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.

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3 subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition. rational drug design ͉ aspartic proteases ͉ carboranes ͉ x-ray structure analysis ͉ virostatics

BackgroundHIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors.Methods and FindingsWe have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy.ConclusionsHIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

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-...