Synthesis and Biological Evaluation of Thiol-Based Inhibitors of Glutamate Carboxypeptidase II:  Discovery of an Orally Active GCP II Inhibitor

Majer, Pavel; Jackson, Paul F.; Delahanty, Greg; Grella, Brian; Ko, Yao Sen; Li, Weixing; Li, Qun; Maclin, Keith M.; Poláková, Jana; Shaffer, Kathryn A.; Stoermer, Doris; Vitharana, Dilrukshi; Wang, Eric Yanjun; Zakrzewski, Anthony; Rojas, Camilo; Slusher, Barbara S.; Wozniak, Krystyna M.; Burak, Eric; Limsakun, Tharin; Tsukamoto, Takashi

doi:10.1021/jm020515w

Cited by 93 publications

(117 citation statements)

References 35 publications

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“…Whereas decreased glutamate concentration due to GCPII inhibition could certainly contribute to cognitive sparing in EAE, it is hypothesized that NAAG is the crucial signaling messenger that ameliorated cognitive function in the present study. Inhibition of GCPII, which induces a measurable up-regulation in NAAG, has proved to be beneficial in treating various preclinical models of neurological disease (18,35,36). Activation of mGluR3 on presynaptic neurons by NAAG decreases glutamate release (37)(38)(39), potentially promoting the survival of neurons crucial to cognitive function.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Glutamate Carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis

Rahn

Watkins

Alt³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Half of all patients with multiple sclerosis (MS) experience cognitive impairment, for which there is no pharmacological treatment. Using magnetic resonance spectroscopy (MRS), we examined metabolic changes in the hippocampi of MS patients, compared the findings to performance on a neurocognitive test battery, and found that N-acetylaspartylglutamate (NAAG) concentration correlated with cognitive functioning. Specifically, MS patients with cognitive impairment had low hippocampal NAAG levels, whereas those with normal cognition demonstrated higher levels. We then evaluated glutamate carboxypeptidase II (GCPII) inhibitors, known to increase brain NAAG levels, on cognition in the experimental autoimmune encephalomyelitis (EAE) model of MS. Whereas GCPII inhibitor administration did not affect physical disabilities, it increased brain NAAG levels and dramatically improved learning and memory test performance compared with vehicle-treated EAE mice. These data suggest that NAAG is a unique biomarker for cognitive function in MS and that inhibition of GCPII might be a unique therapeutic strategy for recovery of cognitive function.G lutamate carboxypeptidase II (GCPII), previously called Nacetylated-α-linked acidic dipeptidase (NAALADase), is a membrane-bound enzyme expressed on the surface of astrocytes that catalyzes the cleavage of N-acetylaspartylglutamate (NAAG) into N-acetylaspartate (NAA) and glutamate ( Fig. 1) (1, 2). NAAG, the most abundant neuropeptide in the mammalian brain, is a selective agonist for metabotropic glutamate receptor 3 (mGluR3) (1, 2). mGluR3s are expressed on the surface of presynaptic neurons and astrocytes in the CNS. Activation of mGluR3s by NAAG reduces cAMP and cGMP levels, inhibits glutamate release, and stimulates a release of transforming growth factor beta (1, 2). A recent study reported impaired performance on spatial reference and working memory tasks in mGluR2/3 knockout mice (3), suggesting a role for group II mGluRs in cognitive function.Cognitive impairment is a frequent comorbidity of many neurological diseases, including multiple sclerosis (MS). MS affects over 2 million individuals worldwide, and ∼40-65% of all MS patients experience cognitive impairment (4). The most commonly and severely affected facets of cognition in MS patients are anterograde episodic memory, working memory, and processing speed (5-7), and these impairments strongly contribute to the high frequency of unemployment in MS patient populations (8). Global brain atrophy, candidate genetic risk factors, accelerated inflammatory processes (7), and dysregulated signaling pathways (9) have been implicated as contributing factors, but an incomplete understanding of the molecular mechanisms behind cognitive impairment in MS has prevented the development and Food and Drug Administration (FDA) approval of drug therapies.The most widely used animal model of MS, experimental autoimmune encephalomyelitis (EAE), is a valuable tool for developing treatments for MS. Three FDA-approved disease-modifying agents were deve...

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Section: Discussionmentioning

confidence: 99%

Inhibition of Glutamate Carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis

Rahn

Watkins

Alt³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…R,S-2-thioglutaric acid was synthesized from glutaric acid using a literature procedure (26). AdoCbl was purchased from Sigma Chemical Company; solutions containing AdoCbl were kept in light-proofed vials and handled in dim light.…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

et al. 2006

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We have investigated the reaction of glutamate mutase with the glutamate analog, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with K i = 0.05 mM. However, rather than simply binding inertly at the active site, 2-thiolglutarate elicits cobalt-carbon bond homolysis and the formation of 5′-deoxyadenosine. The enzyme exhibits a complicated EPR spectrum in the presence of 2-thiolglutarate that is markedly different from any previously observed with the enzyme. The spectrum was well simulated by assuming that it arises from electron-electron spin coupling between a thioglycolyl radical and low-spin Co 2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical at ∼ 10 Å from the cobalt, and at a tilt angle of ∼ 70° to the normal of the corrin ring. This orientation is in good agreement with that expected from the crystal structure of glutamate mutase complexed with substrate. 2-thiolglutarate appears to react in a manner analogous to glutamate by first forming a thioglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfurstablized thioglycolyl radical. The thioglycolyl radical accumulates on the enzyme suggesting it is too stable to undergo further steps in the mechanism at a detectable rate. Keywordsenzyme; coenzyme-B 12 ; free radicals; isomerization; substrate analog; EPR spectrometry Adenosylcobalamin 1 (Coenzyme B 12 , AdoCbl) is the coenzyme for a group of enzymes that catalyze unusual rearrangement or elimination reactions, as well as for class II ribonucleotide reductases. In these enzymes, the coenzyme serves as a masked form of the 5′-deoxyadenosyl radical that is generated through homolytic fission of the AdoCbl cobalt-carbon bond (1-6). An interesting and still poorly understood aspect of these reactions is how these enzymes catalyze homolysis of the coenzyme because the generation of free radicals from stable, closed shell molecules is energetically highly unfavorable.In all cases, homolysis of AdoCbl is tightly coupled to the formation of substrate-based radicals (or in the case of ribonucleotide reductase, a protein-based thiyl radical). The initially formed 5′-deoxyadenosyl radical is extremely unstable and has never been observed spectroscopically.

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“…Replacement of the phosphonomethyl group with a thioalkyl group led to discovery of 2-MPPA [2-(3-mercaptopropyl) pentanedioic acid] (Majer et al, 2003), which was found to be orally available and was the first GCP II inhibitor to be administered to man (van der Post et al, 2005), where it was well tolerated and reached exposure levels similar to those required for therapeutic effects in animal models. To develop a dosing regimen for clinical use, the pharmacokinetics and pharmacodynamics were examined in more detail in animals to determine the required concentration and duration of exposure.…”

Section: Introductionmentioning

confidence: 99%

Pharmacokinetics and Pharmacodynamics of the Glutamate Carboxypeptidase II Inhibitor 2-MPPA Show Prolonged Alleviation of Neuropathic Pain through an Indirect Mechanism

Vornov

Wozniak

et al. 2013

J Pharmacol Exp Ther

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Glutamate carboxypeptidase II (GCP II) is a therapeutic target in neurologic disorders associated with excessive activation of glutamatergic systems. The potent, orally bioavailable GCP II inhibitor 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA) is effective in preclinical models of diseases where excess glutamate release is implicated, including neuropathic pain, and was the first GCP II inhibitor to be administered to man. The relationships between dosing regimen, pharmacokinetics, and analgesia in a neuropathic pain model were examined in rats to aid development of clinical dosing. The efficacy of oral 2-MPPA in the chronic constrictive injury model was not simply related to plasma concentrations. Even though maximal concentrations were observed within 1 hour of dosing, the analgesic effect took at least 8 days of daily dosing to become significant. The delay was not due to tissue drug accumulation since inhibitory concentrations of the drug were achieved in the nerve within 1 hour of dosing. There was also no accumulation of drug in plasma or tissue after multiple daily dosing. Effects were dependent on reaching a threshold concentration since dividing the daily dose led to a loss of effect. The analgesic effect outlasted plasma exposure and was maintained for days even after daily dosing was halted. The delayed onset, dependence on threshold plasma concentration, and sustained effects after exposure support the hypothesis that an indirect, long-lived mechanism of action exists. Although these longer lasting secondary mechanisms are not yet identified, daily clinical dosing of a GCP II inhibitor seems justified.

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Synthesis and Biological Evaluation of Thiol-Based Inhibitors of Glutamate Carboxypeptidase II: Discovery of an Orally Active GCP II Inhibitor

Cited by 93 publications

References 35 publications

Inhibition of Glutamate Carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis

Inhibition of Glutamate Carboxypeptidase II (GCPII) activity as a treatment for cognitive impairment in multiple sclerosis

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

Pharmacokinetics and Pharmacodynamics of the Glutamate Carboxypeptidase II Inhibitor 2-MPPA Show Prolonged Alleviation of Neuropathic Pain through an Indirect Mechanism

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