MS through reduction of the local inflammatory milieau have been performed, leading to the current “immunomodulatory” treatment of the disease. However, more recently the importance of axonal loss and neurodegeneration even in the earliest stages of MS has been also recognized, and additional or concomitant players have been therefore searched. Evidence is now increasing that excessive glutamate is released at the site of demyelination and axonal degeneration in MS plaques, and the most probable candidates for this cellular release are infiltrating leukocytes and activated microglia. These observations are no longer simply preclinical results obtained in the MS animal model, i.e. experimental allergic encephalomyelitis, but have already been partially confirmed by post-mortem studies and in vivo analysis in MS patients, thus raising the possibility that modulation of glutamate release and transport as well as receptors blockade might be relevant targets for the development of future therapeutic interventions.

Recent years witnessed rapid expansion of our knowledge about structural features of human glutamate carboxypeptidase II (GCPII). There are over thirty X-ray structures of human GCPII (and of its close ortholog GCPIII) publicly available at present. They include structures of ligand-free wild-type enzymes, complexes of wild-type GCPII/GCPIII with structurally diversified inhibitors as well as complexes of the GCPII(E424A) inactive mutant with several substrates. Combined structural data were instrumental for elucidating the catalytic mechanism of the enzyme. Furthermore the detailed knowledge of the GCPII architecture and protein-inhibitor interactions offers mechanistic insight into structure-activity relationship studies and can be exploited for the rational design of novel GCPII-specific compounds. This review presents a summary of structural information that has been gleaned since 2005, when the first GCPII structures were solved.

Glutamate is the major mediator of excitatory signaling in the mammalian central nervous system (CNS) and it has recently been described to have a central role in the transduction of sensory input in the peripheral nervous system (PNS), too. However, functional glutamatergic systems are expressed by peripheral non-neural tissues as well, such as heart, kidney, lungs, ovary, testis, blood and skin. Interestingly, glutamatergic alterations have been repeatedly described in these tissues in various neuropsychiatric diseases. Here we will review evidence suggesting that glutamate measurements obtained from sampling ex vivo peripheral cells can permit the assessment of the dynamics of glutamate release, uptake, receptor-mediated signaling, synthesis and degradation, and mirror homologous dysfunctions operative within the CNS in each single patient. Among all the available cell types we will focus on leukocytes, platelets and fibroblasts that can be easily obtained from patients multiple times without concerns related to post-mortem changes. Finally, we will briefly review another possibility, offered by testing plasma (and CSF) glutamate levels, allowing the indirect investigation of glutamatemediated crosstalk between central and peripheral compartments. Technical pitfalls of these biomarkers will be contextually emphasized.

GCPII Variants, Paralogs and Orthologs by K. Hlouchova (1316-1322).
Glutamate carboxypeptidase II (GCPII) and its splice variants, paralogs and human homologs represent a family of proteins with diverse tissue distribution, cellular localization and largely unknown function which have been explored only recently. While GCPII itself has been thoroughly studied from different perspectives, as clearly documented in this series of reviews, very little is known about other members of its family, even though they might be biologically relevant. Differential expression of individual GCPII splice variants is associated with tumor progression and prognosis of prostate cancer. The best studied GCPII homolog, GCPIII or NAALADase II, may be a valid pharmaceutical target for itself since it may compensate for a lack of normal GCPII enzymatic activity. Detailed molecular characterization of this family of proteins is thus very important not only with respect to the potential therapeutic use of GCPII inhibitors, but also for better understanding of the biological role of GCPII within as well as outside the nervous system.

Glutamate is the predominant excitatory neurotransmitter used by primary afferent synapses and neurons in the spinal cord dorsal horn. Glutamate and glutamate receptors are also located in areas of the brain, spinal cord and periphery that are involved in pain sensation and transmission. Not surprisingly, glutamate receptors have been an attractive target for new pain therapies. However, their widespread distribution and array of function has often resulted in drugs targeting these sites having undesirable side-effects. This chapter will review, in general terms, the current knowledge of glutamate and its effects at various glutamate receptors with regards to nociception. In addition, we will briefly review the glutamatergic drugs currently in use as treatments for pain, as well as known novel candidates in various stages of clinical trial. Lastly, we will summarize the data supporting a novel target for pain intervention by way of GCPII inhibition, which appears devoid of the side-effects associated with direct glutamate receptor antagonists and thus holds major promise for future therapy. GCPII (glutamate carboxypeptidase II) cleaves the prevalent neuropeptide NAAG into NAA and glutamate and there is widespread evidence and belief that targeting the glutamate derived from this enzymatic action may be a promising therapeutic route.

Glutamate, first identified in 1866, is the primary excitatory neurotransmitter in the brain. While it is critically important in many highly regulated cortical functions such as learning and memory, glutamate can be much like the magic the Sorcerer's Apprentice used in Goethe's poem: when conjured under unregulated conditions glutamate can get quickly out of control and lead to deleterious consequences. Two broad types of glutamate receptors, the ionotropic and metabotropic, facilitate glutamatergic neurotransmission in the CNS and play key roles in regulating cognitive function. Excessive activation of these receptors leads to excitotoxicity, especially in brain regions that are developmentally and regionally vulnerable to this kind of injury. Dysregulation of glutamate signaling leads to neurodegeneration that plays a role in a number of neuropsychiatric diseases, prompting the development and utilization of novel strategies to balance the beneficial and deleterious potential of this important neurotransmitter. Inhibition of the enzyme glutamate carboxypeptidase II (GCPII) is one method of manipulating glutamate neurotransmission. Positive outcomes (decreased neuronal loss, improved cognition) have been demonstrated in preclinical models of ALS, stroke, and Multiple Sclerosis due to inhibition of GCPII, suggesting this method of glutamate regulation could serve as a therapeutic means for treating neurodegeneration and cognitive impairment.

GCPII Imaging and Cancer by C. A. Foss (1346-1359).
Glutamate carboxypeptidase II (GCPII) in the central nervous system is referred to as the prostate-specific membrane antigen (PSMA) in the periphery. PSMA serves as a target for imaging and treatment of prostate cancer and because of its expression in solid tumor neovasculature has the potential to be used in this regard for other malignancies as well. An overview of GCPII/PSMA in cancer, as well as a discussion of imaging and therapy of prostate cancer using a wide variety of PSMA-targeting agents is provided.

NAAG, NMDA Receptor and Psychosis by Richard Bergeron (1360-1364).
At central synapses, glutamate is the main excitatory neurotransmitter. Once released from presynaptic terminals, glutamate activates a number of different glutamatergic receptors one of which is the ligand gated ionophore glutamatergic subtype N-methyl-Daspartate receptors (NMDARs). NMDARs play a crucial role in controlling various determinants of synaptic function. Nacetylaspartylglutamate (NAAG) is the most prevalent peptide transmitter in the mammalian central nervous system. NAAG is released upon neuronal depolarization by a calcium-dependent process from glutamatergic and GABAergic neurons. It is cleaved by a specific peptidase located on astrocytes, glutamate carboxypeptidase type II (GCP-II), to N-acetylaspartate (NAA) and glutamate. Current evidence supports the hypothesis that NAAG is an endogenous agonist at G protein coupled mGluR3 receptors and an antagonist at NMDAR. In several disorders and animal models of human diseases, the levels of NAAG and the activity of GCP-II are altered in ways that are consistent with NAAG's role in regulation of glutamatergic neurotransmission. Several lines of evidence suggest that a dysfunction in glutamatergic via the NMDAR might be involved in schizophrenia. This hypothesis has evolved from findings that NMDAR antagonists such as phencyclidine (PCP or “angel dust”), produces a syndrome in normal individuals that closely resembles schizophrenia and exacerbates psychotic symptoms in patients with chronic schizophrenia. Recent postmortem, metabolic and genetic studies have provided evidence that hypofunction of discrete populations of NMDAR can contribute to the symptoms of schizophrenia, at least in some patients. The review outlines the role of endogenous NAAG at NMDAR neurotransmission and its putative role in the pathophysiology of schizophrenia.

Toll-like receptors 7 and 8 (TLR7/8), known as pattern recognition receptors (PRR), are currently viewed as important targets for the development of new therapies for multiple diseases. Therefore, manipulating the immune response by using TLR7/8 agonists or antagonists might be of therapeutic value. Nucleic acid-like structures are well-known TLR7/8 ligands, such as single-stranded RNA (ssRNA), small interfering RNA (siRNA), CpG-oligodeoxynucleotides (ODNs) and nucleoside analogues. However, these nucleic acid TLR7/8 ligands show a variety of pharmacological properties and change of their structures offers a high degree of diversity. Unnatural modified nucleosides have been explored to expand the properties and the applications of nucleic acid. In this regard, chemical modification of nucleosides is very useful for production of specific pharmacological qualities of nucleic acid TLR7/8 ligands. In this review, we will summarize the characteristics of nucleic acid TLR7/8 ligand system and describe the applications of chemical modifications, with a focus on potency and structure-activity relationships (SAR).

Azole antimycotics are a well-known and important class of agents that are used in hospital practice, everyday health care, veterinary medicine and for crop protection. The era of azole fungicides began with the breakthrough of chlormidazole roughly 50 years ago. Since then, more than 20 drugs of this group, including triazoles, have been brought to the market. The specific chemical structure and mechanism of the action of azoles along with the eukaryotic character of fungal pathogens raise several serious issues. Resistance to drugs and disturbance to metabolic pathways are among the most important. On the other hand, these same features are responsible for unique and novel applications of these drugs. As a result, old and ineffective antifungal drugs can be successfully used in the treatment of parasitic diseases, bacterial infections or cancers. Are azoles getting their second wind?

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver function and correlates with central adiposity, obesity, insulin resistance, the metabolic syndrome and type 2 diabetes mellitus. The pathological spectrum of NAFLD ranges from fatty liver to non-alcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis, and even hepatocellular carcinoma. Though NAFLD and NASH are becoming a major public health problem, ethical constraints on obtaining human liver tissue limit the interpretability of the data and the ability to delineate cause and effect from complex, interactive disease pathogenic pathways. Animal models of NASH can provide critical information leading to identify potential drug targets and to understand their molecular mechanisms, and are platforms for compound screening in drug development and for the assessment of novel therapeutic strategies. This review is aimed to offer an updated overview of the nutritional, genetic and pharmacologic animal models of NASH. Though the information derived from these models has clear relevance for the comprehension of the molecular basis of human disease, most of them fail to reproduce the full spectrum of liver pathology and the metabolic context that characterizes human NASH. Consequently, it is necessary to establish animal models that can best mimic the actual etiology, progression, and pathogenesis of the disease, and prove effectiveness for examining and selecting compounds with potential therapeutic benefit in NASH.

The slow delayed rectifier current (IKs) is the slow component of cardiac delayed rectifier current and is critical for the late phase repolarization of cardiac action potential. This current is also an important target for Sympathetic Nervous System (SNS) to regulate the cardiac electivity to accommodate to heart rate alterations in response to exercise or emotional stress and can be up-regulated by β- adrenergic or other signal molecules. IKs channel is originated by the co-assembly of pore-forming KCNQ1 α-subunit and accessory KCNE1 β-subunit. Mutations in any subunit can bring about severe long QT syndrome (LQT-1, LQT-5) as characterized by deliquium, seizures and sudden death. This review summarizes the normal physiological functions and molecular basis of IKs channels, as well as illustrates up-to-date development on its blockers and activators. Therefore, the current extensive survey should generate fundamental understanding of the role of IKs channel in modulating cardiac function and donate some instructions to the progression of IKs blockers and activators as potential antiarrhythmic agents or pharmacological tools to determine the physiological and pathological function of IKs.