Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

Abstract

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog 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.

Fig. 1

GGT reactions with γ-Glutamyl Substrates. Cleavage of glutathione, a physiologic substrate of GGT, is shown. The γ-glutamyl bond between the γ-carbon of glutamate and the amine of cysteine is cleaved by GGT. The γ-carbon of glutamate forms an acyl covalent bond with the hydroxyl group on the side chain of Thr-381 of human GGT. The acyl bond can either be hydrolyzed releasing glutamate (Glu), or an acceptor nucleophile can attack forming a new γ-glutamyl compound (transpeptidation reaction). The reactions proceeds via a Ping-Pong mechanism.

Fig. 2

Synthesis scheme for the OU749 analogs. Synthesis and purification of the synthetic intermediates TDA1-10 (A) was required before synthesis of the OU749 analogs Compounds 2-4 and Compounds 6-20 (B). Synthesis of Compound 5 is derived from Compound 11 (C).

Fig. 3

Initial velocity pattern with D-GpNA or L-GpNA as the substrate and glygly as an accepter. Initial rates were measured at pH 7.4 and 37 °C. Double recipricol plots of the initial velocity versus the glygly concentration with 0.25 mM (◆),0.5 mM (●),1 mM (▲), or 3 mM (■) D-GpNA (A) or L-GpNA (B).

Fig. 4

Kinetic analysis of GGT inhibition by OU749 and SBS. The structure of OU749 (A). Double-reciprocal plot of the initial velocity of the hydrolysis of D-GpNA in the presence of 0 (◆), 15.2 μM (▼), 31.25 μM (▲), 62.5 μM (■), 125 μM (●) OU749 (B). The structure of SBS (C). Double reciprocal plot of the initial velocity of the hydrolysis of D-GpNA in the presence of 0 (◆), 6.25 mM (▼), 12.5 mM (▲), 25 mM (■), 50 mM (●) SBS (D). Double reciprocal plot of the initial velocity of the transpeptidation reaction with varying concentrations of L-GpNA and 40 mM glygly (E) or varying concentrations of glygly with 3 mM L-GpNA (F) in the presence of 0 (◆), 6.25 mM (▼), 12.5 mM (▲), 25 mM (■), 50 mM (●) SBS. Data shown are average triplicate values ± S.D. For many data points the S.D. is smaller than the symbol.

Fig. 5

Kinetic analysis of GGT inhibition by Compound 11. Substrate velocity curve of the hydrolysis reaction versus D-GpNA concentration in the presence of 0 (◆), 15.2 μM (▼), 31.25 μM (▲), 62.5 μM (■), 125 μM (●) Compound 11 (A). Velocity vs Compound 11 concentration (closed symbols) and V/K vs Compound 11 concentration (open symbols) plots illustrate the activation of the hydrolysis reaction (B). Double-reciprocal plots of the initial velocities of the transpeptidation reaction varying L-GpNA with 40 mM glygly (C) or varying glygly with 3 mM L-GpNA (D) in the presence of 0 (◆), 15.2 μM (▼), 31.25 μM (▲), 62.5 μM (■), 125 μM (●) Compound 11. Data shown are average triplicate values ± S.D.

Fig. 6

Time courses for the hydrolysis of D-GpNA in the presence of acivicin. The release of pNA from 0.287mM D-GpNA (A) or 2 mM D-GpNA (B) was monitored over time in the presence of acivicin (concentrations of acivicin noted in A). The reactions were conducted at pH 7.4. The increase in the Km of D-GpNA with increasing concentrations of acivicin is shown (insert 6A).

Fig. 7

Kinetic analysis of GGT inhibition by Compound 20. Double-reciprocal plot of the initial velocity of the hydrolysis of D-GpNA in the presence of 0 (◆), 15.2 μM (▼), 31.25 μM (▲), 62.5 μM (■), 125 μM (●) Compound 20 (A). Double reciprocal plot of the initial velocity of the transpeptidation reaction with varying concentrations of glygly with 3 mM L-GpNA (B) in the presence of 0 (○), 15.2 μM (◆), 31.25 μM (▼), 62.5 μM (▲), 125 μM (▼), 250 μM (●) Compound 20. Data shown are average triplicate values ± S.D. For many data points the S.D. is smaller than the symbol.

Fig. 8

Proposed overall kinetic mechanism of inhibition and activation of GGT. Mechanism of hydrolysis of D-GpNA (A). Enzyme (E) is g-glutamylated by D-GpNA with release of pNA to give a form of the enzyme (F) that can transfer the g-glutamyl moiety to water. D-GpNA is not capable of acting as an acceptor. The ester bond can be hydrolyzed with rate constant k_hyd, and this rate is decreased in the presence of an inhibitor such as OU749 (F-I), or increased in the presence of an activator such as Compound 11 giving rate k'_hyd. Acivicin can bind to the free enzyme at the acceptor site, and is slowly trapped (tightly bound) on enzyme. Mechanism of transpeptidation of GpNA (B). Enzyme (E) is g-glutamylated by GpNA with release of pNA to give a form of the enzyme (F) that can transfer the g-glutamyl moiety to an acceptor (L-GpNA and/or glygly). The ester bond can be hydrolyzed with rate constant k_hyd, but competition between water, L-GpNA and/or glygly decreases the rate of hydrolysis. As in panel A, acivicin can bind to the free enzyme at the acceptor site, and is slowly trapped on enzyme.