Glutamate carboxypeptidase activity in human skin biopsies as a pharmacodynamic marker for clinical studies

<p>Abstract</p> <p>Background</p> <p>Glutamate excitotoxicity is thought to be involved in the pathogenesis of neurodegenerative disease. One potential source of glutamate is N-acetyl-aspartyl-glutamate (NAAG) which is hydrolyzed to glutamate and N-acetyl-aspartate (NAA) in a reaction catalyzed by glutamate carboxypeptidase (GCP). As a result, GCP inhibition is thought to be beneficial for the treatment of neurodegenerative diseases where excess glutamate is presumed pathogenic. Both pharmacological and genetic inhibition of GCP has shown therapeutic utility in preclinical models and this has led to GCP inhibitors being pursued for the treatment of nervous system disorders in human clinical trials. Specifically, GCP inhibitors are currently being developed for peripheral neuropathy and neuropathic pain. The purpose of this study was to develop a pharmacodynamic (PD) marker assay to use in clinical development. The PD marker will determine the effect of GCP inhibitors on GCP enzymatic activity in human skin as measure of inhibition in peripheral nerve and help predict drug doses required to elicit pharmacologic responses.</p> <p>Methods</p> <p>GCP activity was first characterized in both human skin and rat paw pads. GCP activity was then monitored in both rodent paw pads and sciatic nerve from the same animals following peripheral administration of various doses of GCP inhibitor. Significant differences among measurements were determined using two-tailed distribution, equal variance student's t test.</p> <p>Results</p> <p>We describe for the first time, a direct and quantifiable assay to evaluate GCP enzymatic activity in human skin biopsy samples. In addition, we show that GCP activity in skin is responsive to pharmacological manipulation; GCP activity in rodent paws was inhibited in a dose response manner following peripheral administration of a potent and selective GCP inhibitor. Inhibition of GCP activity in rat paw pads was shown to correlate to inhibition of GCP activity in peripheral nerve.</p> <p>Conclusion</p> <p>Monitoring GCP activity in human skin after administration of GCP inhibitors could be readily used as PD marker in the clinical development of GCP inhibitors. Enzymatic activity provides a simple and direct measurement of GCP activity from tissue samples easily assessable in human subjects.</p

Structural basis for potent inhibition of d-amino acid oxidase by thiophene carboxylic acids

A series of thiophene-2-carboxylic acids and thiophene-3-carboxylic acids were identified as a new class of DAO inhibitors. Structure-activity relationship (SAR) studies revealed that small substituents are well-tolerated on the thiophene ring of both the 2-carboxylic acid and 3-carboxylic acid scaffolds. Crystal structures of human DAO in complex with potent thiophene carboxylic acids revealed that Tyr224 was tightly stacked with the thiophene ring of the inhibitors, resulting in the disappearance of the secondary pocket observed with other DAO inhibitors. Molecular dynamics simulations of the complex revealed that Tyr224 preferred the stacked conformation irrespective of whether Tyr224 was stacked or not in the initial state of the simulations. MM/GBSA indicated a substantial hydrophobic interaction between Tyr244 and the thiophene-based inhibitor. In addition, the active site was tightly closed with an extensive network of hydrogen bonds including those from Tyr224 in the stacked conformation. The introduction of a large branched side chain to the thiophene ring markedly decreased potency. These results are in marked contrast to other DAO inhibitors that can gain potency with a branched side chain extending to the secondary pocket due to Tyr224 repositioning. These insights should be of particular importance in future efforts to optimize DAO inhibitors with novel scaffolds

Development of L-γ-Methyleneglutamine-based compounds for cancer

Presenter: Md. Imdadul H. Khanhttps://egrove.olemiss.edu/pharm_annual_posters_2021/1014/thumbnail.jp

Resistance to change in Greek higher education

This thesis is a study of resistance to the changes in Greek higher education that were implemented within the framework of the 1999 Bologna Agreement of the European Union in the period 2007-2008. The changes that occurred were of great significance for Greece’s education system as they introduced important changes in the structure and function of Greek higher education. This thesis argues that the organisational culture that had been created throughout the history of Greek higher education was a powerful factor that provoked resistance to the new policies. Methodologically, the thesis argues that discourse, change and institutional culture are closely tied together, and that this is of crucial importance in creating, modifying, and sustaining change within higher education institutions. The process of resistance is examined through the theoretical framework of Critical Discourse Analysis (CDA) (Fairclough, 1995, 2000, 2001, 2003, 2009; Chouliaraki and Fairclough, 1999), and within this framework by applying the empirical-analytical method of the Discourse Historical Approach (Wodak and Meyer, 2009; Reisigl and Wodak, 2009). The framework and method for the study are also complemented by the discourse theory of Laclau and Mouffe (1985). The narrative of the thesis includes a critical examination of the hegemonic struggles that occurred in the 2007-2008 period, the perceptions and ideologies of the key stakeholders (politicians, university faculty, and student groups), and the ways in which the discourses about Greek higher education have been influenced by social, political, and institutional factors. Finally, the implications of the findings for adding to the existing knowledge about management and change in higher education institutions are discussed

Protection against glucose-induced neuronal death by NAAG and GCP II inhibition is regulated by mGluR3

Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective against long-term neuropathy in diabetic animals. In the current study, we have determined that the GCP II inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) is protective against glucose-induced programmed cell death (PCD) and neurite degeneration in dorsal root ganglion (DRG) neurons in a cell culture model of diabetic neuropathy. In this model, inhibition of caspase activation is mediated through the group II metabotropic glutamate receptor, mGluR3. 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist (S)-α-ethylglutamic acid (EGLU). In contrast, group I and III mGluR inhibitors have no effect on 2-PMPA neuroprotection. Furthermore, we show that two mGluR3 agonists, the direct agonist (2 R ,4 R )-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) and N -acetyl-aspartyl-glutamate (NAAG) provide protection to neurons exposed to high glucose conditions, consistent with the concept that 2-PMPA neuroprotection is mediated by increased NAAG activity. Inhibition of GCP II or mGluR3 may represent a novel mechanism to treat neuronal degeneration under high-glucose conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65724/1/j.1471-4159.2003.02321.x.pd

In vivo characterization of glutamine metabolism identifies therapeutic targets in clear cell renal cell carcinoma

Targeting metabolic vulnerabilities has been proposed as a therapeutic strategy in renal cell carcinoma (RCC). Here, we analyzed the metabolism of patient-derived xenografts (tumorgrafts) from diverse subtypes of RCC. Tumorgrafts from VHL-mutant clear cell RCC (ccRCC) retained metabolic features of human ccRCC and engaged in oxidative and reductive glutamine metabolism. Genetic silencing of isocitrate dehydrogenase-1 or isocitrate dehydrogenase-2 impaired reductive labeling of tricarboxylic acid (TCA) cycle intermediates in vivo and suppressed growth of tumors generated from tumorgraft-derived cells. Glutaminase inhibition reduced the contribution of glutamine to the TCA cycle and resulted in modest suppression of tumorgraft growth. Infusions with [amide-15N]glutamine revealed persistent amidotransferase activity during glutaminase inhibition, and blocking these activities with the amidotransferase inhibitor JHU-083 also reduced tumor growth in both immunocompromised and immunocompetent mice. We conclude that ccRCC tumorgrafts catabolize glutamine via multiple pathways, perhaps explaining why it has been challenging to achieve therapeutic responses in patients by inhibiting glutaminase