International Journal of Neuroscience, 2014; 124(12): 867–873 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2014.890935
REVIEW
Blockade of N-acetylaspartylglutamate peptidases: a novel protective strategy for brain injuries and neurological disorders Chunlong Zhong, Qizhong Luo, and Jiyao Jiang Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress glutamate release mainly through selective activation of presynaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3). Therefore, strategies of inhibition of NAAG peptidases and subsequent NAAG hydrolysis to elevate levels of NAAG could reduce glutamate release under pathological conditions and be neuroprotective by attenuating excitotoxic cell injury. A series of potent inhibitors of NAAG peptidases has been synthesized and demonstrated efficacy in experimental models of ischemic–hypoxic brain injury, traumatic brain injury, inflammatory pain, diabetic neuropathy, amyotrophic lateral sclerosis and phencyclidine-induced schizophrenia-like behaviors. The excessive glutamatergic transmission has been implicated in all of these neurological disorders. Thus, blockade of NAAG peptidases may augment an endogenous protective mechanism and afford neuroprotection in the brain. This review aims to summarize and provide insight into the current understanding of the novel neuroprotective strategy based on limiting glutamate excitotoxicity for a wide variety of brain injuries and neurological disorders. KEYWORDS: N-acetylaspartylglutamate (NAAG), glutamate excitotoxicity, brain injury, neurological disorder, neuroprotection
Introduction The search for novel neuroprotective strategies for brain injuries and neurological disorders attracts great interest because of the absence of truly approved therapies. Glutamate is the primary excitatory neurotransmitter in the mammalian nervous system and plays important roles in a variety of physiological functions [1]. Additionally, glutamate-receptor-mediated cell injury is thought to be an important mechanism of secondary neuronal damage. Excessive glutamatergic transmission immediately after a primary pathological insult can damage or kill neurons and therefore these have been implicated in a number of neurological disorders, such as ischemic–hypoxic brain injury, traumatic brain injury, inflammatory pain, diabetic neuropathy, amyotrophic lateral sclerosis and phencyclidine-induced schizophrenia-like behaviors [2–14]. Although the role Received 6 January 2014; revised 31 January 2014; accepted 31 January 2014 Correspondence: Chunlong Zhong, M.D., Ph.D., Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, China. Tel: +86 21 68383626. Fax: +86 21 58394262. E-mail: [email protected]
of glutamate excitotoxicity under these pathological conditions is well established and several drugs have been designed to attenuate the pathological consequences of this excess, translation of this strategy into clinical application remains overwhelmingly disappointing [15,16]. As an alternative strategy, inhibiting the hydrolysis of N-acetylaspartylglutamate (NAAG) may augment an endogenous protective mechanism in the brain. This inhibition enhances the beneficial effects of a reduction of glutamate release under pathological conditions and therefore represents a novel therapeutic approach to secondary neuronal damage from brain injuries and neurological disorders [17–21].
NAAG and NAAG peptidases NAAG is an abundant peptide neurotransmitter found in millimolar concentrations in the mammalian central nervous system and serves as a co-transmitter with several small amine transmitters, including glutamate, GABA and acetylcholine [17,22,23]. Following its synaptic release, NAAG activates the metabotropic 867
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glutamate receptors (mGluRs) on neurons and glial cells [24–26]. mGluRs have been classified into three groups and eight subtypes according to their second-messenger association, sequence homology and agonist selectivity [27,28]. NAAG selectively activates the Group II metabotropic glutamate receptor subtype 3 (mGluR3), with approximately 10-fold less efficacy at mGluR2 [25,29]. Acting at the presynaptic mGluR3, NAAG has been shown to reduce cAMP levels, decreasing voltageregulated calcium conductance, which is important in the reduction of the synaptic release of glutamate and GABA, thus acting as a negative feedback loop [30–33]. Such an action of NAAG would enable the neuronal circuit to remain functional over a wide range of activities without being overwhelmed by high levels of excitatory input. Synaptically released NAAG is rapidly hydrolyzed to form glutamate and N-acetylaspartate (NAA) by at least two catalytic zinc metalloproteases, glutamate carboxypeptidase II and III (GCPII and III), which are also known as NAALADases or NAAG peptidases [34,35]. Although GCPII appears to be expressed exclusively on the surface of glia, GCPIII is expressed at a higher level in cerebellar and cerebral cortical neurons than in astrocytes, suggesting that they have different sites of action [35]. In comparison to GCPII, GCPIII has lower NAAG-hydrolyzing activity and is also effectively inhibited by several known inhibitors of GCPII [36]. GCP III represents less than 2% of the total brain NAAG peptidase activity, indicating that GCP II is primarily responsible for the inactivation of released NAAG in the CNS [35–37]. However, knockout mice, in which the gene encoding GCPII has been deleted, exhibit normal neurological function and behavior, suggesting that blockade of NAAG peptidase is of limited behavioral consequence under unstressed conditions [20,37].
NAAG peptidase inhibitors During the past decade, a series of potent inhibitors of NAAG peptidases has been synthesized and characterized mainly by two research groups [38–41]. Phosphonate- and thiol-based inhibitors The first potent inhibitor of GCPII, 2-(phosphonomethyl)pentanedioic acid (2-PMPA), is a phosphonate analogue of glutamate and was identified by Jackson’s group in 1996 [38]. 2-PMPA has also shown exquisite selectivity for GCPII and GCPIII with characteristics of low molecular weight and high aqueous solubility/stability, which permits 2-PMPA to serve as a pharmacological tool to study the mechanism and physiological role of NAAG peptidases [36,37,42]. While these studies have provided a compelling therapeutic rationale for NAAG peptidase inhibition, the poor pharmacoki-
netic profile of 2-PMPA, due to the highly polar nature of the molecule, limited its practical value as a therapeutic drug. Subsequently, extensive structure–activity relationship (SAR) studies were conducted using 2PMPA as a template, and its phosphinate derivatives such as GPI5232, VA-033 and the phenylalkylphosphonamidates were designed and synthesized [43–46]. It was assumed that the highly polar phosphonate and phosphinate groups were the primary cause of poor oral bioavailability of this kind of GCP II inhibitors. This assumption prompted the researchers to reduce the polarity of the compounds by substituting the phosphorous group with the less polar thiol group. These efforts led to the discovery of the thiol-based 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, also known as GPI5693). As one of the second-generation NAAG peptidases inhibitors, 2-MPPA was the first reported orally available GCP II inhibitor and the first inhibitor that has been tested in humans [39,47]. Even though 2-MPPA did not provoke any adverse CNS effects and was well-tolerated at plasma concentrations, no additional studies were carried out because of the relatively low potency of 2-MPPA together with concerns over potential immune reactivity common to thiol-containing drugs [21]. Recently, a series of 3-(2mercaptoethyl)-1H-indole-2-carboxylic acid derivatives was reported as potent NAAG peptidase inhibitors [48]. The substituents on the indole nitrogen seem to have a significant influence on the activity of the ligands.
Urea-based inhibitors This class of compounds was designed and developed by Kozikowski’s group from the NAAG-based mimic 4,4 -phosphinicobis(butane-1,3-dicarboxylic acid), i.e. PBDA, which was found to act both as a potent NAAG peptidase inhibitor and as an mGluR3 selective agonist [49]. Although structural modifications were performed for the acidic portions of 2-PMPA to seek more lipophilic compounds, these changes resulted in a more than a 1000-fold decrease in inhibitory activity [50]. On the basis of the GCPII, 2-PMPA and PBDA model, new structures were synthesized in which two amino acids are joined through their NH2 groups by a urea linkage [40]. Based on (S)-glutamate-C(O)-(S)-glutamate as the new lead, a number of more potent NAAG peptidase inhibitors, such as ZJ-43, ZJ-11, ZJ-17 and ZJ-38, have been discovered [51]. Unfortunately, while ureabased compounds are highly potent inhibitors of the enzyme, they exhibit very low bioavailability following oral administration and have only limited ability to penetrate the blood–brain barrier (BBB) due to their strong hydrophilicity. As a result, robust efforts are being carried out in several laboratories to improve on bioavailability of novel GCPII inhibitors [52–54]. International Journal of Neuroscience
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Most recently, the crystal structures of GCP II and III have been examined and their pharmacophore pockets compared [55,56]. The differences associated with their active sites appear important in their interactions with peptidase inhibitors and should provide a further important basis for the rational structure-based design of new GCP II inhibitors [52,57].
Glutamate excitotoxicity models rescued by NAAG peptidase inhibition Ischemic–hypoxic brain injury Excitotoxic glutamatergic transmission is believed to underlie the mechanism of ischemic brain injury [3]. Glutamate-mediated activation of NMDA receptors and the consequent increase of the intracellular Ca2+ levels are major factors in neuronal death in both the area of insult and the penumbra of nervous tissue surrounding the injury [4]. A series of pioneering studies has demonstrated the ability of NAAG and NAAG peptidase inhibitors to significantly mitigate the influence of glutamate release under ischemic–hypoxic conditions and are shown to be neuroprotective [42,58,59]. For example, pre-stroke administration of two NAAG peptidase inhibitors, 2-PMPA and GPI5232, decreased extracellular glutamate, reduced lesion volume and improved behavioral performance following transient middle cerebral artery occlusion (MCAO) in rats [42,59]. Continuous infusion of GPI5232 was effective even when administered 2 h after the ischemic injury. Furthermore, GCPII knockout mice exhibited a significantly smaller infarct volume than control littermates in this stroke model [60]. Long-term neurological deficits and death can result from perinatal hypoxia, a condition that is also considered to be the consequence of excessive activation of glutamate receptors. In an in vivo model of neonatal cerebral hypoxia, NAAG itself was found to be neuroprotective, and this effect was blocked by a Group II mGluR antagonist LY341495. 2-PMPA was also effective in reducing excitotoxicity in this neonatal model [61]. Traumatic brain injury (TBI) TBI produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death [5,6]. In a standardized fluid-percussive TBI model, systemic administration of ZJ-43 significantly reduced neuronal degeneration in the CA3 region of the hippocampus 24 h after TBI. More importantly, it also significantly reduced astrocyte death in the hippocampus, a result that has not been obtained with existing therapeutic approaches to TBI [62]. In addition, recent in vivo microdialysis revealed that a single intraperitoneal injection of ZJ-43 significantly reduces the TBI-induced rise in dialysate glutamate levels and sustains increased levels C
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of NAAG over a longer period of time [63]. Moreover, the Group II mGluR selective antagonist, LY341495, successfully abolished the protective effects of ZJ-43, which is consistent with the conclusion that protection was mediated by activation of Group II mGluRs via increased levels of extracellular NAAG [62,63]. These data support the hypothesis that inhibition of NAAG peptidases may decrease the magnitude and duration of excitotoxic events associated with TBI and represent a new potential therapeutic approach to TBI treatment. Inflammatory pain Glutamate is the primary transmitter used by spinal sensory neurons to convey pain perception from their sensory endings in peripheral tissue to their synaptic endings in the spinal cord [7,8]. As NAAG peptidase inhibition reduces glutamate release generated from NAAG, it might have an analgesic effect on pain conditions. Almost all of the defined potent NAAG peptidase inhibitors (phosphonate-based 2-PMPA, and phosphinate-based GPI5232, thiol-based 2-MPPA and urea-based ZJ-11, ZJ-17 and ZJ-43) have been confirmed to reduce the perception of inflammatory pain in rat models [64–68]. The analgesic efficacy of NAAG peptidase inhibition in these chronic pain models was completely blocked by co-administration of the Group II mGluR antagonist, LY-341495 [68]. These data strongly support the hypothesis that the analgesic effect may be mediated by inhibition of ectopic afferent discharges through decreased glutamate accumulation and increased NAAG at the site of nerve injury via the activation of Group II mGluRs [66]. Moreover, the thiol-based inhibitor 2-MPPA is efficacious in an animal model of peripheral neuropathy by oral administration, offering an approach to the pain management that will probably meet with improved patient compliance [39]. Diabetic neuropathy Although the precise etiology of diabetic neuropathy remains unclear, neuronal ischemia elicited by hyperglycemia is believed to be a prominent mechanism. Ischemia results in perturbed mitochondrial function, which might be mediated by excitotoxicity resulting from excessive glutamate release [10]. Chronic injection with GPI5232 decreased hyperalgesia, atrophy, degeneration and conductance changes that are associated with hyperglycemia in sensory axons in the type 1 diabetic BB/Wor rats [69]. In sensory neuronal cultures, 2-PMPA and NAAG reduced neuron apoptosis induced by elevated glucose levels and promoted neurite growth that had been inhibited by high glucose. The role of NAAG peptidase inhibition in this model was demonstrated to be mediated through mGlu3, most likely by increasing the extracellular NAAG concentration [9]. Moreover, several reports showed that the
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neuroprotective effects of 2-PMPA in the sensory neuron cultures were dependent on the presence of glial cells and mGlu3 activation in glial cells by NAAGreduced neuronal death owing to glial secretion of TGFβ [[9,70,71]. The NAAG-induced regulation of glial cell functions seems to play an important role in the neuroprotective actions of NAAG peptidase inhibition. Amyotrophic lateral sclerosis (ALS) Glutamate excitotoxicity is supposed to be a pathogenic mechanism in the death of spinal motor neurons in sporadic and familial ALS [11,12]. Transgenic mice that express a mutant form of the superoxide dismutase 1(SOD1) gene are considered to be a model for human familial ALS because these mice undergo a progressive loss of motor neurons that parallels neuron loss in patients with ALS [71]. Long-term oral administration of the NAAG peptidase inhibitor 2-MPPA to the transgenic mice demonstrated dramatic neuroprotection and significantly delayed the onset of neurological symptoms and motor-neuron loss, while prolonging the survival of these mice [72]. 2-PMPA also reduced motor-neuron death in spinal cord cells that were transfected with SOD1 in culture [73]. These data suggest that NAAG peptidase inhibition might represent an attractive target for the treatment of ALS [73]. Schizophrenia Although dysfunctional dopamine-mediated neurotransmission has long been considered a central element in schizophrenia, a growing body of evidence suggests that dysfunctional glutamate-mediated neurotransmission plays an important role in the development of this disorder [13,14]. The observation that animals injected with phencyclidine (PCP), an open-channel blocker of the NMDA receptor, exhibit positive (motor activity and stereotypies), negative (social withdrawal) and cognitive (paired-pulse facilitation) schizophrenia-like behaviors, which is a widely accepted model for the study of schizophrenia, gives further support to the “glutamate theory” of schizophrenia [74,75]. The report that some PCP-induced symptoms are reduced by treatment with Group II mGluR agonists provided a major impetus to test the hypothesis that increasing the synaptic NAAG level by the inhibition of NAAG peptidase activity would similarly moderate the schizophrenialike symptoms evoked by PCP [76,77]. Recent results demonstrated that the urea-based inhibitor ZJ-43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior and head movements. These effects were shown to be mediated by an increased activation of Group II mGluRs [78,79]. These data indicate that NAAG peptidase inhibitors could represent a new therapeutic approach to modulating the schizophrenia-like symptoms evoked by PCP.
The possible protective mechanisms of NAAG peptidase inhibition Excess glutamate release has been implicated in each of the aforementioned models of human pathological conditions. These applications have supported the contention that NAAG peptidase is a significant therapeutic target. The strategy of inhibiting NAAG peptidase and subsequent NAAG hydrolysis seems to afford protection from secondary brain pathology in several ways [63]. First, NAAG acts as a potent agonist of the presynaptic mGluR3, which reduces presynaptic glutamate release from neurons. Thus, inhibition of NAAG peptidase activity resulting in the prolonged presence of endogenous NAAG could provide protection through prolonged activation of presynaptic mGluR3 and subsequent reduction in the damaging glutamate release [18]. The present findings showing that the selective Group II mGluR antagonist, LY341495, was similarly effective in blocking the therapeutic effects of NAAG peptidase inhibitors in animal models of traumatic brain injury, pain and schizophrenia strongly suggest that the protection afforded by the strategy of inhibiting NAAG peptidase is mainly mediated by the activation of Group II mGluRs [62,68,78]. Second, as glutamate is one of the byproducts of the hydrolysis of NAAG, blockade of NAAG peptidases could have neuroprotective effects by reducing a secondary source of excessive glutamate [41]. However, it seems not to be the primary therapeutic effect, as the NAAG levels are up to 10-fold lower than the glutamate levels and thus possibly not responsible for the changes in the glutamate levels [23]. Third, astrocytes also express Group II mGluRs [26,80,81]. Selective activation of Group II mGluRs on astrocytes stimulates the release of transforming growth factorβ, which acts as a neuroprotective agent [70,82]. Moreover, activation of Group II mGluRs on astrocytes also positively modulates the expression of glutamate transporters GLAST and GLT1, thereby increasing the ability of astrocytes to remove glutamate from the synapse [81]. Finally, inhibition of NAAG peptidases would also decrease a synaptic source of NAA, which is transported into astrocytes along with sodium. Thus, reduced transport of NAA would likely reduce the sodium load on astrocytes and limit potential mechanisms of astrocyte edema [62]. Therefore, NAAG peptidase inhibitors could have the potential to provide protection for a variety of neurological diseases in which glutamate excitotoxicity is a significant determinant [21,83].
Conclusion and perspectives Although most of the currently identified NAAG peptidase inhibitors are highly polar compounds with limited ability to cross the BBB and do not appear to be International Journal of Neuroscience
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sufficiently bioavailable to be used as drugs in humans under conditions where the BBB is intact, it is important to note that the efficacy of the urea-based NAAG peptidase inhibitor ZJ-43 in the TBI model suggests that there might be sufficient BBB breakdown at the site of the trauma to permit entry of this inhibitor [19,62,63]. To bring these NAAG peptidase inhibitors to the marketplace, it will be equally important to optimize their pharmacokinetic profiles [19]. As reported previously, this new approach to modulate glutamate levels via NAAG peptidase inhibition does not affect basal glutamate but selectively decreases the excitotoxic rise in extracellular glutamate following a pathological insult [42,84]. Several studies finding that GCPII knockout mice exhibit normal neurological function and behavior strengthen the potential of NAAG peptidase inhibition to serve as a therapeutic strategy, and this might act without the known side-effects associated with conventional glutamate receptor antagonists[37,60]. Data from the first clinical evaluation of the NAAG peptidase inhibitor GPI 5693 for the treatment of neuropathic pain supports this possibility [47]. In summary, blockade of NAAG peptidases may represent a new therapeutic approach to secondary neuronal damage from brain injuries and neurological disorders. In addition, NAAG peptidase inhibitors should prove to be important tools in understanding the physiological role of NAAG in the brain, perhaps deepening our understanding of glutamate neurotransmission [21,63,85,86].
Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. This research was supported in part by a basic research program of project 973 by the Ministry of Science and Technology of China (2011CB013304) and by grants from the National Natural Science Foundation of China (Nos. 30500525, 30670719 and 81070990), the Shanghai Science and Technology Commission (Nos. 10QH1401700, 09140901300 and 05QMX1428) and the Shanghai Municipal Education Commission (No. 09SG20). The authors would also like to express their heartfelt thanks to Professor Bruce G. Lyeth in the Department of Surgical Neurology, University of California at Davis, for his professional revisions and valuable editorial comments.
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REVIEW
Blockade of N-acetylaspartylglutamate peptidases: a novel protective strategy for brain injuries and neurological disorders Chunlong Zhong, Qizhong Luo, and Jiyao Jiang Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress glutamate release mainly through selective activation of presynaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3). Therefore, strategies of inhibition of NAAG peptidases and subsequent NAAG hydrolysis to elevate levels of NAAG could reduce glutamate release under pathological conditions and be neuroprotective by attenuating excitotoxic cell injury. A series of potent inhibitors of NAAG peptidases has been synthesized and demonstrated efficacy in experimental models of ischemic–hypoxic brain injury, traumatic brain injury, inflammatory pain, diabetic neuropathy, amyotrophic lateral sclerosis and phencyclidine-induced schizophrenia-like behaviors. The excessive glutamatergic transmission has been implicated in all of these neurological disorders. Thus, blockade of NAAG peptidases may augment an endogenous protective mechanism and afford neuroprotection in the brain. This review aims to summarize and provide insight into the current understanding of the novel neuroprotective strategy based on limiting glutamate excitotoxicity for a wide variety of brain injuries and neurological disorders. KEYWORDS: N-acetylaspartylglutamate (NAAG), glutamate excitotoxicity, brain injury, neurological disorder, neuroprotection
Introduction The search for novel neuroprotective strategies for brain injuries and neurological disorders attracts great interest because of the absence of truly approved therapies. Glutamate is the primary excitatory neurotransmitter in the mammalian nervous system and plays important roles in a variety of physiological functions [1]. Additionally, glutamate-receptor-mediated cell injury is thought to be an important mechanism of secondary neuronal damage. Excessive glutamatergic transmission immediately after a primary pathological insult can damage or kill neurons and therefore these have been implicated in a number of neurological disorders, such as ischemic–hypoxic brain injury, traumatic brain injury, inflammatory pain, diabetic neuropathy, amyotrophic lateral sclerosis and phencyclidine-induced schizophrenia-like behaviors [2–14]. Although the role Received 6 January 2014; revised 31 January 2014; accepted 31 January 2014 Correspondence: Chunlong Zhong, M.D., Ph.D., Department of Neurosurgery, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 1630 Dongfang Road, Shanghai 200127, China. Tel: +86 21 68383626. Fax: +86 21 58394262. E-mail: [email protected]
of glutamate excitotoxicity under these pathological conditions is well established and several drugs have been designed to attenuate the pathological consequences of this excess, translation of this strategy into clinical application remains overwhelmingly disappointing [15,16]. As an alternative strategy, inhibiting the hydrolysis of N-acetylaspartylglutamate (NAAG) may augment an endogenous protective mechanism in the brain. This inhibition enhances the beneficial effects of a reduction of glutamate release under pathological conditions and therefore represents a novel therapeutic approach to secondary neuronal damage from brain injuries and neurological disorders [17–21].
NAAG and NAAG peptidases NAAG is an abundant peptide neurotransmitter found in millimolar concentrations in the mammalian central nervous system and serves as a co-transmitter with several small amine transmitters, including glutamate, GABA and acetylcholine [17,22,23]. Following its synaptic release, NAAG activates the metabotropic 867
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glutamate receptors (mGluRs) on neurons and glial cells [24–26]. mGluRs have been classified into three groups and eight subtypes according to their second-messenger association, sequence homology and agonist selectivity [27,28]. NAAG selectively activates the Group II metabotropic glutamate receptor subtype 3 (mGluR3), with approximately 10-fold less efficacy at mGluR2 [25,29]. Acting at the presynaptic mGluR3, NAAG has been shown to reduce cAMP levels, decreasing voltageregulated calcium conductance, which is important in the reduction of the synaptic release of glutamate and GABA, thus acting as a negative feedback loop [30–33]. Such an action of NAAG would enable the neuronal circuit to remain functional over a wide range of activities without being overwhelmed by high levels of excitatory input. Synaptically released NAAG is rapidly hydrolyzed to form glutamate and N-acetylaspartate (NAA) by at least two catalytic zinc metalloproteases, glutamate carboxypeptidase II and III (GCPII and III), which are also known as NAALADases or NAAG peptidases [34,35]. Although GCPII appears to be expressed exclusively on the surface of glia, GCPIII is expressed at a higher level in cerebellar and cerebral cortical neurons than in astrocytes, suggesting that they have different sites of action [35]. In comparison to GCPII, GCPIII has lower NAAG-hydrolyzing activity and is also effectively inhibited by several known inhibitors of GCPII [36]. GCP III represents less than 2% of the total brain NAAG peptidase activity, indicating that GCP II is primarily responsible for the inactivation of released NAAG in the CNS [35–37]. However, knockout mice, in which the gene encoding GCPII has been deleted, exhibit normal neurological function and behavior, suggesting that blockade of NAAG peptidase is of limited behavioral consequence under unstressed conditions [20,37].
NAAG peptidase inhibitors During the past decade, a series of potent inhibitors of NAAG peptidases has been synthesized and characterized mainly by two research groups [38–41]. Phosphonate- and thiol-based inhibitors The first potent inhibitor of GCPII, 2-(phosphonomethyl)pentanedioic acid (2-PMPA), is a phosphonate analogue of glutamate and was identified by Jackson’s group in 1996 [38]. 2-PMPA has also shown exquisite selectivity for GCPII and GCPIII with characteristics of low molecular weight and high aqueous solubility/stability, which permits 2-PMPA to serve as a pharmacological tool to study the mechanism and physiological role of NAAG peptidases [36,37,42]. While these studies have provided a compelling therapeutic rationale for NAAG peptidase inhibition, the poor pharmacoki-
netic profile of 2-PMPA, due to the highly polar nature of the molecule, limited its practical value as a therapeutic drug. Subsequently, extensive structure–activity relationship (SAR) studies were conducted using 2PMPA as a template, and its phosphinate derivatives such as GPI5232, VA-033 and the phenylalkylphosphonamidates were designed and synthesized [43–46]. It was assumed that the highly polar phosphonate and phosphinate groups were the primary cause of poor oral bioavailability of this kind of GCP II inhibitors. This assumption prompted the researchers to reduce the polarity of the compounds by substituting the phosphorous group with the less polar thiol group. These efforts led to the discovery of the thiol-based 2-(3-mercaptopropyl)pentanedioic acid (2-MPPA, also known as GPI5693). As one of the second-generation NAAG peptidases inhibitors, 2-MPPA was the first reported orally available GCP II inhibitor and the first inhibitor that has been tested in humans [39,47]. Even though 2-MPPA did not provoke any adverse CNS effects and was well-tolerated at plasma concentrations, no additional studies were carried out because of the relatively low potency of 2-MPPA together with concerns over potential immune reactivity common to thiol-containing drugs [21]. Recently, a series of 3-(2mercaptoethyl)-1H-indole-2-carboxylic acid derivatives was reported as potent NAAG peptidase inhibitors [48]. The substituents on the indole nitrogen seem to have a significant influence on the activity of the ligands.
Urea-based inhibitors This class of compounds was designed and developed by Kozikowski’s group from the NAAG-based mimic 4,4 -phosphinicobis(butane-1,3-dicarboxylic acid), i.e. PBDA, which was found to act both as a potent NAAG peptidase inhibitor and as an mGluR3 selective agonist [49]. Although structural modifications were performed for the acidic portions of 2-PMPA to seek more lipophilic compounds, these changes resulted in a more than a 1000-fold decrease in inhibitory activity [50]. On the basis of the GCPII, 2-PMPA and PBDA model, new structures were synthesized in which two amino acids are joined through their NH2 groups by a urea linkage [40]. Based on (S)-glutamate-C(O)-(S)-glutamate as the new lead, a number of more potent NAAG peptidase inhibitors, such as ZJ-43, ZJ-11, ZJ-17 and ZJ-38, have been discovered [51]. Unfortunately, while ureabased compounds are highly potent inhibitors of the enzyme, they exhibit very low bioavailability following oral administration and have only limited ability to penetrate the blood–brain barrier (BBB) due to their strong hydrophilicity. As a result, robust efforts are being carried out in several laboratories to improve on bioavailability of novel GCPII inhibitors [52–54]. International Journal of Neuroscience
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Most recently, the crystal structures of GCP II and III have been examined and their pharmacophore pockets compared [55,56]. The differences associated with their active sites appear important in their interactions with peptidase inhibitors and should provide a further important basis for the rational structure-based design of new GCP II inhibitors [52,57].
Glutamate excitotoxicity models rescued by NAAG peptidase inhibition Ischemic–hypoxic brain injury Excitotoxic glutamatergic transmission is believed to underlie the mechanism of ischemic brain injury [3]. Glutamate-mediated activation of NMDA receptors and the consequent increase of the intracellular Ca2+ levels are major factors in neuronal death in both the area of insult and the penumbra of nervous tissue surrounding the injury [4]. A series of pioneering studies has demonstrated the ability of NAAG and NAAG peptidase inhibitors to significantly mitigate the influence of glutamate release under ischemic–hypoxic conditions and are shown to be neuroprotective [42,58,59]. For example, pre-stroke administration of two NAAG peptidase inhibitors, 2-PMPA and GPI5232, decreased extracellular glutamate, reduced lesion volume and improved behavioral performance following transient middle cerebral artery occlusion (MCAO) in rats [42,59]. Continuous infusion of GPI5232 was effective even when administered 2 h after the ischemic injury. Furthermore, GCPII knockout mice exhibited a significantly smaller infarct volume than control littermates in this stroke model [60]. Long-term neurological deficits and death can result from perinatal hypoxia, a condition that is also considered to be the consequence of excessive activation of glutamate receptors. In an in vivo model of neonatal cerebral hypoxia, NAAG itself was found to be neuroprotective, and this effect was blocked by a Group II mGluR antagonist LY341495. 2-PMPA was also effective in reducing excitotoxicity in this neonatal model [61]. Traumatic brain injury (TBI) TBI produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death [5,6]. In a standardized fluid-percussive TBI model, systemic administration of ZJ-43 significantly reduced neuronal degeneration in the CA3 region of the hippocampus 24 h after TBI. More importantly, it also significantly reduced astrocyte death in the hippocampus, a result that has not been obtained with existing therapeutic approaches to TBI [62]. In addition, recent in vivo microdialysis revealed that a single intraperitoneal injection of ZJ-43 significantly reduces the TBI-induced rise in dialysate glutamate levels and sustains increased levels C
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of NAAG over a longer period of time [63]. Moreover, the Group II mGluR selective antagonist, LY341495, successfully abolished the protective effects of ZJ-43, which is consistent with the conclusion that protection was mediated by activation of Group II mGluRs via increased levels of extracellular NAAG [62,63]. These data support the hypothesis that inhibition of NAAG peptidases may decrease the magnitude and duration of excitotoxic events associated with TBI and represent a new potential therapeutic approach to TBI treatment. Inflammatory pain Glutamate is the primary transmitter used by spinal sensory neurons to convey pain perception from their sensory endings in peripheral tissue to their synaptic endings in the spinal cord [7,8]. As NAAG peptidase inhibition reduces glutamate release generated from NAAG, it might have an analgesic effect on pain conditions. Almost all of the defined potent NAAG peptidase inhibitors (phosphonate-based 2-PMPA, and phosphinate-based GPI5232, thiol-based 2-MPPA and urea-based ZJ-11, ZJ-17 and ZJ-43) have been confirmed to reduce the perception of inflammatory pain in rat models [64–68]. The analgesic efficacy of NAAG peptidase inhibition in these chronic pain models was completely blocked by co-administration of the Group II mGluR antagonist, LY-341495 [68]. These data strongly support the hypothesis that the analgesic effect may be mediated by inhibition of ectopic afferent discharges through decreased glutamate accumulation and increased NAAG at the site of nerve injury via the activation of Group II mGluRs [66]. Moreover, the thiol-based inhibitor 2-MPPA is efficacious in an animal model of peripheral neuropathy by oral administration, offering an approach to the pain management that will probably meet with improved patient compliance [39]. Diabetic neuropathy Although the precise etiology of diabetic neuropathy remains unclear, neuronal ischemia elicited by hyperglycemia is believed to be a prominent mechanism. Ischemia results in perturbed mitochondrial function, which might be mediated by excitotoxicity resulting from excessive glutamate release [10]. Chronic injection with GPI5232 decreased hyperalgesia, atrophy, degeneration and conductance changes that are associated with hyperglycemia in sensory axons in the type 1 diabetic BB/Wor rats [69]. In sensory neuronal cultures, 2-PMPA and NAAG reduced neuron apoptosis induced by elevated glucose levels and promoted neurite growth that had been inhibited by high glucose. The role of NAAG peptidase inhibition in this model was demonstrated to be mediated through mGlu3, most likely by increasing the extracellular NAAG concentration [9]. Moreover, several reports showed that the
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neuroprotective effects of 2-PMPA in the sensory neuron cultures were dependent on the presence of glial cells and mGlu3 activation in glial cells by NAAGreduced neuronal death owing to glial secretion of TGFβ [[9,70,71]. The NAAG-induced regulation of glial cell functions seems to play an important role in the neuroprotective actions of NAAG peptidase inhibition. Amyotrophic lateral sclerosis (ALS) Glutamate excitotoxicity is supposed to be a pathogenic mechanism in the death of spinal motor neurons in sporadic and familial ALS [11,12]. Transgenic mice that express a mutant form of the superoxide dismutase 1(SOD1) gene are considered to be a model for human familial ALS because these mice undergo a progressive loss of motor neurons that parallels neuron loss in patients with ALS [71]. Long-term oral administration of the NAAG peptidase inhibitor 2-MPPA to the transgenic mice demonstrated dramatic neuroprotection and significantly delayed the onset of neurological symptoms and motor-neuron loss, while prolonging the survival of these mice [72]. 2-PMPA also reduced motor-neuron death in spinal cord cells that were transfected with SOD1 in culture [73]. These data suggest that NAAG peptidase inhibition might represent an attractive target for the treatment of ALS [73]. Schizophrenia Although dysfunctional dopamine-mediated neurotransmission has long been considered a central element in schizophrenia, a growing body of evidence suggests that dysfunctional glutamate-mediated neurotransmission plays an important role in the development of this disorder [13,14]. The observation that animals injected with phencyclidine (PCP), an open-channel blocker of the NMDA receptor, exhibit positive (motor activity and stereotypies), negative (social withdrawal) and cognitive (paired-pulse facilitation) schizophrenia-like behaviors, which is a widely accepted model for the study of schizophrenia, gives further support to the “glutamate theory” of schizophrenia [74,75]. The report that some PCP-induced symptoms are reduced by treatment with Group II mGluR agonists provided a major impetus to test the hypothesis that increasing the synaptic NAAG level by the inhibition of NAAG peptidase activity would similarly moderate the schizophrenialike symptoms evoked by PCP [76,77]. Recent results demonstrated that the urea-based inhibitor ZJ-43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior and head movements. These effects were shown to be mediated by an increased activation of Group II mGluRs [78,79]. These data indicate that NAAG peptidase inhibitors could represent a new therapeutic approach to modulating the schizophrenia-like symptoms evoked by PCP.
The possible protective mechanisms of NAAG peptidase inhibition Excess glutamate release has been implicated in each of the aforementioned models of human pathological conditions. These applications have supported the contention that NAAG peptidase is a significant therapeutic target. The strategy of inhibiting NAAG peptidase and subsequent NAAG hydrolysis seems to afford protection from secondary brain pathology in several ways [63]. First, NAAG acts as a potent agonist of the presynaptic mGluR3, which reduces presynaptic glutamate release from neurons. Thus, inhibition of NAAG peptidase activity resulting in the prolonged presence of endogenous NAAG could provide protection through prolonged activation of presynaptic mGluR3 and subsequent reduction in the damaging glutamate release [18]. The present findings showing that the selective Group II mGluR antagonist, LY341495, was similarly effective in blocking the therapeutic effects of NAAG peptidase inhibitors in animal models of traumatic brain injury, pain and schizophrenia strongly suggest that the protection afforded by the strategy of inhibiting NAAG peptidase is mainly mediated by the activation of Group II mGluRs [62,68,78]. Second, as glutamate is one of the byproducts of the hydrolysis of NAAG, blockade of NAAG peptidases could have neuroprotective effects by reducing a secondary source of excessive glutamate [41]. However, it seems not to be the primary therapeutic effect, as the NAAG levels are up to 10-fold lower than the glutamate levels and thus possibly not responsible for the changes in the glutamate levels [23]. Third, astrocytes also express Group II mGluRs [26,80,81]. Selective activation of Group II mGluRs on astrocytes stimulates the release of transforming growth factorβ, which acts as a neuroprotective agent [70,82]. Moreover, activation of Group II mGluRs on astrocytes also positively modulates the expression of glutamate transporters GLAST and GLT1, thereby increasing the ability of astrocytes to remove glutamate from the synapse [81]. Finally, inhibition of NAAG peptidases would also decrease a synaptic source of NAA, which is transported into astrocytes along with sodium. Thus, reduced transport of NAA would likely reduce the sodium load on astrocytes and limit potential mechanisms of astrocyte edema [62]. Therefore, NAAG peptidase inhibitors could have the potential to provide protection for a variety of neurological diseases in which glutamate excitotoxicity is a significant determinant [21,83].
Conclusion and perspectives Although most of the currently identified NAAG peptidase inhibitors are highly polar compounds with limited ability to cross the BBB and do not appear to be International Journal of Neuroscience
Blockade of NAAG peptidases provides neuroprotection
sufficiently bioavailable to be used as drugs in humans under conditions where the BBB is intact, it is important to note that the efficacy of the urea-based NAAG peptidase inhibitor ZJ-43 in the TBI model suggests that there might be sufficient BBB breakdown at the site of the trauma to permit entry of this inhibitor [19,62,63]. To bring these NAAG peptidase inhibitors to the marketplace, it will be equally important to optimize their pharmacokinetic profiles [19]. As reported previously, this new approach to modulate glutamate levels via NAAG peptidase inhibition does not affect basal glutamate but selectively decreases the excitotoxic rise in extracellular glutamate following a pathological insult [42,84]. Several studies finding that GCPII knockout mice exhibit normal neurological function and behavior strengthen the potential of NAAG peptidase inhibition to serve as a therapeutic strategy, and this might act without the known side-effects associated with conventional glutamate receptor antagonists[37,60]. Data from the first clinical evaluation of the NAAG peptidase inhibitor GPI 5693 for the treatment of neuropathic pain supports this possibility [47]. In summary, blockade of NAAG peptidases may represent a new therapeutic approach to secondary neuronal damage from brain injuries and neurological disorders. In addition, NAAG peptidase inhibitors should prove to be important tools in understanding the physiological role of NAAG in the brain, perhaps deepening our understanding of glutamate neurotransmission [21,63,85,86].
Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. This research was supported in part by a basic research program of project 973 by the Ministry of Science and Technology of China (2011CB013304) and by grants from the National Natural Science Foundation of China (Nos. 30500525, 30670719 and 81070990), the Shanghai Science and Technology Commission (Nos. 10QH1401700, 09140901300 and 05QMX1428) and the Shanghai Municipal Education Commission (No. 09SG20). The authors would also like to express their heartfelt thanks to Professor Bruce G. Lyeth in the Department of Surgical Neurology, University of California at Davis, for his professional revisions and valuable editorial comments.
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