Still NAAG’ing After All These Years

CHAPTER NINE

Still NAAG’ing After All These Years: The Continuing Pursuit of GCPII Inhibitors J.J. Vornov*,{,1, K.R. Hollinger*,1, P.F. Jackson†,1, K.M. Wozniak*, M.H. Farah*, P. Majer§, R. Rais*, B.S. Slusher*,2 *

Johns Hopkins School of Medicine, Baltimore, MD, United States Janssen Pharmaceuticals, San Diego, CA, United States Medpace, Cincinnati, OH, United States § Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic 2 Corresponding author: e-mail address: [email protected] † {

Contents 1. Introduction 1.1 NAAG Is a Widely Distributed Cotransmitter 1.2 NAAG Is Produced by NAAG Synthetase and Packaged in Vesicles by Sialin 1.3 NAAG Is Released from Synapses and Axons 1.4 The Answer to the Nagging Question: GCPII Controls the Dual Function of NAAG 1.5 Under Basal Conditions, NAAG Provides Negative Presynaptic Feedback and Glial Trophic Effects via mGlu3 Receptors and Antagonism of NR2B NMDA Receptors 1.6 Under High Levels of Synaptic Activity, NAAG Release and GCPII Activity Are Enhanced Resulting in Excess Glutamate Release 1.7 NAAG’s Role in Glutamate Supply and Energy Metabolism 2. Design of GCPII Inhibitors 2.1 First Potent and Selective GCPII Inhibitor Discovered 2.2 Second-Generation Phosphinic Acid-Based GCPII Inhibitors 2.3 Thiol-Based GCPII Inhibitors 2.4 Urea-Based GCPII Inhibitors 2.5 Hydroxamic Acid-Based GCPII Inhibitors 2.6 GCPII Structural Studies 2.7 Other Classes of GCPII Inhibitors 2.8 Strategies for Improving the Pharmacokinetic Properties of GCPII Inhibitors 2.9 Intranasal Delivery for Enhancing Brain Penetration of GCPII Inhibitors

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These authors contributed equally to this manuscript.

Advances in Pharmacology, Volume 76 ISSN 1054-3589 http://dx.doi.org/10.1016/bs.apha.2016.01.007

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3. Therapeutic Utility of GCPII Inhibitors in CNS and PNS Disorders 3.1 Stroke and TBI 3.2 Amyotrophic Lateral Sclerosis 3.3 Schizophrenia 3.4 Multiple Sclerosis 3.5 Drug Addiction 3.6 Alzheimer's Disease 3.7 Pain 3.8 Peripheral Neuropathy 4. Conclusion Conflict of Interest Acknowledgments References

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Abstract Nearly two decades ago, Joe Coyle published a single-authored review with the provocative title, The Nagging Question of the Function of N-Acetylaspartylglutamate (Coyle, 1997). In this review, Coyle documented NAAG's localization to subpopulations of glutamatergic, cholinergic, GABAergic, and noradrenergic neurons, Ca2+-dependent release, mGlu3 receptor agonist and NMDA receptor antagonist activity, and cleavage by the glial enzyme glutamate carboxypeptidase II (GCPII). However, at the time of his review, NAAG's physiological function as a neurotransmitter remained elusive. Ironically his review was published months following the discovery of the first potent and selective GCPII inhibitor, 2-(phosphonomethyl)pentanedioc acid (2-PMPA) ( Jackson et al., 1996). Over the ensuing decades, over a dozen independent laboratories used 2-PMPA and other GCPII inhibitors to elucidate two distinct neurotransmitter functions for NAAG. Under basal conditions, when GCPII activity is relatively low, intact NAAG dampens synaptic activity via presynaptic mGlu3 receptor activation and NMDA receptor blockade. However, under stimulated conditions, NAAG release and GCPII activity are enhanced resulting in excess glutamate generation, activating NMDA and other glutamate receptors, often pathologically. Diverse classes of GCPII inhibitors have been synthesized and shown to increase NAAG, decrease glutamate, and provide robust efficacy in many disease models wherein abnormal glutamatergic transmission is presumed pathogenic. In addition, over the past 20 years, basic questions regarding NAAG's synthesis, packaging into vesicles, and receptor selectivity profile have been eloquently elucidated. The purpose of this chapter is to summarize these advances and the promise of regulating NAAG metabolism through GCPII inhibition as a therapeutic strategy.

1. INTRODUCTION Studies performed over the three decades following Coyle’s initial characterization of NAAG as a glutamate receptor ligand (Zaczek, Koller,

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Cotter, Heller, & Coyle, 1983) have confirmed that NAAG meets all of the classical requirements of a neurotransmitter. As discussed in subsequent sections, NAAG is synthesized in specific neurons, stored in presynaptic vesicles, released upon stimulation, acts at specific glutamatergic receptors, and is cleared by a catabolic enzyme. In addition, NAAG can serve as a source of neurotransmitter glutamate.

1.1 NAAG Is a Widely Distributed Cotransmitter NAAG was initially described in the mid-1960s by two different groups in the horse (Curatolo, D Arcangelo, Lino, & Brancati, 1965) and bovine (Miyamoto, Kakimoto, & Sano, 1966) brain and confirmed in the human nervous system (Auditore, Olson, & Wade, 1966). NAAG brain concentrations were found to be in the high micromolar to millimolar range, far exceeding the levels of other neurotransmitters identified at that time and making it the most prevalent neuropeptide in the brain (Miyamoto & Tsujio, 1967). NAAG shows a 10-fold variation in concentration across the CNS, with the highest levels in the spinal cord and brain stem. Concentrations are somewhat higher in white matter compared to gray matter (Koller, Zaczek, & Coyle, 1984) and 100-fold higher in brain than in other organs (Miyake, Kakimoto, & Sorimachi, 1981). More recently, magnetic resonance spectroscopy (MRS) has permitted absolute quantification of NAAG in the living human brain, confirming the biochemical estimates, with NAAG concentrations of 1.5–2.7 and 0.6–1.5 mmol/L in the white and gray matter, respectively (Agarwal & Renshaw, 2012). By immunocytochemistry, NAAG is localized as a cotransmitter in important brain pathways including ascending and descending spinal axons, spinal motoneurons, retinal ganglion cells, geniculo-cortical neurons, nigrostriatal neurons, cerebellar afferent neurons, neurons of the deep cerebellar nuclei, and large spinal sensory neurons (Cangro, Namboodiri, Sklar, Corigliano-Murphy, & Neale, 1987; Forloni, Grzanna, Blakely, & Coyle, 1987; Frondoza, Logan, Forloni, & Coyle, 1990; Passani, Vonsattel, Carter, & Coyle, 1997). NAAG is also present in oligodendrocyte cultures and activated microglia (Passani, Elkabes, & Coyle, 1998), suggesting an additional role in glial signaling.

1.2 NAAG Is Produced by NAAG Synthetase and Packaged in Vesicles by Sialin NAAG synthetase (NAAGS) was identified in 2010 (Becker, Lodder, Gieselmann, & Eckhardt, 2010; Collard et al., 2010) using a bioinformatics

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approach by identifying gene sequences of amino acid ligases localized to the brain, confirming earlier studies that NAAG is synthesized enzymatically and not via protein synthesis (Arun, Madhavarao, Moffett, & Namboodiri, 2006; Cangro et al., 1987; Gehl, Saab, Bzdega, Wroblewska, & Neale, 2004). Only a few peptides, such as glutathione, are similarly synthesized in a ribosome independent manner. Two NAAGS genes were eventually identified from members of the ribosomal modification protein RIMK-like family members, RIMKLA and RIMKLB (Collard et al., 2010). Expression of either of these genes in cell culture showed NAAGS activity, but only when NAA and glutamate were present. In situ hybridization demonstrated localization of gene expression in neurons as had been predicted by previous immunochemical studies (Passani, Vonsattel, Carter, et al., 1997). NAAG was first localized to synaptic vesicles using immunocytochemistry in 1988 (Williamson & Neale, 1988), but the mechanism of its vesicular packaging was not understood until 2013, when vesicular uptake catalyzed by the lysosomal membrane protein sialin (Lodder-Gadaczek, Gieselmann, & Eckhardt, 2013) was shown to be responsible for the ATP-dependent vesicular packaging of NAAG for synaptic release. It is noteworthy that this mechanism is distinct from the H+-dependent vesicular packaging of glutamate and catecholamine neurotransmitters. Interestingly, the concentration of brain NAAG is significantly reduced in sialin-deficient mice, suggesting that most brain NAAG is likely destined for synaptic release.

1.3 NAAG Is Released from Synapses and Axons NAAG’s calcium-dependent release following synaptic activation was initially characterized by microdialysis studies using both radiolabeled (Tsai, Forloni, Robinson, Stauch, & Coyle, 1988) and unlabeled (Tsai, Stauch, Vornov, Deshpande, & Coyle, 1990) NAAG. More recently, Walder et al. (2013) showed at the lizard neuromuscular junction that NAAG is depleted by potassium-induced depolarization and by electrical stimulation of motor axons providing additional support of NAAG’s synaptic release and confirming its localization to synaptic vesicles. The medial giant nerve fibers (MGNFs) of the crayfish have proved to be a valuable system for the study of NAAG synthesis and release (Urazaev, Grossfeld, & Lieberman, 2005) as these axons preferentially release NAAG, not glutamate, as a transmitter. In this model, when [3H]-glutamate was provided in the bath, NAAG was found to contain approximately 50% of the label. Subsequent axonal

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stimulation resulted in release of the NAAG from the axon, glutamate generation from NAAG via GCPII, activation of glial NMDA receptors, and the generation of a calcium-mediated glial depolarization signal. It is not known whether this axonal release of NAAG occurs in the mammalian CNS. A recent MRS study in man (Castellano, Dias, Foerster, Li, & Covolan, 2012) for the first time provided evidence of NAAG release in the human brain. During a simple visual stimulation paradigm, MRS measurements showed a 200% increase in the NAAG MRS signal, while the NAA signal decreased by 20%. One interpretation of this finding is that synaptic activity dramatically increases NAAG synthesis. Based on our understanding of NAAG storage in vesicles, it is also possible that this NAAG signal increase arises from release of NAAG from synaptic vesicles into the extracellular space, where the MRS signal is enhanced by change in local environment.

1.4 The Answer to the Nagging Question: GCPII Controls the Dual Function of NAAG GCPII is an extracellular, glial enzyme with its active site in the extrasynaptic space, making it ideally positioned to control whether NAAG functions to block or drive glutamatergic transmission. As detailed below, under basal conditions GCPII activity appears low, permitting NAAG to function as an intact dipeptide. However, under conditions of high synaptic activity, NAAG release and its cleavage by GCPII is enhanced, serving to liberate glutamate that subsequently activates extrasynaptic glutamatergic receptors on surrounding neurons and glia (Fig. 1). As detailed in later sections discussing pathological conditions, GCPII inhibitors appear to reverse this activated state, decreasing glutamate release and increasing NAAG, returning the system to its basal state.

1.5 Under Basal Conditions, NAAG Provides Negative Presynaptic Feedback and Glial Trophic Effects via mGlu3 Receptors and Antagonism of NR2B NMDA Receptors As first described by Wroblewska, Wroblewski, Saab, and Neale (1993) using cerebellar granule cells, intact NAAG is an agonist at the group II metabotropic glutamate receptors (Conn & Pin, 1997). Unlike glutamate, NAAG is highly selective for group II mGluRs, having no effect on cAMP-coupled group III mGluRs or the phosphoinositide-coupled group I mGluRs (Conn & Pin, 1997; Schoepp, Johnson, & Monn, 1992). Using

Fig. 1 (A) Components of the NAAG/GCPII neurotransmitter system. NAAG is synthesized presynaptically by NAAG synthetase and packaged into vesicles by sialin. After release, intact NAAG interacts with mGlu3 and NMDA receptors and/or is hydrolyzed by glial GCPII to release glutamate outside the synaptic cleft. (B) Under basal conditions, GCPII activity and synaptic NAAG and glutamate concentrations are relatively low. NAAG modulates synaptic activity by activating presynaptic mGlu3 receptors and postsynaptic GluN2A-rich NMDA receptors. Further, NAAG that reaches the extrasynaptic space inhibits GluN2B-rich NMDA receptor-mediated EPSCs and stimulates glial mGlu3 receptors which induces trophic effects. (C) Under pathologic conditions with high synaptic activity, elevated levels of glutamate and NAAG flood the synapse. NAAG that reaches the extrasynaptic space is rapidly cleaved by GCPII to liberate glutamate. The excess glutamate and NAAG activate both GluN2A- and GluN2B-rich NMDA receptors, increasing EPSCs. (D) Blockade of GCPII under activated conditions prevents the breakdown of NAAG lowering overall glutamate levels. The resulting increased NAAG further decreases glutamate release through feedback inhibition via presynaptic mGlu3 receptors and induces trophic effects via activation of glial mGlu3 receptors. Overall, inhibition of GCPII increases NAAG and lowers glutamate, returning the system toward its basal state.

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selective expression of mGluR genes in cell culture and selective knockouts in vivo, it is now established that within the group II mGluR class NAAG preferentially activates the mGlu3 receptor subtype (Wroblewska et al., 1997) with an EC50 of 65 μM (Wroblewska et al., 1997). The specificity for mGlu3 was confirmed in vivo using a mouse model of schizophrenia in which GCPII inhibition efficacy was observed only when mGlu3 receptor but not mGlu2 receptor was expressed (Olszewski, Bzdega, & Neale, 2012). Failures to replicate mGlu3 activation in vitro (Fricker et al., 2009; Johnson, 2011) may be due to expression system used and presence of the proper G protein coupling mechanism (Ghose et al., 1997). As an agonist of presynaptic mGlu3 receptors, NAAG provides negative feedback to limit cotransmitter release (Adedoyin, Vicini, & Neale, 2010; Bischofberger & Schild, 1996; Wroblewska, Wegorzewska, Bzdega, Olszewski, & Neale, 2006) with demonstrated physiological effects at glutamatergic synapses (Lea, Wroblewska, Sarvey, & Neale, 2001), the neuromuscular junction (Malomouzh et al., 2005; Walder et al., 2013), and spinal cord glycenergic synapses (Romei, Raiteri, & Raiteri, 2013). mGlu3 receptors are also believed to be the major glutamate receptors on astrocytes and oligodendrocytes (Sun et al., 2013), and glial mGlu3 receptor stimulation has been shown to enhance the activity of neurotrophic factors (Battaglia et al., 2015; Durand, Carniglia, Caruso, & Lasaga, 2013), such as TGF-beta (Thomas, Olkowski, & Slusher, 2001). It is important to note that many of these experiments had to be performed by exposing systems to NAAG in the presence of GCPII blockers. If GCPII were active, hydrolysis of NAAG would result in effects due to glutamate, not NAAG. Intact NAAG also serves as an agonist at GluN2A-containing NMDA receptors and an antagonist at GluN2B-containing NMDA receptors (Khacho, Wang, Ahlskog, Hristova, & Bergeron, 2015). However, at low pH, as can occur during ischemia or injury, NAAG acts as an antagonist at both GluN2A- and GluN2B-containing receptors. Thus, under normal conditions, NAAG within the synapse would be expected to activate synaptic GluN2A-containing NMDA receptors while providing negative feedback on further release through presynaptic mGlu3 receptors. If NAAG diffuses from the synapse without being hydrolyzed by GCPII, it is expected to block extrasynaptic NMDA receptors, which are known to be enriched in GluN2B subunits. These recent subtype specificity studies may explain why the initial electrophysiological explorations of NAAG’s effects on NMDA receptors were conflicting showing both mild agonist

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(Westbrook, Mayer, Namboodiri, & Neale, 1986) and antagonist (Puttfarcken, Handen, Montgomery, Coyle, & Werling, 1993; Sekiguchi, Okamoto, & Sakai, 1989) effects. Several lines of evidence suggest that under basal conditions, when GCPII activity is low, NAAG is released into the synapse and serves as a neurotransmitter without rapid cleavage to glutamate. First, blockade of GCPII has no effect on extracellular glutamate as measured by microdialysis (Nagel et al., 2006; Slusher et al., 1999), although NAAG increases as expected. Second, blockade of GCPII produces no signs of sedation, ataxia, or stereotypical behavior, as would be expected if synaptic glutamate levels were altered (Slusher et al., 1999). Finally, GCPII knockout animals develop normally (Bacich et al., 2005; Gao et al., 2015) and actually show enhanced performance in a novel object recognition task ( Janczura et al., 2013), suggesting no deleterious effect on normal synaptic glutamate neurotransmission. In man, only a lack of both NAA and NAAG measured by MRS have been described in a single case report of a child with reduced myelination and developmental delays (Martin, Capone, Schneider, & Hennig, 2001). A follow-up report (Boltshauser et al., 2004) showed that the patient had seizures and no language ability and confirmed that the deficit was due to a lack of L-aspartate N-acetyltransferase, making the patient unable to synthesize NAA, but leading to a lack of NAAG, confirming the metabolic pathway in man (Fig. 2).

Fig. 2 NAAG synthesis and catabolism. NAA is synthesized by aspartate N-acetyltransferase which is a necessary precursor along with glutamate for the enzyme NAAG synthetase to form NAAG. Glutamate carboxypeptidase II catabolizes NAAG back to NAA and glutamate.

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1.6 Under High Levels of Synaptic Activity, NAAG Release and GCPII Activity Are Enhanced Resulting in Excess Glutamate Release In marked contrast, under pathological conditions in which excessive glutamate has been implicated, GCPII inhibitors have profound effects on neuronal injury, behavior, and glutamate release. This strongly suggests that high levels of stimulation increase NAAG hydrolysis by GCPII resulting in excess glutamate release and activation of glutamatergic receptors. The release of glutamate from NAAG was first shown directly in a rat model of cerebral ischemia (Slusher et al., 1999). Brain ischemia is accompanied by a large increase in extracellular glutamate which activates NMDA receptors and causes neuronal injury. Administration of the GCPII inhibitor 2-PMPA was shown to attenuate the elevation in extracellular glutamate and subsequent neurotoxicity. Importantly, the decrease in glutamate observed in this study was accompanied by an, reciprocal rise in extracellular NAAG, although the extent to which extracellular glutamate is derived from NAAG is not known. A similar reciprocal decrease in glutamate and increase in NAAG was recently reported using GCPII inhibitors in a traumatic brain injury (TBI) model (Zhong et al., 2006). GCPII inhibition has shown similar therapeutic effects in a wide variety of preclinical models linked to excessive glutamate, as discussed later. Similar to ischemia, the effects are likely due, in part, to decreasing glutamate derived from NAAG. In addition, the increased NAAG concentrations have additional protective effects through mGlu3 receptor agonism, causing inhibition of glutamate release. The positive contribution of NAAG-mediated mGlu3 activation has been confirmed by blockade of therapeutic effects of GCPII inhibition by mGlu3 antagonists in many preclinical models (Adedoyin et al., 2010; Olszewski et al., 2004; Yamamoto et al., 2004; Zhong et al., 2006; Zuo, Bzdega, Olszewski, Moffett, & Neale, 2012). The question arises whether glutamate released from NAAG is of importance only under pathological conditions. The switch from mGlu3 receptor activation to NMDA receptor activation upon stimulation can be directly observed in the MGNF system (Urazaev et al., 2005). At low levels of axonal stimulation, NAAG is detected in the extracellular space and glia show an mGlu3-mediated hyperpolarization response. However, after action potential generation, GCPII activity increases, NAAG hydrolysis occurs resulting in glutamate release and an augmentation of the glial response due to NMDA receptor activation in addition to mGlu3 activation.

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The mechanism by which GCPII is activated by axonal stimulation is unexplored, but it is known that GCPII is activated by low phosphate (Robinson, Blakely, Couto, & Coyle, 1987; Slusher et al., 1990), providing a theoretical mechanism for short-term regulation of its activity. In astrocytes, GCPII protein levels are under long-term control by HDAC acetylation (Arun et al., 2006). GCPII activity is chronically elevated in some pathological conditions, such as genetically epileptic prone rats (Meyerhoff, Carter, Yourick, Slusher, & Coyle, 1992), chronic phencyclidine (PCP) exposure in rats (Flores & Coyle, 2003), and in motor cortex and spinal cord of amyotrophic lateral sclerosis patients (Tsai et al., 1991). Activation of GCPII and release of glutamate from NAAG provides a possible mechanism for extrasynaptic glutamate receptor activation on neurons and glia. Normally, glutamate concentrations in the synapse are tightly controlled by excitatory amino acid transporters (EAATs), preventing synaptically released glutamate from activating extrasynaptic receptors. However, under conditions of high synaptic activity, NAAG should escape from the synapse as it is not a substrate for EAATs, releasing glutamate after hydrolysis by GCPII. The NAAG/GCPII system is therefore an excellent candidate as the prime transmitter for what has been called the “Tripartite Glutamatergic Synapse” (Machado-Vieira, Manji, & Zarate, 2009). NAAG can leave the synapse, permitting sensing of ongoing synaptic activity by nearby neurons and glia for coordinated control of glutamatergic neurotransmission. Unfortunately, under pathological conditions, this extrasynaptic glutamate release drives abnormal behavior and may cause neurodegeneration.

1.7 NAAG’s Role in Glutamate Supply and Energy Metabolism It has been speculated that under stimulated conditions, NAAG also plays a central role in energy metabolism and homeostasis in the brain by shuttling glutamate from neurons to glia (Baslow, 2015). It is now well established that the neurons and glia act as a single metabolic unit with the glutamate– glutamine cycle (Shetty, Galeffi, & Turner, 2012). As shown in Fig. 3, NAAG released as a cotransmitter provides an alternative glutamate source to glia that, unlike glutamate itself avoids direct activation of the full complement of ionotropic and metabotropic glutamate receptors. There is evidence from the crayfish model that NAAG cycling is a significant component of glutamate–glutamine cycle (Urazaev et al., 2001), but NAAG’s direct contribution to glutamate and energy homeostasis is

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Fig. 3 Role of NAAG in glutamate supply. After NAAG is released into the synapse, it is cleaved by GCPII to liberate glutamate and NAA in the extracellular space. Glutamate is transported into glia where it enters the glutamate–glutamine cycle to supply glutamine to neurons for glutamate synthesis. NAA is transported back into neurons where it may be, at least in part, recycled back to NAAG.

currently only theoretical. It has been reported that the GCPII inhibitor 2PMPA administered systemically can block the BOLD signal on MRI (Baslow, Dyakin, Nowak, Hungund, & Guilfoyle, 2005), suggesting that glutamate derived from NAAG mediates neurovascular coupling and provides the link between synaptic activity and local increases in blood flow. In summary, GCPII inhibitors can potentially dampen excessive glutamate effects through multiple mechanisms involving both neurons and glia. GCPII inhibition will increase NAAG, activating presynaptic mGlu3 receptors, and blocking NMDA receptors particularly under acidic extracellular conditions of synaptic activation. GCPII inhibition will directly decrease glutamate derived from NAAG and indirectly inhibit further glutamate release through NAAG’s action at mGlu3 receptors. Finally, GCPII inhibition will decrease glutamate availability through NAAG sequestration, potentially slowing the glutamate/glutamine cycle. Remarkably, the effects of GCPII inhibition under basal conditions appear modest, suggesting that GCPII inhibitors are ideal therapies that selectively block excessive glutamate effects without altering normal function. Perhaps under normal signaling conditions, release of NAAG is minimal as is typical of many peptide neurotransmitters. For example, the potent opiate receptor antagonist

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naloxone has little effect under normal conditions presumably because of minimal basal activation of the endorphin system. Given its potential therapeutic utility, several independent laboratories have synthesized and characterized small-molecule inhibitors of GCPII from multiple structural classes. Furthermore, several inhibitor–enzyme crystal structures have been elucidated. The section below details the progress in the design of GCPII inhibitors over the past two decades.

2. DESIGN OF GCPII INHIBITORS 2.1 First Potent and Selective GCPII Inhibitor Discovered The first reported inhibitors of GCPII were a series of glutamate derivatives and quisqualic acid (Subasinghe et al., 1990; Fig. 4, 1). Unfortunately these derivatives lacked the potency and selectivity required for an effective GCPII inhibitor tool compound. In 1988 Barbara Slusher, one of Joe Coyle’s former students, began working at Zeneca Pharmaceuticals in the neuroscience drug discovery unit. She described her work in the Coyle lab to a group of colleagues and began to generate interest in the potential of inhibiting GCPII for use in the treatment of neurological disorders. As a result, a small team began to design GCPII inhibitors with the goal of delivering a tool compound that could permit a more systematic evaluation of the role of NAAG and GCPII inhibition both in vitro and in vivo. Although there was little information regarding the structural requirements of the enzyme, divalent metal ions were known to be required for activity, suggesting it was a metallopeptidase. Based upon previous metalloprotease inhibitor design, it was suggested that a chelator in addition to a recognition unit for the enzyme would lead to a series of potent inhibitors (Rich, 1990). Of the available metal chelators, the group began to examine a series of phosphoric and phosphinic acids. This led to the design of 2-PMPA (Fig. 4, 2), which contained a phosphonic acid and the glutamic acid recognition portion of NAAG. This compound was shown to be very potent with a Ki of 0.4 nM ( Jackson et al., 1996). In addition, the molecule was highly selective with no activity reported against a large number of enzymes, transporters, and receptors (Slusher et al., 1999). This represented the first effective tool compound to study the mechanistic and physiological inhibition of GCPII and has been extensively utilized to explore the role of the GCPII enzyme by laboratories all over the world including the United States, Italy, Czech Republic, United Kingdom, Poland, and Germany (Barinka, Rojas, Slusher, & Pomper, 2012).

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Fig. 4 Inhibitors of GCPII. Compounds 2–6 are representative of the phosphonatebased inhibitors, while compounds 7–9 are members of the thiol-based class. The urea-based compounds shown with structures 10–12 represent nontraditional metalloenzyme motifs. Other inhibitors such as hydroxamic acids and indole derivatives are shown with structures 12–14. Compound 15 represents a radiolabeled GCPII inhibitor.

2.2 Second-Generation Phosphinic Acid-Based GCPII Inhibitors As can be seen from its structure (2), 2-PMPA is highly polar with a calculated log D > –10. In order to decrease the polarity and improve the inherent difficulties in developing such a polar molecule, modifications were made to

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its structure. The original SAR suggested that the S10 pocket of the enzyme was more restrictive and required the glutamate configuration (Fig. 4, 3). However, the S1 pocket appeared to have more room as shown with compound 4. Because initial microdialysis studies showed that 2-PMPA did not alter basal glutamate in the brain, Zeneca Pharmaceuticals decided not to pursue the target and permitted the team to publish their work ( Jackson et al., 1996). In 1994 a small biotech company, called Guilford Pharmaceuticals in Baltimore, MD, recruited Slusher and Jackson from Zeneca and the pair decided to continue their exploration of the utility of GCPII inhibition. Although they repeated the finding that 2-PMPA has no effect on basal glutamate, they found that it selectively decreased injury-induced glutamate release in the brain and produced robust neuroprotective effects, which led to their landmark Nature Medicine article in 1999 and triggered the company to pursue a full drug discovery effort around the target (Slusher et al., 1999). The company’s first attempts were made to develop phosphinic acid derivatives with a goal to improve the highly polar nature and poor pharmacokinetic properties of 2-PMPA. Hundreds of analogs were synthesized such as compounds 5 and 6 (GPI 5232) ( Jackson et al., 2001). Although they had encouraging potency, their pharmacokinetic properties were not significantly improved to provide a viable path to a brain penetrant, orally available compound.

2.3 Thiol-Based GCPII Inhibitors In order to decrease the log D further and in line with the experience in the ACE inhibitor field, a series of thiol replacements for the phosphorous in 2-PMPA were synthesized. These compounds showed good activity in vitro. The most potent compound in the series was 2-(3-mercaptopropyl)pentanedioc acid, 2-MPPA, 7 (Majer et al., 2003). This level of potency was not expected, as the chain length is longer than in 2-PMPA; compound 8, which is directly analogous to 2-PMPA, was predicted to be more potent. It has been suggested that this differential potency may be due to the fact that GCPII has two zincs at the binding site and the thiol may be interacting with a different zinc than the phosphonate-based compounds (Ferraris, Shukla, & Tsukamoto, 2012). Although not as potent as 2-PMPA, this compound demonstrated good oral bioavailability in rats (Majer et al., 2003). Though high brain levels were not achieved, 2-MPPA showed reasonable PNS exposure and exhibited robust efficacy

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following oral administration in several neuropathic pain and peripheral neuropathy studies as detailed later (Potter, Wozniak, Callizot, & Slusher, 2014; Vornov et al., 2013). The company took the compound into two Phase I safety studies in normal volunteers and patients with chronic diabetic neuropathy, where it was shown to be safe with no adverse events reported (van der Post et al., 2005). The group at Guilford then began to perform SAR studies around this compound in order to increase its potency. In contrast to the phosphonate-based compounds, it was possible to modify the glutarate portion and increase potency (Majer et al., 2006). For example, compound 9 is seven times more potent than the parent compound. This discovery of the thiol series of inhibitors represented a significant advance in the search for clinically efficacious compounds as this series has the advantage of good oral bioavailability and potency. Although 2-MPPA was safe and well tolerated in two Phase 1 studies (up to 14-day dosing) at presumed therapeutic doses, subsequent immunological toxicities observed in chronic GLP primate studies halted its development. The primate toxicology seen with 2-MPPA was immune complex formation in the kidneys and was attributed to the thiol nature of the compound, not its GCPII inhibiting activity. It is well documented that thiol-containing drugs have a propensity to elicit immune hypersensitivity reactions (Katsutani & Shionoya, 1992). In addition, 2-MPPA was not an ideal drug candidate because of its racemic nature, poor chemical stability, low melting point, and a reactive sulfhydryl group making process chemistry and formulation difficult.

2.4 Urea-Based GCPII Inhibitors In 2001 Kozikowski reported a new class of inhibitors based upon a urea replacement for the phosphorous group of the phosphinic acid class of compounds (Kozikowski et al., 2001). This group has been widely used in the past as an amide surrogate. By examining the SAR and modeling of a series of dipeptide analogs, in addition to the phosphinic acid derivatives, the group hypothesized that a compound containing the glutamic acid recognition motion followed by a spacer and another carboxylic acid it would be possible to inhibit the enzyme. They synthesized the urea-based series shown in Fig. 4 (compounds 10 and 11) and demonstrated that they were potent inhibitors of GCPII (Kozikowski et al., 2004; Zhou, Neale, Pomper, & Kozikowski, 2005). This was important as it represented the first series of compounds that did not contain a traditional chelating group.

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As with the phosphinic acid series of compounds, the S1 site of GCPII is amenable to SAR modifications. Specifically, a series of alkyl- and aryl-based derivatives were made and shown to be potent inhibitors of GCPII (Kozikowski et al., 2004). In addition, isosteres of one of the carboxylic acids also showed good inhibition. However, similar to the phosphorous-based inhibitors, the presence of multiple highly polar groups has limited the brain penetration and thus the development of these compounds for CNS disease.

2.5 Hydroxamic Acid-Based GCPII Inhibitors There are several chelating groups that are known to serve as useful zincbinding groups in metalloprotease design. In addition to phosphorousand thiol-based inhibitors, the hydroxamic acid-based compounds have been extensively explored. However, unlike other metalloprotease systems, the potency between phosphinic acid-based derivatives and hydroxamic acid-based molecules is not similar when comparing the 2-PMPA and 2-MPPA templates. The most potent compound described to date is compound 12 which has an IC50 ¼ 220 nM (Stoermer et al., 2003). The reason for the decreased potency vs phosphonates is not clear but again may be due to the differences between enzymes with one or two metals at the active site. However, in this case, replacement of the glutarate functionality led to more potent compounds. By incorporating an aryl group into the side chain, potent inhibitors were synthesized as shown with compound 13, which has an IC50 of 30 nM (Tsukamoto et al., 2002). Although these compounds exhibit good efficacy in inhibiting the enzyme, similar to the phosphinic acid and urea-based compounds, they exhibit poor oral bioavailability and brain penetration (B. S. Slusher, unpublished observation).

2.6 GCPII Structural Studies There has been a number of crystallography studies published showing both the binding interactions of the phosphorous-based compounds and the ureabased derivatives (Barinka et al., 2007). The first structures of GCPII in complex with phosphorous-based inhibitors were published in 2006 (Mesters et al., 2006). This and subsequent work indicated the expected mode of action of the inhibitors. Specifically, there was a requirement for two carboxylic acid groups in the S10 region. In addition, there is a stereochemical preference for one enantiomer. This was demonstrated by the synthesis of both enantiomers of 2-PMPA and subsequent crystallization with human GCPII (Tsukamoto et al., 2005).

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The majority of the structural work around GCPII inhibitors has been done with the urea-based compounds. In 2014 Barinka reported on the computational and X-ray analysis of six inhibitors with GCPII (Barinka et al., 2008). In all of this work there is a crucial interaction with the α-carboxylate of the glutarate moiety found in the inhibitors and Arg210. This interaction was confirmed with site-directed mutation of this residue (Mlcochova et al., 2007). The Arg210Ala derivative protein shows a fivefold decrease in the IC50 for 2-PMPA. Surprisingly, this work demonstrated that the S10 pocket can accommodate larger groups through conformational changes, with flexibility around the Leu259-Gly263 segment. This finding may aid in the design of new compounds that may be able to address the issue of brain permeability.

2.7 Other Classes of GCPII Inhibitors In addition to the compounds outlined earlier, a number of other GCPII inhibitors have been reported. A series of indole derivatives was reported by Guilford, all of which have good potency (compound 14; Grella et al., 2010). Berkman has also described a series of phosphoramidate inhibitors and has extensively explored the S1 pocket of the enzyme with these compounds (Ley et al., 2015; Mendes, Wong-On-Wing, & Berkman, 2015). In addition, X-ray analysis of these inhibitors with GCPII has been recently reported in conjunction with extensive modeling work (Novakova et al., 2015). More recently, a novel series of carborane containing ureabased inhibitors have been synthesized which has allowed for further refinement of the spatial requirements of the S1 pocket (Youn et al., 2015). There have also been a number of groups that have taken the urea-based and phosphoramidate-based compounds and developed PET tracers for the potential use of diagnosing this metastatic prostate cancer such as compound 15 (Lapi et al., 2009; Maresca et al., 2009). This approach and potential therapeutic applications have been recently reviewed by Pomper (Mease, Foss, & Pomper, 2013). In summary, many classes of potent and selective GCPII inhibitors have been synthesized, but to date all have had one or more issues related to poor oral availability, instability, and limited brain penetration that hampers their development as a CNS therapeutic. As a result there is a need to develop new compounds that have alternative chemical templates or utilize drug delivery or prodrug technologies, as detailed below, that will allow progression of GCPII inhibitors into the clinic.

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2.8 Strategies for Improving the Pharmacokinetic Properties of GCPII Inhibitors There has recently been an increased focus on using prodrug strategies to improve the poor pharmacokinetic profile of GCPII inhibitors. Given that 2-PMPA remains the most potent and efficacious GCPII inhibitor yet discovered, our group has focused on improving its oral and tissue availability by masking its polar ionizable groups. These less polar prodrugs are anticipated to have enhanced intestinal permeability and once in plasma, will be cleaved to active drug via plasma or liver enzymes. The initial strategy to conceal the phosphonate, while keeping the two carboxylates unsubstituted was unsuccessful due to the instability of the derivatives, likely due to the neighboring group participation effect of the α-carboxylate, which facilitates hydrolysis of the phosphonate substituents via a five-membered cyclic mechanism. The phosphonate was then systematically masked by various hydrophobic moieties, while simple alkyl esters were used to functionalize the carboxylates (Fig. 5A). Compounds of the (e) class exhibited the best oral exposure in mice, but the carboxylic esters

Fig. 5 (A) Phosphonate prodrugs of 2-PMPA. Bisphosphonates were covered with salicylate (a), phe-phosphoramidate (b), long-chain alkyl ester (c), polyethylene glycol ester (d), pivaloyloxymethyl (POM) (e), and pivaloyloxy-carbonyloxymethyl (POC) (f ), while simple alkyl esters were used to cover the carboxylates. Tris-POM-2-PMPA (ee) and Tris-POC-2-PMPA (ff ). (B) Oral availability of Tris-POC-2-PMPA in dog. Tris-POC-2PMPA dosed at 10 mg/kg equivalent 2-PMPA via oral route gave >20-fold enhanced exposures compared to oral 2-PMPA. (C) Brain-to-plasma ratio of 2-PMPA following i.n. vs i.p. administration in rats. Brain tissue-to-plasma ratio in olfactory bulb and cortex was 150% and 71% following i.n. administration vs 2% when given by i.p. route.

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were found to be too stable and the parent 2-PMPA was released only in minimal quantities. Both α,γ-diesters and α-monoesters were also tested with the same result. To overcome this limitation, a hydrophobic promoiety was introduced on the α-carboxylate. The resulting Tris-POC-2-PMPA was determined to be chemically stable in both simulated gastric fluid and buffer at pH 7.4. The Tris-POC-2-PMPA (ff ) was then evaluated in both rodent (mice) and nonrodent species (dog), and shown to provide excellent oral exposure with >20-fold enhancement vs 2-PMPA (Fig. 5B). These compounds are currently undergoing further analysis and may provide a viable avenue for the oral delivery of a GCPII inhibitor.

2.9 Intranasal Delivery for Enhancing Brain Penetration of GCPII Inhibitors Another strategy for selectively enhancing brain delivery of 2-PMPA was recently reported by our laboratory (Rais et al., 2015). The intranasal (i.n.) route has been employed for drug delivery of a number of small molecules, macromolecules, gene vectors, and cells, and has been shown to be successful in animal and clinical studies (Chen, Fawcett, Rahman, Ala, & Frey, 1998; Dhuria, Hanson, & Frey, 2009; Frey et al., 1997; Johnson, Hanson, & Frey, 2010; Lochhead & Thorne, 2011; Stevens, Ploeger, van der Graaf, Danhof, & de Lange, 2011; Vaka, Sammeta, Day, & Murthy, 2009). Using this strategy, we showed that compared to intraperitoneal (i.p.) administration, equivalent doses of i.n.-administered 2-PMPA resulted in similar plasma exposures (AUC0–t, i.n./AUC0–t, i.p. ¼ 1.0 in plasma), but the AUC ratio was >50-fold in brain. Moreover, the brain-to-plasma ratio based on AUC0–t in the olfactory bulb and cortex were 149%, and 71% following i.n. administration and in contrast only 2% via i.p. route (Fig. 5C). Lastly, due to anatomical differences in rodent and human nose, we examined i.n. delivery of 2-PMPA in a nonhuman primate, where it also showed a selective brain permeation with 1.5 μM concentrations in primate cerebrospinal fluid (CSF) and undetectable levels in plasma (<10 nM) at 30-min postdose. These new drug delivery and prodrug strategies are now being employed with other potent GCPII inhibitors and analogs in an attempt to achieve therapeutic concentrations in plasma and brain. These strategies may finally enable the translation of GCPII inhibitors back into the clinic so that the enormous amounts of preclinical efficacy data may be confirmed or disproved in clinical studies.

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3. THERAPEUTIC UTILITY OF GCPII INHIBITORS IN CNS AND PNS DISORDERS GCPII inhibitors have been shown to be effective and well tolerated in several animal models of neurological and psychiatric disorders where excessive glutamatergic receptor activation is implicated. As discussed earlier, it appears their effectiveness arises through the dual mechanisms of increasing NAAG’s actions as a mGlu3 receptor agonist and NMDA receptor antagonist, while simultaneously decreasing NMDA receptor activation by attenuating the release of glutamate derived from NAAG without major effects on normal function. GCPII is recruited as a source of glutamate in pathological conditions (Gafurov, Urazaev, Grossfeld, & Lieberman, 2001; Urazaev et al., 2001, 2005), making inhibition of the enzyme a desirable therapeutic strategy for conditions that report low NAAG and elevated glutamate levels. Over a dozen independent laboratories have published studies demonstrating the therapeutic utility of GCPII inhibition in over 20 in vivo animal models. These findings are summarized later and presented historically in a timeline (Fig. 6).

3.1 Stroke and TBI The utility of 2-PMPA to treat conditions dependent on excessive glutamate and NMDA receptor activation was first described in 1999 in preclinical models of stroke (Slusher et al., 1999). Prior to this study, potent NMDA receptor antagonists had demonstrated promise in traumatic CNS injury models by blunting the excess NMDA receptor activation caused by the injury-induced enormous increase of extracellular glutamate (Bernards & Akers, 2006; Faden, Demediuk, Panter, & Vink, 1989). Translation of these antagonists (eg, Selfotel, Cerestat; Narayan et al., 2002) to the clinic, however, failed due to intolerable drug side effects. In the stroke model, microdialysis studies revealed no effect of 2-PMPA on basal glutamate but selective attenuation of the stroke-induced rise in extracellular glutamate and a concomitant rise in NAAG, along with robust levels of neuroprotection (Slusher et al., 1999) similar to those reported for NMDA receptor antagonists (Small & Buchan, 1997) but with no overt toxicities observed. Inspired by these results, other laboratories began evaluating GCPII inhibitors in ischemic and traumatic CNS models (Harada et al., 2000; Kozikowski et al., 2004; Williams, Lu, Slusher, & Tortella, 2001). In a rat model of spinal cord injury, 2-PMPA decreased the spike in CSF glutamate levels and

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Ischemia/stroke/TBI Pain/peripheral neuropathy

Slusher et al. (1999)

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Harada et al. (2000)

Shippenberg et al. (2000)

Williams et al. (2001)

Jackson et al. (2001)

Jackson et al. (2001)

Zhang et al. (2002)

Chen et al. (2002)

Witkin et al. (2002)

Carpenter et al. (2003)

Majer et al. (2003)

Popik et al. (2003)

Kozikowski et al. (2004)

Yamamoto et al. (2004)

Olszewski et al. (2004)

Long et al. (2005)

Zhong et al. (2005)

Kozela et al. (2005)

Zhong et al. (2006)

Zhang et al. (2006)

Nagel et al. (2006)

Yamamoto, Nozaki-Taguchi, Yamamoto, Nozaki-Taguchi, Sakashita, and Inagaki (2001) and Sakashita (2001)

ALS Schizophrenia MS

Ghadge et al. (2003)

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Olszewski et al. (2008)

Carozzi et al. (2010)

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Feng et al. (2011)

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Feng et al. (2012) Gurkoff et al. (2013)

Peng et al. (2010)

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Wozniak et al. (2012)

Yamada et al. (2012)

Olszewski, Bzdega, et al. (2012)

Vornov et al. (2013)

Janczura et al. (2013)

Olszewski, Janczura, et al. (2012)

Zuo et al. (2012)

Rahn et al. (2012)

Gao et al. (2015) Hollinger et al. (2016)

Fig. 6 Therapeutic utility of GCPII inhibitors observed over the past 20 years in various preclinical models of neurological and psychiatric disease.

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attenuated the development of motor deficits and neuropathological damage (Long, Yourick, Slusher, Robinson, & Meyerhoff, 2005). Administration of a structurally distinct GCPII inhibitor, ZJ-43, protected against TBIinduced neurological damage (Zhong et al., 2005), which was blocked through coadministration of a mGlu2/3 receptor antagonist (Zhong et al., 2006). GCPII inhibition can both prevent and treat neurological damage, as a ZJ-43 prodrug delivered 30 min post-TBI with hypoxia decreased hippocampal neurodegeneration and astrocytic death (Feng et al., 2012). Cognitive impairment is a frequent and debilitating side effect of TBI, and administration of the GCPII inhibitor PGI-02776 (a prodrug of ZJ-43) improved TBI-induced spatial memory deficits (Feng et al., 2011; Gurkoff et al., 2013). Not only were small-molecule GCPII inhibitors effective, but transgenic mice lacking the GCPII gene were also protected against ischemic and TBI (Bacich et al., 2005; Gao et al., 2015).

3.2 Amyotrophic Lateral Sclerosis Glutamate has long been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), as high CSF glutamate levels and deficiencies in glutamate metabolism and transport have been reported in ALS patients (Plaitakis & Caroscio, 1987; Rothstein, Martin, & Kuncl, 1992; Rothstein et al., 1990; Shaw, Forrest, Ince, Richardson, & Wastell, 1995). In 1995 the FDA approved the first treatment for ALS, riluzole, which is thought to work in part by inhibiting the release of presynaptic glutamate. In 1990 ALS patients were found to have selective abnormalities in the NAAG/GCPII system. Specifically, reductions in spinal cord tissue NAAG, elevations in CSF NAAG, and increased GCPII activity were reported (Rothstein et al., 1990; Tsai et al., 1991). It is not surprising, therefore, that GCPII inhibition was tested as a treatment for ALS (Ghadge et al., 2003). Ghadge and colleagues found that daily administration of 2-MPPA to SOD1 mutant mice prolonged survival and decreased physical and neuropathological signs of disease. Interestingly, 2-MPPA effects were unaffected by mGlu2/3 receptor antagonism, suggesting preferential involvement of NMDA receptor blockade as the beneficial mechanism of action of GCPII inhibition in ALS.

3.3 Schizophrenia The glutamate hypothesis of schizophrenia was initially proposed based on the observation that NMDA receptor antagonists induced positive and

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negative symptoms in healthy individuals that resembled schizophrenia (Halberstadt, 1995; Krystal et al., 1994). In 1995 the Coyle laboratory reported increased NAAG levels in the brains of patients with schizophrenia, positing the theory that the elevated NAAG could serve as an endogenous NMDA receptor blocker, thereby inducing a hypoglutamatergic state (Tsai et al., 1995). Predictably, many laboratories then chose to study the role of GCPII, NAAG, and glutamate in schizophrenia. GCPII inhibitors have shown promising efficacy in several animal models of schizophrenia. Similar to as in humans, schizophrenia can be induced in mice by injecting glutamate receptor antagonists, such as dizocilpine (MK-801) and PCP. GCPII expression is increased in the PCP model (Flores & Coyle, 2003), and neuroprotection and amelioration of motor activation are afforded by GCPII inhibitor administration, first reported by Neale and colleagues in 2004 (Olszewski et al., 2004). Both 2-PMPA and ZJ-43 induced an increase in extracellular NAAG levels while reversing motor activation caused by PCP (Zuo et al., 2012), and cognitive impairment was dose dependently ameliorated by GCPII inhibition (Olszewski, Janczura, et al., 2012). The benefits of GCPII inhibition in schizophrenia models are at least partially due to NAAG-mediated group II metabotropic receptor activation (Olszewski et al., 2008), specifically the mGlu3 receptor. 2-PMPA is effective in treating PCP-induced motor activation in mGlu2 receptor knockout mice but ineffective in mGlu3 receptor knockout mice (Olszewski, Bzdega, et al., 2012). The GCPII inhibitor 2-MPPA dose dependently inhibits behavioral signs of schizophrenia in the MK-801 and D-amphetamine rodent models, and these effects are reversed by mGlu2/3 receptor antagonist coadministration (Takatsu, Fujita, Tsukamoto, Slusher, & Hashimoto, 2011; Zuo et al., 2012). Interestingly, administration of a mGluR2/3 agonist alone has limited efficacy as compared to the GCPII inhibitor ZJ-43, suggesting that activation of mGlu3 receptor by NAAG contributes but is not completely responsible for its beneficial effects (Takatsu et al., 2011). A number of human studies have examined changes in NAAG, GCPII, and the mGlu3 receptor in schizophrenia. Reported activity and expression levels of brain GCPII are conflicting due to different methodologies employed and different brain regions analyzed (Ghose et al., 2009, 2004; Guilarte et al., 2008). Polymorphisms in GRM3, the gene for mGlu3 receptor, are associated with changes in glutamatergic neurotransmission, decreased cognitive function, and heightened risk for developing schizophrenia (Egan et al., 2004; Ripke et al., 2014). Because mGlu3, but not

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mGlu2, has emerged as a genetic marker, and because a lack of efficacy has been observed for nonspecific group II metabotropic glutamate receptor agonists or mGlu2 PAMs (Ellaithy, Younkin, Gonza´lez-Maeso, & Logothetis, 2015; Li, Hu, Li, & Gao, 2015), therapies selectively targeting the mGlu3 receptor, such as pharmacological upregulation of NAAG, are of great interest. In fact, higher brain NAAG levels are associated with improved cognition in patients with schizophrenia ( Jessen et al., 2013). These data, along the efficacy of structurally diverse GCPII inhibitors in a wide range of schizophrenia preclinical models, suggest that GCPII inhibition may be a promising new therapeutic strategy for schizophrenia.

3.4 Multiple Sclerosis Glutamate excitotoxicity has long been implicated in the pathogenesis of multiple sclerosis (MS), with elevated glutamate levels in active CNS lesions and in the CSF of patients during relapse (Stover et al., 1997). Studies in experimental autoimmune encephalomyelitis (EAE) animal models reveal amelioration of physical signs of disease following treatment with glutamate receptor antagonists (Sulkowski, Dabrowska-Bouta, & Struzynska, 2013; Wallstrom et al., 1996). These results have not translated to the clinic, however, as a trial evaluating the glutamate receptor antagonist memantine did not improve symptoms, but rather caused reversible neurological deficits (Villoslada, Arrondo, Sepulcre, Alegre, & Artieda, 2009). Cognitive impairment affects approximately half of all MS patients. Recently our group reported positive correlations between hippocampal NAAG levels and cognition in relapsing-remitting MS patients (Rahn et al., 2012), showing that higher NAAG is associated with better cognition. Subsequent EAE studies demonstrated no change in physical disability, but a dose-dependent reversal of cognitive impairment and restoration of brain NAAG levels due to daily treatment with a GCPII inhibitor (Hollinger, Alt, Riehm, Slusher, & Kaplin, 2016; Rahn et al., 2012). Based on these findings, GCPII inhibition may not be an efficacious treatment for the physical signs of MS, but could provide relief for those patients suffering from learning and memory impairments.

3.5 Drug Addiction While the majority of drug addiction research is focused on dopaminergic signaling, the importance of glutamate pathways in drug addiction, relapse, and tolerance is also widely recognized (Madayag et al., 2007; Pulvirenti,

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Maldonado-Lopez, & Koob, 1992; Uys & LaLumiere, 2008). Inhibition of GCPII with 2-PMPA attenuates the development of cocaine-induced behavioral sensitization in rats (Shippenberg, Rea, & Slusher, 2000) and causes a dose-dependent decrease in cocaine-kindled seizures (Witkin et al., 2002). Inhibition of GCPII with either 2-MPPA or 2-PMPA also decreases cocaine relapse in rats (Peng et al., 2010; Xi et al., 2010), and this effect was blocked with a mGluR2/3 antagonist (Xi et al., 2010). The significance of NAAG and mGlu3 receptor in mediating drug-related behavior is further highlighted in rodent studies that show attenuation of cocaine-seeking behavior and reinstatement of addiction following administration of a mGluR2/3 agonist (Cannella et al., 2013; Peters & Kalivas, 2006). Favorable effects of GCPII inhibition have also been observed in rodent models of morphine tolerance (Kozela et al., 2005; Popik, Kozela, Wrobel, Wozniak, & Slusher, 2003).

3.6 Alzheimer's Disease Reductions in brain NAAG were reported in Alzheimer’s disease (AD) patients vs healthy controls nearly two decades ago ( Jaarsma, Veenmavan der Duin, & Korf, 1994; Passani, Vonsattel, Carter, et al., 1997; Passani, Vonsattel, & Coyle, 1997). GCPII was dismissed as a viable therapeutic target for AD, however, due to a report that GCPII had amyloid-beta (Aβ)-degrading properties, and that GCPII inhibition led to increased cerebral Aβ load (Kim, Chae, Koh, Lee, & Jo, 2010). However, two independent laboratories could not reproduce these findings (Alt, Stathis, Rojas, & Slusher, 2013; Sedlak et al., 2013). Our laboratory has confirmed the postmortem changes in NAAG and extended the finding to several AD mouse models (unpublished observation). The reductions in brain NAAG and the lack of involvement of GCPII in Aβ degradation, along with reports of elevated glutamate in AD (Kaiser et al., 2010), provide justification to evaluate GCPII inhibition as a treatment for AD in future studies.

3.7 Pain Abnormal glutamate transmission has been well implicated in acute and chronic pain (Bleakman, Alt, & Nisenbaum, 2006; Zhou, Bonasera, & Carlton, 1996). NMDA receptor antagonists are effective in preclinical models of pain (Zhou, Chen, & Pan, 2011). AMPA/kainate (Zhou et al., 1996) and metabotropic receptors (Zhou et al., 2011) blockers also modulate pain, although their role is less defined (Fisher & Coderre, 1996; Goudet et al., 2009). Multiple GCPII inhibitors have shown efficacy in acute and

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inflammatory pain models, with 2-PMPA being the most extensively studied (Yamamoto, Nozaki-Taguchi, & Sakashita, 2001; Zhou et al., 2005). 2-PMPA applied intrathecally (i.t.) reduces nociceptive behaviors in the rat formalin and carrageenan models (Yamamoto, Nozaki-Taguchi, & Sakashita, 2001; Yamamoto, Nozaki-Taguchi, Sakashita, & Inagaki, 2001). Intracerebroventricular (i.c.v.) 2-PMPA produces analgesia to formalin (Yamamoto, Kozikowski, Zhou, & Neale, 2008) and spinal application reduces wind-up sensitization phenomena after carrageenan inflammation (Carpenter et al., 2003). Systemic administration of 2-PMPA reduces allodynia as well as ectopic discharges in both the chronic constrictive and partial sciatic nerve ligation neuropathic pain models (Chen, Wozniak, Slusher, & Pan, 2002; Nagel et al., 2006). The urea-based inhibitors have also proven efficacious following direct brain microinjection, i.c.v. or i.t. administration (Kozikowski et al., 2004; Yamamoto et al., 2004, 2008) as well as following systemic or local injection (Adedoyin et al., 2010; Yamada et al., 2012; Yamamoto et al., 2007). Thiol-based GCPII inhibitors (eg, 2-MPPA and E2072) are also effective in reducing pain in chronic neuropathy models (Majer et al., 2003; Vornov et al., 2013; Wozniak et al., 2012). The majority of studies support a spinal or local mediated site of action (Chen et al., 2002; Kozikowski et al., 2004; Yamamoto, Nozaki-Taguchi, & Sakashita, 2001), but there is also evidence to suggest a brain-mediated component (Adedoyin et al., 2010; Yamamoto et al., 2008). Multiple studies have shown that the analgesic effects of GCPII inhibitors can be attenuated by mGlu3 receptor antagonists (Thomas et al., 2006; Yamamoto et al., 2004, 2007); however, the possibility of mediation of GCPII analgesic effects via NMDA receptor antagonism has not been well explored.

3.8 Peripheral Neuropathy In addition to acute and inflammatory pain, damage to the PNS can result in a chronic peripheral neuropathy, which leads to a range of neuronal functional, morphological, and perceptual abnormalities. Causes of peripheral neuropathy include infection, traumatic injury, medication (eg, chemotherapy), and disease (eg, diabetes). Because excessive glutamatergic signaling is present in peripheral neuropathy (Yogeeswari, Semwal, Mishra, & Sriram, 2009) and GCPII is abundant in the PNS (Marmiroli, Slusher, & Cavaletti, 2012), GCPII inhibitors have also been explored in this condition. In preclinical models, GCPII inhibitors have provided functional improvement in

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nerve conduction velocity deficits as well as protection against morphological deficits encountered in multiple chemotherapy-induced neuropathy models (Carozzi et al., 2010; Wozniak et al., 2012; Zhang, Murakawa, Wozniak, Slusher, & Sima, 2006). GCPII inhibitors are also effective in reducing thermal hyperalgesia, nerve conduction slowing, as well as morphological abnormalities in the BBW rat model of diabetic neuropathy (Zhang et al., 2006, 2002). The mechanism for these beneficial effects is not clearly defined, but the effects of GCPII inhibition suggest a role for excessive glutamate. For example, it is known that hyperglycemia caused by diabetic neuropathy leads to ischemia in nerves, and ischemia results in mitochondrial function perturbation, possibly resulting in glutamate excitotoxicity (Tomiyama et al., 2005; Zhong, Luo, & Jiang, 2014). In addition, our laboratory found that inhibiting GCPII activity in Schwann cells in vitro dramatically increases myelin formation of dorsal root ganglion axons (unpublished observation), suggesting potential glial trophic effects of increased NAAG. Not only do myelin levels increase, but axons are myelinated throughout their length with several internodes, comparable to a normal myelination pattern observed in vivo. Similar evaluations conducted in mice with sciatic nerve crush reveal that treatment with 2-PMPA produces a significantly thicker myelin sheath postcrush, without apparent changes in the rate of axonal regeneration. These findings are particularly exciting in that this has important therapeutic implications for chemotherapy and diabetic-induced peripheral neuropathy, where axonal degeneration and myelination abnormalities are common.

4. CONCLUSION Joe Coyle’s “nagging question” of the function of NAAG has in large part been answered by the development of specific and selective GCPII inhibitors, since these compounds can attenuate excess glutamate release and enhance brain NAAG, resulting in decreased NMDA receptor activation, increased mGlu3 receptor activation, and robust neuroprotection. Under normal conditions, GCPII activity is low and NAAG serves as an agonist at mGlu3 receptors, causing feedback inhibition and release of trophic factors. Under pathological conditions, however, GCPII activity and NAAG release is increased, leading to excess extracellular glutamate, hyperstimulation of NMDA receptors, and excitotoxic damage. Thus, GCPII inhibitors remain an extremely promising therapy for a wide range of

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neurological and psychiatric diseases involving excess glutamate. Unfortunately, only one GCPII inhibitor has ever been tested in man (van der Post et al., 2005) but was discontinued due to off-target side effects. The new delivery approaches and prodrug development discussed herein promise to provide a future path for drug development so that the enormous amounts of preclinical efficacy data may finally be confirmed or disproved in clinical studies.

CONFLICT OF INTEREST The authors have no conflicts of interest to declare.

ACKNOWLEDGMENTS The authors thank Jennifer Fairman, CMI, FAMI, for her Figs. 1 and 3 artwork, and Angela Rubin for her technical assistance with figures and references. This work was supported, in part, by NIH Grant R01 CA161056 awarded to B.S.S.

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