SYNOPSIS Gamma-glutamyl transpeptidase (GGT) is an essential enzyme for maintaining cysteine homeostasis, leukotriene synthesis, metabolism of glutathione-conjugates and catabolism of extracellular glutathione. Overexpression of GGT has been implicated in many pathologies, and clinical inhibitors of GGT are under development for use in the treatment of asthma, cancer and other diseases. Inhibitors are generally characterized using synthetic GGT substrates. This study of uncompetitive inhibitors of GGT, has revealed that the potency with which compounds inhibit GGT activity in the standard biochemical assay does not correlate with the potency with which they inhibit the physiological reaction catalyzed by GGT. Kinetic studies provided insight into the mechanism of inhibition. Modifications to the sulfobenzene or distal benzene ring of the uncompetitive inhibitor, OU749, affected activity. One of the most potent inhibitors was identified among a novel group of analogs with an amine group para on the benzosulfonamide ring. New, more potent uncompetitive inhibitors of the physiological GGT reaction were found to be less toxic than the glutamine-analogs that have been tested clinically. Development of non-toxic inhibitors is essential for exploiting GGT as a therapeutic target.

A novel class of inhibitors of the enzyme gamma-glutamyl transpeptidase (GGT) were evaluated. OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.

Objective: This study aimed to compare provider gestalt to assigned Narx score, a common prescription drug monitoring program (PDMP) component that gauges the patient's risk of misuse or abuse.Design: This is a prospective, anonymous survey from advanced practice providers (APPs), emergency medicine residents, and emergency medicine attendings. Setting: Data from two emergency departments (EDs) within the OhioHealth network were included. One hospital is a 213-bed academic, community hospital. The other hospital is a 434-bed academic level I trauma center.Patients, Participants: The survey was open to all providers. Exclusion criteria for patients included prior knowledge of the patient and/or their Narx score, or cancer-related pain.Interventions: Surveys were collected over a 3-month period. Variables included provider type and level of experience, participant demographics, provider gestalt, and the patient's actual Narx score.Main Outcome Measure(s): Primary outcome was the ability of providers to accurately estimate a patient's Narx score. Groups were defined as Match = No (gestalt and actual score did not match) and Match = Yes (gestalt and actual score matched). Various characteristics were compared between these two groups.Results: Providers were able to accurately estimate actual Narx score (72.7 percent). The Match = Yes group was younger (p = 0.01). Dental pain was more common in the Match = No group, 11.5 percent versus 0 percent (p = 0.02). Match = No group also had a higher incidence of triggers. Specifically, any trigger (p = 0.006), explicitly asking for pain medication (p = 0.03), and asking for opioids by name (p = 0.03). Every 10-year decrease in age showed a 1.5 times increased likelihood of accurately estimating Narx score (p = 0.02). Having no triggers showed a three times increased likelihood of accurately estimating Narx score (p = 0.02). Prescribing was largely unchanged after viewing the actual Narx score. Conclusions: Providers are able to accurately estimate Narx score, though there are limiting factors. Older patients, those with dental pain, and those who give specific triggers are more difficult to estimate. Providers did not change their prescribing patterns after viewing the actual Narx score. Overestimation versus underestimation of Narx score was not directly studied.

Resistance to chemotherapy is a major impediment to successful cancer treatment. To enhance therapeutic intervention, we are developing novel compounds that target the redox-adaptive response in tumors, a process that increases intracellular glutathione levels. Elevated levels of intracellular glutathione render tumors resistant to chemotherapy and radiation therapy by protecting against reactive oxygen species, inactivating electrophilic metabolites of chemotherapy drugs, and blocking Bcl-2-mediated apoptosis. Glutathione synthesis requires free intracellular cysteine. Cells under oxidative stress synthesize high levels of glutathione and the supply of cysteine becomes rate limiting for glutathione synthesis. We have developed a novel strategy to deplete the free cysteine available to tumor cells, thereby blocking the drug resistance in tumor cells that have undergone redox-adaption. To accomplish this in humans, two enzymes must be inhibited, gamma-glutamyl transpeptidase (aka gamma-glutamyl transferase, GGT1) and gamma-glutamyl leukotrienase (aka, GGT-rel, GGT5). In humans, both GGT1 and GGT5 cleave the gamma-glutamyl bond of extracellular glutathione initiating the breakdown of glutathione into its constituent amino acids, glutamate, cysteine and glycine. Inhibiting GGT1 and GGT5 depletes the body of cysteine by blocking the catabolism of glutathione in the kidney resulting in the excretion of glutathione in the urine. Inhibiting these enzymes also blocks the tumor's ability to use extracellular glutathione as a source of cysteine.Our approach not only reduces the ability of the tumor to synthesize glutathione, it also reduces the cysteine (and the total amount of reduced thiol) in the tumor. Previously we used high-throughput screening and identified OU749 (N-[5-(4-methoxybenzyl)-1,3,4-thiadiazol-2-yl]benzenesulfonamide), a novel inhibitor of GGT1. We have subsequently developed a new assay system, which provides an unambiguous method to evaluate the inhibition of the physiologic reactions catalyzed by both GGT1 and GGT5. Kinetic data obtained from the assay can be used to directly evaluate the potency and mechanism of action of each inhibitor. Synthesis of an extensive array of OU749 analogues and evaluation of the inhibitory activity of these analogues in our new assay system has resulted in the identification of two new lead compounds, HL-6 and HL-7. These new leads are uncompetitive inhibitors of both human GGT1 and GGT5. These compounds are orders of magnitude less toxic than the currently available GGT1 inhibitors. Continued optimization of these lead compounds will provide new drugs for potentiating cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 818. doi:1538-7445.AM2012-818