GCPII and its close homolog GCPIII: from a neuropeptidase to a cancer marker and beyond.

Glutamate carboxypeptidases II and III (GCPII and GCPIII) are highly homologous di-zinc metallopeptidases belonging to the M28 family. These enzymes are expressed in a variety of tissues, including the brain, prostate, kidney, testis and jejunum. GCPII has been recognized as a neuropeptidase in the central nervous system, as a folate hydrolase participating in absorption of folates in the jejunum and, most importantly, as a prostate-specific membrane antigen that is highly expressed in prostate adenocarcinoma. Furthermore, it has been identified in the neovasculature of most human solid tumors. In contrast, GCPIII has not been associated with any specific physiological function or pathology, and its expression, activity and inhibition have not been as well-studied. In this review, we provide an overview of the current understanding of the structure, enzymatic activity, substrate specificity, and tissue distribution of these two homologous enzymes. We discuss their potential physiological functions and describe the available animal models, including genetically modified mice. We also review the potential use of specific monoclonal antibodies and small-molecule inhibitors recognizing GCPII/III for diagnosis, imaging and experimental therapy of human cancers and other pathologies.

A novel PSMA/GCPII-deficient mouse model shows enlarged seminal vesicles upon aging.

BACKGROUND: Prostate-specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II (GCPII), is an important diagnostic and therapeutic target in prostate cancer. PSMA/GCPII is also expressed in many healthy tissues, but its function has only been established in the brain and small intestine. Several research groups have attempted to produce PSMA/GCPII-deficient mice to study the physiological role of PSMA/GCPII in detail. The outcomes of these studies differ dramatically, ranging from embryonic lethality to production of viable PSMA/GCPII-deficient mice without any obvious phenotype. METHODS: We produced PSMA/GCPII-deficient mice (hereafter also referred as Folh1-/- mice) by TALEN-mediated mutagenesis on a C57BL/6NCrl background. Using Western blot and an enzyme activity assay, we confirmed the absence of PSMA/GCPII in our Folh1-/- mice. We performed anatomical and histopathological examination of selected tissues with a focus on urogenital system. We also examined the PSMA/GCPII expression profile within the mouse urogenital system using an enzyme activity assay and confirmed the presence of PSMA/GCPII in selected tissues by immunohistochemistry. RESULTS: Our Folh1-/- mice are viable, breed normally, and do not show any obvious phenotype. Nevertheless, aged Folh1-/- mice of 69-72 weeks exhibit seminal vesicle dilation, which is caused by accumulation of luminal fluid. This phenotype was also observed in Folh1+/- mice; the overall difference between our three cohorts (Folh1-/- , Folh1+/- , and Folh1+/+ ) was highly significant (P < 0.002). Of all studied tissues of the mouse urogenital system, only the epididymis appeared to have a physiologically relevant level of PSMA/GCPII expression. Additional experiments demonstrated that PSMA/GCPII is also present in the human epididymis. CONCLUSIONS: In this study, we provide the first evidence characterizing the reproductive tissue phenotype of PSMA/GCPII-deficient mice. These findings will help lay the groundwork for future studies to reveal PSMA/GCPII function in human reproduction.

Mouse glutamate carboxypeptidase II (GCPII) has a similar enzyme activity and inhibition profile but a different tissue distribution to human GCPII.

Glutamate carboxypeptidase II (GCPII), also known as prostate-specific membrane antigen (PSMA) or folate hydrolase, is a metallopeptidase expressed predominantly in the human brain and prostate. GCPII expression is considerably increased in prostate carcinoma, and the enzyme also participates in glutamate excitotoxicity in the brain. Therefore, GCPII represents an important diagnostic marker of prostate cancer progression and a putative target for the treatment of both prostate cancer and neuronal disorders associated with glutamate excitotoxicity. For the development of novel therapeutics, mouse models are widely used. However, although mouse GCPII activity has been characterized, a detailed comparison of the enzymatic activity and tissue distribution of the mouse and human GCPII orthologs remains lacking. In this study, we prepared extracellular mouse GCPII and compared it with human GCPII. We found that mouse GCPII possesses lower catalytic efficiency but similar substrate specificity compared with the human protein. Using a panel of GCPII inhibitors, we discovered that inhibition constants are generally similar for mouse and human GCPII. Furthermore, we observed highest expression of GCPII protein in the mouse kidney, brain, and salivary glands. Importantly, we did not detect GCPII in the mouse prostate. Our data suggest that the differences in enzymatic activity and inhibition profile are rather small; therefore, mouse GCPII can approximate human GCPII in drug development and testing. On the other hand, significant differences in GCPII tissue expression must be taken into account when developing novel GCPII-based anticancer and therapeutic methods, including targeted anticancer drug delivery systems, and when using mice as a model organism.

Malonate-based inhibitors of mammalian serine racemase: kinetic characterization and structure-based computational study.

Overactivation of NMDA receptors has been implicated in various neuropathological conditions, including brain ischaemia, neurodegenerative disorders and epilepsy. Production of d-serine, an NMDA receptor co-agonist, from l-serine is catalyzed in vivo by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine racemase. Specific inhibition of this enzyme has been proposed as a promising strategy for treatment of neurological conditions caused by NMDA receptor dysfunction. Here we present the synthesis and activity analysis of a series of malonate-based inhibitors of mouse serine racemase (mSR). The compounds possessed IC50 values ranging from 40 ± 11 mM for 2,2-bis(hydroxymethyl)malonate down to 57 ± 1 µM for 2,2-dichloromalonate, the most effective competitive mSR inhibitor known to date. The structure-activity relationship of the whole series in the human orthologue (hSR) was interpreted using Glide docking, WaterMap analysis of hydration and quantum mechanical calculations based on the X-ray structure of the hSR/malonate complex. Docking into the hSR active site with three thermodynamically favourable water molecules was able to discern qualitatively between good and weak inhibitors. Further improvement in ranking was obtained using advanced PM6-D3H4X/COSMO semiempirical quantum mechanics-based scoring which distinguished between the compounds with IC50 better/worse than 2 mM. We have thus not only found a new potent hSR inhibitor but also worked out a computer-assisted protocol to rationalize the binding affinity which will thus aid in search for more effective SR inhibitors. Novel, potent hSR inhibitors may represent interesting research tools as well as drug candidates for treatment of diseases associated with NMDA receptor overactivation.

Inhibition of human serine racemase, an emerging target for medicinal chemistry.

Proteins of glutamatergic NMDA receptor signaling pathways have been studied as targets for intervention in a variety of neuropathological conditions, including neurodegenerations, epilepsy, neuropathic pain, drug addiction, and schizophrenia. High activity NMDA-blocking agents have been designed to treat some of these disorders; however, their effect is often compromised by undesirable side effects. Therefore, alternative ways of modulating NMDA receptor function need to be sought after. The opening of the NMDA receptor ion channel requires occupation of two distinct binding sites, the glutamate site and the glycine site. It has been shown that D-serine, rather than glycine, can trigger the physiological NMDA receptor function. D-serine is a product of the activity of a specific enzyme, serine racemase (SR), which was identified a decade ago. SR has therefore emerged as a new potential target for the NMDA-receptor-based diseases. There is evidence linking increased levels of D-Ser to amyotrophic lateral sclerosis and Alzheimer's disease and decreased concentrations of D-serine to schizophrenia. SR is a pyridoxal-5'-phosphate dependent enzyme found in the cytosol of glial and neuronal cells. It is activated by ATP, divalent cations like Mg(2+) or Ca(2+), and reducing agents. This paper reviews the present literature on the activity and inhibition of mammalian SRs. It summarizes approaches that have been applied to design SR inhibitors and lists the known active compounds. Based on biochemical and docking analyses, i) we delineate for the first time the ATP binding site of human SR, and ii) we suggest possible mechanisms of action for the active compounds. In the end, we discuss the SR features that make the discovery of its inhibitors a challenging, yet very important, task of medicinal chemistry.