Membrane-bound glutamate carboxypeptidase II (GCPII) is a zinc metalloenzyme that catalyzes the hydrolysis of the neurotransmitter N-acetyl-L-aspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate and L-glutamate (which is itself a neurotransmitter). Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. Here, we report crystal structures of the extracellular part of GCPII in complex with both potent and weak inhibitors and with glutamate, the product of the enzyme's hydrolysis reaction, at 2.0, 2.4, and 2.2 Å resolution, respectively. GCPII folds into three domains: protease-like, apical, and C-terminal. All three participate in substrate binding, with two of them directly involved in C-terminal glutamate recognition. One of the carbohydrate moieties of the enzyme is essential for homodimer formation of GCPII. The three-dimensional structures presented here reveal an induced-fit substratebinding mode of this key enzyme and provide essential information for the design of GCPII inhibitors useful in the treatment of neuronal diseases and prostate cancer.

Conflict of interest: JDP, BSS, RR, and PM are scientific founders of Dracen Pharmaceuticals and possess equity. Technology arising in part from the studies described herein were patented by Johns Hopkins University and subsequently licensed to Dracen Pharmaceuticals.

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44±750) in Drosophila Schneider's cells and its puri®-cation to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on¯uorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate speci®city of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identi®ed as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity. Keywords: enzyme glycosylation, glutamate carboxypeptidase II, NAALADase, neuroprotection, zinc metallopeptidase. Membrane glutamate carboxypeptidase II (GCPII) is a metalloproteinase expressed in various tissues and organs (Berger et al. 1995;Chang et al. 1999). Its proteolytic activity was ®rstly recognized in brain cells, where it has been shown to be responsible for the cleavage of N-acetyl-Laspartyl-L-glutamate (NAAG) yielding free glutamate in the extracellular space. Based on the initial characterization of its substrate speci®city, the enzyme was termed N-acetylated alpha-linked-acidic-dipeptidase (NAALADase; Robinson et al. 1987).NAAG is an abundant neuropeptide found in millimolar concentrations in the brain (Coyle 1997). It is a mixed agonist/antagonist at N-methyl-D-aspartate ionotropic receptors and an agonist at the group II metabotropic glutamate receptors (mGlu; Wroblewska et al. 1993Wroblewska et al. , 1997. After release from pre-synaptic terminals, NAAG diffuses from the synaptic cleft and is rapidly hydrolysed by GCPII located on adjacent astrocytes. Free glutamate subsequently acts at the various glutamate receptor subtypes. Excessive receptor activation by glutamate is thought to be at least partially responsible for the neuronal injury caused by stroke (Fagg and Foster 1986). Inhibition of NAAG metabolism was suggested as a possible strategy to attenuate excitatory amino Abbreviations used: Ac, acetyl group; AMC, 7-amino-4-methylcoumarine; DIEA, diisopropylethylamine; DPM, disintegrations per minute; DPP IV, dipeptidyl peptidase IV; FAB-MS, fast atom bombardment mass spectrometry; FBS, foetal bovine serum; Fmoc, 9-...

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including d-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[ d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them, 5-chloro-benzo[ d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50 in the submicromolar range. Oral administration of CBIO in conjunction with d-serine enhanced the plasma and brain levels of d-serine in rats compared to the oral administration of d-serine alone.