Another example of medicinally relevant protease that recently became the major topic in my laboratory is glutamate carboxypeptidase II, an enzyme implicated in the liberation of glutamate in brain synapsis. Glutamate is an activator of ionotropic glutamate receptors such as N-methyl D-aspartate receptors (NMDA); their overactivation has been implicated in neurodegenerative disorders including stroke and amyotrophic lateral sclerosis (ALS). Conventional therapy tries to block post-synaptic glutamate receptors (mostly NMDA receptors) with small molecules. Unfortunately, NMDA receptor antagonists cause severe side effects, probably as a result of insufficient specificity of these compounds.
An alternative to the blocking of glutamate receptors is to reduce the levels of presynaptic glutamate. One important source of presynaptic glutamate is N-acetyl-L-aspartyl-L-glutamate (NAAG). This neurotransmitter is found in millimolar concentrations in the brain, where it can undergo hydrolysis to N-acetyl-L-aspartate (NAA) and L-glutamate by glutamate carboxypeptidase II (GCPII). GCPII inhibition was shown to be a promising approach for the treatment of stroke, ALS, chronic pain, diabetic neuropathy, and other neurological disorders associated with glutamate excitotoxicity.
Interestingly, GCPII is identical to prostate-specific membrane antigen (PSMA), a tumor marker in prostate cancer. GCPII is also found in the membrane brush border of the small intestine, where it acts as a folate hydrolase. GCPII inhibitors could be thus helpful in the imaging and treatment of tumors in the cases where folate is required for their growth.
From the biochemical perspective, GCPII is a 750-residue, membrane-associated, heavily glycosylated dinuclear zinc peptidase. In collaboration with R. Hilgenfeld’s group in Luebeck we were able to determine the crystal structure of the fully glycosylated extracellular domains of GCPII in complex with a specific inhibitor, with phosphate ion, and with glutamate, the product of substrate cleavage (Mesters et al. 2006). We show that the binding of the specific inhibitor, and very likely that of substrate, is associated with a major rearrangement of a substrate-binding loop, revealing an induced-fit mechanism for the enzyme. These findings provide the basis for the design of the next generation inhibitors of GCPII, especially in view of the fact that all previously published structural models of the enzyme-inhibitor complex are incorrect.
These tools and preliminary findings enable us to address a number of
specific questions, namely: what is the mechanism of action of GCPII?
What are the residues critical for its activity, substrate specificity,
and inhibition? Can we design more potent inhibitors of the enzyme?
What is its role outside the cells of the central nervous system,
namely in tumor progression? Could GCPII be used as a molecular address
for specific targeting of cytostatics? Is GCPII activity necessary
for tumour growth? Are there any physiological ligand(s) of GCP II both
in the prostate and in the central nervous system?
Selected papers: