A Novel, Species-specific Class of Uncompetitive Inhibitors of γ-Glutamyl Transpeptidase

Abstract

Expression of ␥-glutamyl transpeptidase (GGT) in tumors contributes to resistance to radiation and chemotherapy. GGT is inhibited by glutamine analogues that compete with the substrate for the ␥-glutamyl binding site. However, the glutamine analogues that have been evaluated in clinical trials are too toxic for use in humans. We have used high throughput screening to evaluate small molecules for their ability to inhibit GGT and have identified a novel class of inhibitors that are not glutamine analogues. These compounds are uncompetitive inhibitors, binding the ␥-glutamyl enzyme complex. OU749, the lead compound, has an intrinsic K i of 17.6 M. It is a competitive inhibitor of the acceptor glycyl-glycine, which indicates that OU749 occupies the acceptor site while binding to the ␥-glutamyl substrate complex. OU749 is more than 150-fold less toxic than the GGT inhibitor acivicin toward dividing cells. Inhibition of GGT by OU749 is species-specific, inhibiting GGT isolated from human kidney with 7-10-fold greater potency than GGT isolated from rat or mouse kidney. OU749 does not inhibit GGT from pig cells. Human GGT expressed in mouse fibroblasts is inhibited by OU749 similarly to GGT from human cells, which indicates that the species specificity is determined by differences in the primary structure of the protein rather than species-specific, post-translational modifications. These studies have identified a novel class of inhibitors of GGT, providing the basis for further development of a new group of therapeutics that inhibit GGT by a mechanism distinct from the toxic glutamine analogues.

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

“…Both livers were frozen within 12 h post-mortem. Membrane fractions were prepared as described previously (30). A 0.5% (v/v) Triton X-100 extract of the membrane fraction was used for SDS-PAGE and Western blot analysis, and a 0.5% (w/v) CHAPS extract was used for immunopurification of GGT and mass spectrometric analysis.…”

Novel Insights into Eukaryotic γ-Glutamyltranspeptidase 1 from the Crystal Structure of the Glutamate-bound Human Enzyme

“…To express the soluble ectodomain of the enzyme, the transmembrane domain (residues 1-27) was omitted and replaced with a tobacco etch virus (TEV) proteasecleavable polyhistidine tag. The N-terminal His 6 -tagged hGGT1 (P19440; amino acids 28 -569) open reading frame was integrated into the genome of the Pichia pastoris strain X-33 (15). Two 40-ml volumes of BMGY medium (Invitrogen) containing 100 g ml Ϫ1 of Zeocin were inoculated with the transformed X-33 strain and cultured at 30°C to high density overnight at 250 rpm.…”

“…Rational improvements on this design have been hampered by unresolved differences in the binding mechanism between human and bacterial GGTs that are not readily discernable from their primary structures. We have recently identified a novel class of uncompetitive species-specific hGGT1 inhibitors that are several orders of magnitude less toxic than the canonical substrate analog, acivicin (15)(16)(17). Strategic refinements of the structure of the founding member of this class of hGGT1 inhibitors, OU749, have also been impeded by the lack of an accurate structural model for the human enzyme (or any eukaryotic GGT).…”

Human GGT2 Does Not Autocleave into a Functional Enzyme: A Cautionary Tale for Interpretation of Microarray Data on Redox Signaling

“…The soluble fractions contained GGT eluted from the membrane. Transpeptidation assays contained 3 mM L-c glutamylparanitroanalide (LGpNA; Sigma, St. Louis, MO) as the substrate and 40 mM glycylglycine (glygly; Sigma) as the acceptor and were conducted as previously described (27). For hydrolysis assays Dc-glutamyl-paranitroanalide (D-GpNA; Bachem, Torrance, CA) was used as the substrate (48).…”