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

Article abstract of DOI:10.3109/14756366.2011.597748

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.

Full text of DOI:10.3109/14756366.2011.597748

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(4): 476–489
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.597748
ReseaRch aRtIcle
Divergent effects of compounds on the hydrolysis and
transpeptidation reactions of γ-glutamyl transpeptidase
Stephanie Wickham1, Nicholas Regan2, Matthew B. West1, Vidya Prasanna Kumar3, Justin ai2,
Pui Kai Li2, Paul F. Cook3, and Marie H. Hanigan1
1Department of Cell Biology, University of Oklahoma Health Sciences Centre, Oklahoma City, OK, USA, 2Division of
Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, OH, USA, and
3Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA
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.
Keywords: Gamma-glutamyl substrates, OU749, Enzyme, Inhibition
Introduction
are the cleavage of the γ-glutamyl bond of the substrate
in the enzyme-substrate complex (ES) and the forma-
tion of a transient enzyme-glutamyl substrate complex
(F-form of the enzyme). As the substrate is cleaved,
the γ-glutamyl group forms a transient acyl bond with
the enzyme, and the remainder of the substrate is
released (Figure 1)15–17. In human GGT, the acyl bond
forms between the γ-carbon of the γ-glutamyl sub-
strate and the hydroxyl (β-oxygen) on the side chain
of Thr-38118. In the hydrolysis reaction, water hydro-
lyzes the acyl bond between the γ-glutamyl group and
the nucleophilic residue, releasing both glutamate
and the enzyme19,20. In the transpeptidation reaction,
the γ-glutamyl group is transferred to the amine of
an acceptor, thereby forming a new γ-glutamyl com-
pound21. The transpeptidation reaction occurs by
a modified ping-pong mechanism16,22. The pH and
amino acid concentrations in extracellular fluids
where GGT is localized favor the hydrolysis reaction,
γ-Glutamyl transpeptidase (GGT) is a cell surface enzyme
that catalyzes the cleavage of the γ-glutamyl bond of glu-
tathione (GSH) and other γ-glutamyl compounds1. In
humans, the expression of GGT is restricted predominantly
to the apical surface of ducts and glands where fluids leave
the body2. e highest concentration of GGT is on the api-
cal surface of the proximal tubule cells in the kidney where
it prevents excretion of GSH into the urine by cleaving GSH
present in the glomerular filtrate2,3. Aberrant expression
and localization of GGT is observed in many disease states
including cancer4. Inhibiting GGT would sensitize tumours
to chemotherapy and may be therapeutic in other diseas-
es5–8. However, GGT inhibitors that have been evaluated
clinically are glutamine analogs and are neurotoxic9–13. We
have previously reported the discovery of a novel inhibitor
of GGT, OU749, which is not a glutamine analog14.
GGT can catalyze a hydrolysis reaction or a trans-
peptidation reaction. The first steps in both reactions
Address for Correspondence: Biomedical Research Centre, Rm. 264, 975 N.E. 10th St., Oklahoma City, OK 73104, USA. Tel: 405-271-3832.
Fax: 405-271-3758. E-mail: marie-hanigan@ouhsc.edu
(Received 15 April 2011; revised 07 June 2011; accepted 13 June 2011)
476

Hydrolysis and transpeptidation reactions 477
GpNA,
glygly,
DMSO,
d- or l-glutamic acid γ-4-nitroanalide HCl
glycyl-glycine
dimethyl sulfoxide
abbreviations
GGT,
γ-glutamyl transpeptidase
and previous studies have indicated that the hydro-
lysis reaction is the predominant reaction catalyzed
e characteristics of each intermediate are as follows:
TDA-1: 283 mg (64%), off-white crystals, mp 195–197°C;
1H NMR (300 MHz, DMSO) δ 3.81 (s, 3H), 4.19 (s, 2H),
5.01 (br s, 2H), 6.88 (d, 2H, 8.4), 7.22 (d, 2H, 8.4). MS
(m/z) = 244.05 [M+Na]+; TDA-2: 203 mg (53%), off-white
by GGT in vivo22,23
.
We previously identified OU749, a novel inhibitor
of the GGT transpeptidation reaction14. Studies of its
inhibition revealed that it is uncompetitive against l-γ-
glutamyl paranitroanilide (l-GpNA) in the transpepti-
dation reaction, binding the enzyme-glutamyl substrate
complex (F-form) and competing with the acceptor14.
Our hypothesis is that OU749 and its structural analogs
inhibit both the hydrolysis and transpeptidation reac-
tions catalyzed by GGT. In this study, we performed
in-depth kinetic analyses of the inhibition of both reac-
tions by OU749 and a series of new structural analogs.
We evaluated the potency with which the compounds
inhibited the reactions and the mechanisms of inhibi-
tion. ese data were compared to the inhibition by the
glutamine analog, acivicin, a slow binding inhibitor with
a slow rate of release. Our studies of both the hydrolysis
and transpeptidation reactions have been conducted at
physiologic pH. e standard GGT assay used by other
investigators is conducted at pH 8.0 or higher, which
may alter the physiologic cleavage mechanism due to
decreased protonation of the amino acid side chains
within the active site24. In addition, in the transpepti-
dation reaction, the presence of high concentrations
of acceptor may induce conformational changes in the
enzyme similar to the effects of hippurate24. e data
from this study provide insights into the essential fea-
tures of both the acceptors and inhibitors of the GGT
reaction.
1
crystals, mp 182–184°C; H NMR (300 MHz, DMSO) δ
4.14 (s, 2H), 7.04 (s, 2H), 7.31 (m, 5H). MS (m/z) = 214.04
[M+Na]+; TDA-3: 317 mg (61%), off-white crystals, mp
182°C; 1H NMR (300 MHz, DMSO) δ 4.19 (s, 2H), 7.10 (s,
2H), 7.28 (d, 1H, 8.1), 7.58 (s, 1H), 7.60 (d, 1H, 8.1). MS
(m/z) = 281.96 [M+Na]+; TDA-4: 307 mg (68%), off-white
1
crystals, mp 196–198°C; H NMR (300 MHz, DMSO) δ
4.16 (s, 2H), 7.06 (s, 2H), 7.30 (d, 2H, 8.1), 7.39 (d, 2H,
8.1). MS (m/z) = 248.00 [M+Na]+; TDA-5: 205 mg (50%),
off-white crystals, mp 204–206°C; 1H NMR (300 MHz,
DMSO) δ 2.27 (s, 3H), 4.09 (s, 2H), 7.02 (s, 2H), 7.14 (s,
4H). MS (m/z) = 228.06 [M+Na]+; TDA-6: 217 mg (52%),
off-white crystals, mp 206–208°C; 1H NMR (300 MHz,
DMSO) δ 4.15 (s, 2H), 7.04 (s, 2H), 7.16 (t, 2H, 8.4) 7.32
(t, 2H, 8.4). MS (m/z) = 232.03 [M+Na]+; TDA-7: 368 mg
1
(78%), off-white crystals, mp 187°C; H NMR (300 MHz,
DMSO) δ 4.34 (s, 2H), 7.12 (s, 2H), 7.57 (d, 2H, 8.7), 8.20
(d, 2H, 8.7). MS (m/z) = 259.03 [M+Na]+; TDA-8: 216 mg
1
(48%), off-white crystals, mp 193–195°C; H NMR (300
MHz, DMSO) δ 4.18 (s, 2H), 7.07 (s, 2H), 7.25 (d, 1H, 7)
7.34 (m, 3H). MS (m/z) = 248.00 [M+Na]+; TDA-9: 194 mg
1
(41%), off-white crystals, mp 185–187°C; H NMR (300
MHz, DMSO) δ 2.86 (s, 6H), 3.99 (s, 2H), 6.68 (d, 2H, 8.4),
6.97 (s, 2H), 7.07 (2, 2H, 8.4). MS (m/z) = 257.08 [M+Na]+;
TDA-10: 207 mg (50%), off-white crystals, mp 194–196°C;
1H NMR (300 MHz, DMSO) δ2.26 (s, 3H), 4.10 (s, 2H), 7.02
(s, 2H), 7.06 (m, 3H), 7.21 (t, 1H, 7.5). MS (m/z) = 228.05
[M+Na]+.
Methods
Inhibitors
Synthesis of compounds 2–4 and compounds 6–20
(Figure 2B)
OU749 was purchased from ChemBridge Corp (San
Diego, CA). Sodium benzosulfonamide was purchased
from Sigma (St. Louis, MO).
One mmol of the appropriate thiodiazole compound
was dissolved in 1 mL of pyridine and cooled to 0°C.
e appropriate benzenesulfonyl chloride (1.1 mmol)
was then dissolved in 0.5 mL of pyridine and added to
the reaction mixture. e reaction was allowed to warm
to room temperature and stirred overnight. e reac-
tion was diluted with 10 mL of water and acidified with
dilute HCl. e solid was then filtered and washed with
water. e product was recrystallized from ethanol.
e characteristics of each OU749 analogs is as follows:
OU749: 153 mg (42%), off-white crystals, mp 122–124°C;
1H NMR (300 MHz, DMSO) δ 3.75 (s, 3H), 4.12 (s, 2H),
6.93 (d, J= 8.6 Hz, 2H), 7.25 (d, J= 8.5 Hz, 2H), 7.64− 7.45
(m, 3H), 7.76 (dd, J= 6.9, 1.5 Hz, 2H), 14.06 (s, 1H). MS
(m/z) = 384.04 [M+Na]+; Compound 2: 259 mg (69%),
Synthesis of compounds 2–20 (refer to Tables 2 and 3
for structures)
Synthesis of synthetic intermediates TDA1-10
(Figure 2A). Two mmol of the appropriate phenyl acetic
acid and 2 mmol of thiosemicarbazide were dissolved in
1 mL of POCl3 and refluxed for 45 minutes. e reaction
was then cooled to room temperature, and 3 mL of water
were added carefully. e solution was then refluxed
for 4 hours. e reaction mixture was filtered hot, and
the solid was washed with warm water. e filtrate was
basified with saturated KOH, and the solid was isolated
by filtration. e solid was recrystallized from ethanol.
© 2012 Informa UK, Ltd.

478 S. Wickham et al.
off-white crystals, mp 143–145°C; 1H NMR (300 MHz,
CDCl3) δ 2.41 (s, 3H), 3.84 (s, 3H), 4.07 (s, 2H), 6.91 (d,
J= 8.8 Hz, 2H), 7.19 (d, J= 8.8 Hz, 2H), 7.25 (d, J= 7.9 Hz,
2H),7.79(d,J= 8.3Hz,2H),11.57(s,1H).MS(m/z) = 398.06
[M+Na]+; Compound 10: 155 mg (39%), off-white crys-
(s, 3H), 4.05 (s, 2H), 6.90 (d, J= 8.6 Hz, 2H), 7.17 (d, J= 8.4
Hz, 2H), 7.41 (d, J= 8.4 Hz, 2H), 7.81 (d, J= 8.4 Hz, 2H),
11.46 (s, 1H). MS (m/z) = 418.01 [M+Na]+; Compound 8:
208 mg (48%), off-white crystals, mp 135–137°C; 1H NMR
(300 MHz, DMSO) δ 3.75 (s, 3H), 4.14 (s, 2H), 6.92 (d,
J= 8.5 Hz, 2H), 7.25 (d, J= 8.5 Hz, 2H), 7.71 (dd, J= 8.4, 2.1
1
tals, mp 124–126°C; H NMR (300 MHz, CDCl3) δ 3.82
Figure 1. GGT reactions with γ-Glutamyl Substrates. Cleavage of glutathione, a physiologic substrate of GGT, is shown. e γ-glutamyl
bond between the γ-carbon of glutamate and the amine of cysteine is cleaved by GGT. e γ-carbon of glutamate forms an acyl covalent
bond with the hydroxyl group on the side chain of r-381 of human GGT. e acyl bond can either be hydrolyzed releasing glutamate
(Glu), or an acceptor nucleophile can attack forming a new γ-glutamyl compound (transpeptidation reaction). e reactions proceeds via
a Ping-Pong mechanism.
Figure 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).
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis and transpeptidation reactions 479
Hz, 1H), 7.82 (d, J= 8.4 Hz, 1H), 7.91 (d, J= 2.1 Hz, 1H),
14.25 (s, 1H). MS (m/z) = 451.97 [M+Na]+; Compound
9: 178 mg (45%), off-white crystals, mp 168–170°C; H
2H), 7.63 (d, J= 8.7 Hz, 2H), 8.02 (d, J= 9.0 Hz, 2H), 8.23
(d, J= 8.8 Hz, 2H), 8.36 (d, J= 9.0 Hz, 2H), 14.55 (s, 1H).
MS (m/z) = 422.02 [M+H]+; Compound 15: 194 mg (47%),
off-white crystals, mp 185–188°C; 1H NMR (300 MHz,
DMSO) δ 4.26 (s, 2H), 7.34− 7.28 (m, 1H), 7.39 (dd, J= 5.4,
2.9 Hz, 2H), 7.45 (t, J= 1.9 Hz, 1H), 8.02 (d, J= 8.8 Hz, 2H),
8.36 (d, J= 8.8 Hz, 2H), 14.41 (s, 1H). MS (m/z) = 432.98
[M+Na]+; Compound 18: 211 mg (54%), off-white crys-
tals, mp 178–180°C; 1H NMR (300 MHz, DMSO) δ 2.30 (s,
3H), 4.18 (s, 2H), 7.12 (d, J= 7.9 Hz, 3H), 7.26 (t, J= 7.6 Hz,
1H), 8.01 (d, J= 8.8 Hz, 2H), 8.35 (d, J= 8.8 Hz, 2H), 14.34
(s, 1H). MS (m/z) = 413.04 [M+Na]+.
1
NMR (300 MHz, CDCl3) δ 3.82 (s, 3H), 3.84 (s, 3H), 4.04
(s, 2H), 6.89 (d, J= 6.9 Hz, 2H), 6.92 (d, J= 7.2 Hz, 2H),
7.17 (d, J= 8.5 Hz, 2H), 7.82 (d, J= 8.8 Hz, 2H), 11.00 (s,
1H). MS (m/z) = 414.06 [M+Na]+; Compound 6: 27 mg
1
(7%), off-white crystals, mp 170°C; H NMR (300 MHz,
CDCl3) δ 3.81 (s, 3H), 4.01 (s, 2H), 6.88 (d, J= 8.6 Hz, 2H),
7.15 (d, J= 8.6 Hz, 2H), 7.48 (t, J= 7.8 Hz, 1H), 7.51 - 7.60
(m, 2H), 7.93− 7.80 (m, 1H), 8.01 (d, J= 8.2 Hz, 1H), 8.24
(d, J= 7.3 Hz, 1H), 8.80− 8.60 (m, 1H), 10.60 (s, 1H). MS
(m/z) = 434.06 [M+Na]+; Compound 3: 185mg (47%), off-
white crystals, mp 134–136°C; 1H NMR (300 MHz, CDCl3)
δ 3.83 (s, 3H), 4.09 (s, 2H), 6.91 (d, J= 8.5 Hz, 2H), 7.19 (d,
J= 8.5 Hz, 2H), 7.39 (t, J= 7.8 Hz, 1H), 7.51 (d, J= 7.8 Hz,
1H), 7.78 (d, J= 7.8 Hz, 1H), 7.91 (s, 1H). MS (m/z) = 418.01
[M+Na]+; Compound 4: 113 mg (26%), off-white crystals,
Synthesis of compound 5 (Figure 2C)
Compound 11 (130 mg, 0.32 mmol) and 10%Pd/C
(15.4 mg) were dissolved in EtOAc (7 mL). e reaction
was degassed five times and stirred at RT for 24 hours
under H2. e reaction was filtered through celite and
concentrated in vacu. Product was recrystallized from
EtOAc and Hexanes. e characteristics of Compound 5
1
mp 104–106; H NMR (300 MHz, DMSO) δ 3.74 (s, 3H),
4.13 (s, 2H), 6.92 (d, J= 8.4 Hz, 2H), 7.24 (d, J= 8.5 Hz,
2H), 7.60 (dd, J= 8.3, 2.3 Hz, 1H), 7.82 (d, J= 2.1 Hz, 1H),
7.99 (d, J= 8.4 Hz, 1H), 14.27 (s, 1H). MS (m/z) = 451.97
[M+Na]+; Compound 11: 214 mg (53%), off-white crys-
tals, mp 187–190°C; 1H NMR (300 MHz, DMSO) δ 3.74 (s,
3H), 4.14 (s, 2H), 6.92 (d, J= 8.7 Hz, 2H), 7.25 (d, J= 8.7 Hz,
2H), 8.00 (d, J= 9.0 Hz, 2H), 8.35 (d, J= 9.0 Hz, 2H), 14.34
(s, 1H). MS (m/z) = 429.03 [M+Na]+; Compound 7: 110mg
(26%), off-white crystals, mp 178–179°C; 1H NMR (CDCl3)
(δ, ppm; J, hertz) 1.27 (s, 9H), 3.75 (s, 3H), 4.12 (s, 2H),
6.93 (d, 2H, 8.7), 7.25 (d, 2H, 8.7), 7.56 (d, 2H, 8.4), 7.67
(d, 2H, 8.4). MS (m/z) = 440.15 [M+Na]+; Compound 12:
254mg (59%), off-white crystals, mp 169–170°C; 1H NMR
(CDCl3) (δ, ppm; J, hertz) 3.75 (s, 3H), 4.14 (s, 2H), 6.93
(d, 2H, 8.7), 7.25 (d, 2H, 8.7), 7.93 (d, 2H, 8.4), 7.97 (d, 2H,
8.4). MS (m/z) = 452.03 [M+Na]+; Compound 13: 223 mg
1
are: 112 mg (89%), off-white crystals, mp 167–168°C; H
NMR (300 MHz, DMSO) δ 3.74 (s, 3H), 4.10 (s, 2H), 6.83
(d, J= 8.8 Hz, 2H), 6.92 (d, J= 8.6 Hz, 2H), 7.24 (d, J= 8.6
Hz, 2H), 7.52 (d, J= 8.7 Hz, 2H), 8.65 (s, 1H), 8.93 (s, 1H),
13.87 (s, 1H). MS (m/z) = 415.05 [M+Na]+.
Enzyme isolation
Human GGT1 (P19440), lacking the transmembrane
domain, was expressed in Pichia pastoris and isolated as
previously described14. e specific activity of the puri-
fied enzyme was 406.5 units/mg. One unit of GGT activ-
ity was defined as the amount of enzyme that released
1 µmol of paranitroaniline/min at 37°C at pH 7.4 in the
transpeptidation reaction.
1
(59%), off-white crystals, mp 198–200°C; H NMR (300
Hydrolysis reaction
MHz, DMSO) δ 4.23 (s, 2H), 7.52− 7.24 (m, 5H), 8.01 (d,
J= 8.7 Hz, 2H), 8.36 (d, J= 8.7 Hz, 2H), 14.36 (s, 1H). MS
(m/z) = 399.02[M+Na]+;Compound20:219 mg(49%),off-
white crystals, mp 199–201°C; 1H NMR (300 MHz, DMSO)
δ 4.26 (s, 2H), 7.35 (dd, J= 8.1, 1.7 Hz, 1H), 7.63 (d, J= 8.2
Hz, 1H), 7.66 (d, J= 1.5 Hz, 1H), 8.03 (d, J= 8.9 Hz, 2H),
8.36 (d, J= 8.8 Hz, 2H), 14.41 (s, 1H). MS (m/z) = 466.94
[M+Na]+; Compound 16: 201 mg (49%), off-white crys-
tals, mp 156–158°C; 1H NMR (300 MHz, DMSO) δ 4.24 (s,
2H), 7.37 (d, J= 8.4 Hz, 2H), 7.43 (d, J= 8.4 Hz, 2H), 8.02 (d,
J= 8.9 Hz, 2H), 8.36 (d, J= 8.8 Hz, 2H), 13.86 (s, 1H). MS
(m/z) = 432.98 [M+Na]+; Compound 17: 256 mg (66%),
off-white crystals, mp 207–209°C; 1H NMR (300 MHz,
DMSO) δ 2.29 (s, 3H), 4.16 (s, 2H), 7.17 (d, J= 8.4 Hz, 2H),
7.22 (d, J= 8.2 Hz, 2H), 8.00 (d, J= 8.6 Hz, 2H), 8.35 (d,
J= 8.5 Hz, 2H), 14.34 (s, 1H). MS (m/z) = 413.04 [M+Na]+;
Compound 19: 215mg (55%), off-white crystals, mp 189–
192°C; 1H NMR (300 MHz, DMSO) δ 4.22 (s, 2H), 7.19 (t,
J= 8.8 Hz, 2H), 7.47− 7.33 (m, 2H), 8.01 (d, J= 8.8 Hz, 2H),
8.35 (d, J= 8.8 Hz, 2H), 14.37 (s, 1H). MS (m/z) = 417.01
[M+Na]+; Compound 14: 153 mg (36%), off-white crys-
tals, mp 180–183°C; 1H NMR (300 MHz, DMSO) δ 4.42 (s,
e assay buffer contained: 100 mM Na2HPO4, 3.2 mM
KCl, 1.8 mM KH2PO4, and 27.5 mM NaCl pH 7.4. e
concentration of the d-GpNA (Bachem,Torrance, CA)
substrate was varied from 0.25 mM to 3 mM d-GpNA. e
reaction was initiated with the addition of 19 mU GGT.
e reaction was incubated at 37°C and monitored con-
tinuously at 405 nm by a Bio-Rad model 680 microplate
reader with Microplate Manager 5.2 software (Bio-Rad,
Hercules, CA).
Transpeptidation reaction
e same assay buffer was used for both the hydrolysis
reaction and the transpeptidation reaction. e trans-
peptidation reaction included 40 mM glycylglycine
(glygly, Sigma, St. Louis, MO) as the acceptor. e con-
centration of the substrate for the transpeptidation reac-
tion, l-GpNA (Sigma) was varied from 0.25mM to 3 mM.
e concentration of l-GpNA was 3 mM for experiments
in which the concentration of glygly was varied. To initi-
ate the transpeptidation reaction, 4 mU GGT were added.
e reaction was incubated at 37°C and monitored con-
tinuously at 405 nm.
© 2012 Informa UK, Ltd.

480 S. Wickham et al.
infinite concentrations of X, the activator/inhibitor. e
product a(KID/KIN) is the value of Vmax or Vmax/KD-GpNA at
infinite activator/inhibitor25.
For irreversible inhibition studies, time courses were
fit to eq. 6.
Data Analysis
Samples in each experiment were run in triplicate. Each
inhibitor was evaluated in two or more independent
experiments. Double reciprocal plots were generated to
assess data quality and determine the correct rate equa-
tion for data fitting. Data were fitted using the proper rate
equation and the Marquardt-Levenberg algorithm sup-
plied with the Enzfitter program (BIOSOFT, Cambridge,
UK). Kinetic parameters with standard errors were esti-
mated using a simple weighting method.
Data adhering to Michaelis-Menten Kinetics were fit-
ted to eq. 1. Data for initial rate patterns with d-, l-GpNA
varied at different fixed levels of glygly were fitted to eq.
2, which describes a ping pong kinetic mechanism. Data
for competitive and uncompetitive inhibition were fitted
P = A(1-e−kt
)
(6)
In eq. 6, A is the burst amplitude, t is time, and k is the
first order rate constant for formation of the inactivated
enzyme. Graphs, averages, and standard error between
experiments were calculated using Prism GraphPad
Software (San Diego, CA).
Results
to eqs. 3 and 4. Data for the dependence of Vmax and Vmax
/
Initial Rate Studies with D- and L-GpNA
K
D-GpNA on the concentration of activator were fitted using
In this study, we examined the inhibition of both the GGT
hydrolysis and transpeptidation reactions by OU749, and
a new series of structural analogs. e hydrolysis and
transpeptidation reactions were analyzed separately
by using different stereo-isomers of the γ-glutamyl-p-
nitroanilide (GpNA) substrate. We first characterized the
kinetics of both reactions in the absence of inhibitors.
Cleavage of the γ-glutamyl bond of either d- or l-GpNA
releases p-nitroaniline, which has a ε405 of 7,680 M−1cm−1,
allowing for the reactions to be monitored continuously
with high sensitivity. e d-glutamyl isomer of GpNA
was used as the substrate for the hydrolysis reaction, in
which water serves as the acceptor. e l-glutamyl iso-
mer of GpNA, with glycylglycine (glygly) as an acceptor,
was used for the transpeptidation reaction. l-GpNA can
serve as both a substrate and a low affinity acceptor, while
d-GpNA is unable to serve as an acceptor18,21,26. erefore,
in the absence of an added acceptor, GGT exclusively
catalyzes a hydrolysis reaction with d-GpNA as the sub-
strate. When l-GpNA is used as a substrate, the GGT
reaction is a mixture of both the hydrolysis and transpep-
tidation reactions. Under these conditions, the rate of the
reaction is the sum of hydrolysis and transpeptidation
reactions, with the hydrolysis reaction contributing more
at low l-GpNA concentrations and transpeptidation
dominating at saturating substrate concentrations. For
the hydrolysis reaction, the second order rate constants,
V/KGpNA, were similar for d-GpNA and l-GpNA (13.2 0.1
vs 7.8 0.2 min−1nM−1), indicating that, at limiting sub-
strate concentrations, the rate of the hydrolysis reaction
is similar with d-GpNA and l-GpNA (Table 1). However,
the maximum rate (Vmax) obtained with l-GpNA was
eq. 525.
V
max A

(1)
(2)
Ka + A
Vmax AB

KaB+ Ka A + AB
Vmax A


a 




(3)
(4)
1
K
1+
+ A
Kis
Vmax A





1
K + A 1+

a
Kis

X
a +
KIN
X
(5)

1+
KID
In eqs 1–5, v and Vmax are the initial and maximum rates,
respectively, Ka and Kb are Michaelis constants for sub-
strates A and B, respectively, and Kis and Kii are slope and
intercept inhibition constants. In eq. 5, a is the value of
Vmax or Vmax/KD-GpNA at zero activator/inhibitor, KID is the
activation or inhibition constant, and KIN is a constant
that causes the parameter to go to a constant value at
Table 1. Kinetic parameters for reaction with d- and l-GpNA.
Kinetic parameter
V (mM/min/nM)
V/Kglygly (min−1nM−1)
V/ KGpNA (min−1nM−1)
Kglygly (mM)
d-GpNA minus acceptor
d-GpNA plus acceptor
53 11
l-GpNA minus acceptor
l-GpNA plus acceptor
2.1 0.2
6.5 0.2
141 11
7.6 1.5
14
1
9
13.2 0.1
12.7 2.5
7.8 0.2
117
7.0 0.3
9.9 0.9
1.2 0.1
KGpNA (mM)
0.16 0.02
4.2 0.1
0.83 0.06
ae acceptor is glygly.
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis and transpeptidation reactions 481
Figure 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 0.25mM (◆),0.5ꢀmMꢀ(•),1ꢀmMꢀ(▲), or 3mM (■)
d-GpNA (A) or l-GpNA (B).
approximately three times faster than that measured
with d-GpNA, and the KGpNA for l-GpNA was five times
higher than that measured for d-GpNA. is is at least
partially due to the ability of l-GpNA to serve as an
acceptor, resulting in a transpeptidation reaction, which
is faster than the hydrolysis reaction. erefore, to study
the hydrolysis reaction in isolation, we used d-GpNA as
the substrate without any added acceptor.
data gave rise to a series of parallel lines (Figure 4B). is
pattern is indicative of uncompetitive inhibition and
binding of OU749 to the covalent E-γ-glutamyl complex
(the F-form of the enzyme). is is the same mode of
inhibition observed previously for OU749 versus l-GpNA
with glygly as an acceptor14. A Kii of 73 µM was obtained
for OU749 in the hydrolysis reaction, which is similar to
the Kii of 68 µM that was obtained in the transpeptidation
reaction with the l-GpNA substrate plus acceptor glygly
(Table 2). ese values are comparable to the Kii value of
73 µM for OU749 measured previously in the transpep-
tidation reaction14. Maintaining the l-GpNA substrate
concentration at 3 mM while varying the concentration
of the acceptor, glygly, gave rise to a competitive inhi-
bition profile with a Kis of 54 µM, confirming previously
reported data with glygly as an acceptor14. us, OU749
inhibits GGT by binding to the acceptor site.
Characterization of the transpeptidation reaction
with both d- and l-GpNA showed that addition of the
acceptor, glygly, accelerates the reaction with both sub-
strates (Table 1). In the presence of glygly, the kinetic
mechanism is ping-pong with d-GpNA or l-GpNA as the
donor and glygly acting as an acceptor21,27. In both cases,
data fit well to the rate equation for a ping-pong mecha-
nism, eq. 2, as demonstrated by the parallel lines in the
double reciprocal plots (Figure 3A and B). e measured
Vmax for d- and l-GpNA reactions increased in the pres-
ence of glygly by 25- and 22-fold, respectively (Table 1).
However, some notable differences were observed in the
kinetics of the transpeptidation reaction with d- versus
l-GpNA. e ratio of the maximum rates of the two reac-
tions was almost 3:1 in favor of l-GpNA over d-GpNA in
the presence of glygly (141 11 vs 53 11 mM/min·nM).
While there was no change in the V/K for d-GpNA in the
presence of an acceptor, the V/K for l-GpNA increased
by about 15-fold in the presence of glygly. Insights into
the mechanism of the reaction with l-GpNA provided
by these data are included in the discussion. To evaluate
the affect of the inhibitors on the transpeptidation reac-
tion, we used the physiologic l-isomer as a substrate with
glygly as the acceptor.
OU749 substructure inhibition of GGT
Sodium benzosulfonamide (SBS, Figure 4C) is a sub-
structural component of OU749. e effect of SBS on
both the hydrolysis and transpeptidation reactions was
evaluated. e data show that SBS is an uncompeti-
tive inhibitor verus d-GpNA in the hydrolysis reaction
(Figure 4D). is is the same mechanism observed for
OU749 (Figure 4B). However, in the hydrolysis reaction,
SBS was more than 2,000-fold less effective than OU749
with a Kii of 160 81 mM (Table 2). ese data suggest
that, although the SBS moiety provides some of the bind-
ing energy of OU749, it is a minor contributor. SBS also
qualitatively mimics OU749 as an inhibitor of transpep-
tidation. It is uncompetitive versus l-GpNA (Figure 4E)
and competitive versus glygly (Figure 4F). SBS was
several orders of magnitude less potent than OU749 at
inhibiting the transpeptidation reaction. For SBS, the Kii
and Kis values were, 28.8 1.6 mM and 2.28 0.07 mM,
respectively, compared with 68 µM and 54 µM for OU749.
e difference in the potency of SBS versus OU749 as an
inhibitor of both the hydrolysis and transpeptidation
reactions implies that the benzosulfonamide portion of
OU749 contributes to the inhibition of GGT by blocking
Inhibition of the GGT hydrolysis and transpeptidation
reactions by OU749
OU749 (Figure 4A) is an uncompetitive inhibitor of GGT
transpeptidation14. To determine the mechanism by
which OU749 inhibits the hydrolysis reaction, the rate
of the hydrolysis reaction was determined as a function
of d-GpNA concentration and different concentrations
of OU749, including zero. Double reciprocal plots of the
© 2012 Informa UK, Ltd.

482 S. Wickham et al.
Figure 4. Kinetic analysis of GGT inhibition by OU749 and SBS. e 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 (■),ꢀ125ꢀµMꢀ(•)ꢀOU749ꢀ(B).ꢀeꢀstructureꢀofꢀSBSꢀ(C).ꢀ
Double reciprocal plot of the initial velocity of the hydrolysis of d-GpNA in the presence of 0 (◆), 6.25 mM (▼), 12.5mM (▲), 25mM (■),
50ꢀmMꢀ(•)ꢀSBSꢀ(D).ꢀDoubleꢀreciprocalꢀplotꢀofꢀtheꢀinitialꢀvelocityꢀofꢀtheꢀtranspeptidationꢀreactionꢀwithꢀvaryingꢀconcentrationsꢀofꢀl-GpNA and
40mM glygly (E) or varying concentrations of glygly with 3mM l-GpNA (F) in the presence of 0 (◆), 6.25mM (▼), 12.5mM (▲), 25mM (■),
50ꢀmMꢀ(•)ꢀSBS.ꢀDataꢀshownꢀareꢀaverageꢀtriplicateꢀvaluesꢀ±ꢀS.D.ꢀForꢀmanyꢀdataꢀpointsꢀtheꢀS.D.ꢀisꢀsmallerꢀthanꢀtheꢀsymbol.
access of the acceptor to the acyl bond, although SBS is
much less effective than OU749 in this regard. To deter-
mine the impact of the benzosulfonamide ring on GGT
inhibition, we modified this substructure in the context
of OU749 and assessed the effect of these modifications
in the hydrolysis and transpeptidation reactions.
of both the hydrolysis and transpeptidation reactions
of GGT (Table 2). e compounds fall into a number
of classes of inhibitory potency compared to OU749.
Compounds 2–4, with p-methyl, m-chloro, or p, o-dichloro
substitutions had no effect on inhibition of the hydroly-
sis reaction but were three to five times more effective
inhibitors of the transpeptidation reaction. ese data
suggest that the aforementioned substitutions do not
alter the stability of the ES complex. ese compounds
are equivalent to OU749 in their ability to block access of
water to the acyl bond but are more effective than OU749
Modification of the benzosulfonamide ring of OU749
A series of structural analogs of OU749 with substitu-
tions at the para (X), meta (W), or ortho (V) positions on
the benzosulfonamide ring were evaluated as inhibitors
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis and transpeptidation reactions 483
Table 2. Inhibition of GGT by structural analogs of OU749.
Compound #
Substitution
None
Hydrolysis d-GpNA Kii (µM)
73.2 8.9
Transpeptidation l-GpNA Kii (µM)
Transpeptidation glygly Kis (µM)
54.1 3.9
OU749
67.6 6.3
22.7 0.5
22.0 0.7
15.7 0.7
64.4 5.1
74.9 3.6
149.9 13.0
18.2 0.7
149 23
2
X=CH3
73.3 4.0
10.0 0.4
3
W=Cl
74.9 2.2
11.9 1.1
4
X=Cl; V=Cl
X= NHOH
WV=Benzene
X=C(CH3)3
X=Cl; W=Cl
X=OCH3
X=Cl
75.6 7.2
3.7 1.0
5
149.4 13.6
161.0 4.5
341.5 6.4
3160 90
25.7 1.3
6
45.7 3.4
7
143.2 8.7
10.8 0.8
8
9
3800 720
133.3 10.3
13.4 0.8
10
11
12
5530 1460
Activator
32.5 0.9
92.8 4.3
116.7 7.4
X=NO2
95.7 7.6
X=CF3
Activator
46.3 3.5
in blocking access of the dipeptide acceptors to the acyl
bond in the transpeptidation reaction. Compounds 5–7
with the hydrophilic p-hydroxylamino, the bulky l-naph-
thylsulfonamide ring, or p-tBu substitutions are two to five
times less effective than OU749 in inhibiting the hydrolysis
reaction but have smaller effects as inhibitors of transpep-
tidation. e latter is also true of compounds 8–10 with p-,
m-dichloro, p-methoxy or p-chloro substituents, which are
40 to 75 times poorer inhibitors of the hydrolysis reaction
but, with the exception of the bulkier p-methoxy, are better
inhibitors of the transpeptidation reaction. Comparison of
the data obtained with compounds 3 and 4 with the data
for compounds 8 and 10 reveals that the combination of
Cl substituents alters the ability of the inhibitor to block
hydrolysis of the acyl bond but has little effect on the
transfer of the acyl bond to an acceptor. For the hydrolysis
reaction, the detrimental effect of having a Cl substituent
in the para position can be ameliorated by ortho, but not
meta, substitution in the same molecule, while meta
substitution alone generates no effect.
Introduction of a highly electron-withdrawing group,
-NO2 or -CF3, in the para (X) position (Compounds 11
and 12, Table 2) resulted in activation of the hydrolysis
reaction. However, addition of these groups maintained
inhibition of the transpeptidation reaction, with less
than a 2-fold effect on the Kii relative to OU749. Data are
shown for Compound 11 (Figure 5). A detailed analysis
of the kinetics of the hydrolysis reaction is shown
in Figure 5A and B. At each of the concentrations of
Compound 11, including zero, data were first fitted to eq.
1 to obtain estimates of Vmax and Vmax/KD-GpNA. e limit-
ing rate constants were fitted to eq. 5, which allows for
an estimate of the values of the rate constant at zero and
infinite concentration of the compound and a determi-
nation of its activation constant (Kact). Note that activa-
tion is observed on Vmax, while inhibition is observed for
Vmax/KD-GpNA (Figure 5B). e compound is thus a V-type
activator in the hydrolysis reaction. GGT inhibition by
Compound 11 was also analyzed in the transpeptidation
reaction. Compound 11 is an uncompetitive inhibitor
versus l-GpNA in the transpeptidation reaction, indicat-
ing that it binds to the l-γ-glutamyl intermediate, pre-
sumably at the acceptor site (Figure 5C). e Kis was 93
µM. e Kii of Compound 11 while varying glygly in the
transpeptidation reaction was 96 µM (Figure 5D). e
affinity of Compound 11 for the d-γ-glutamyl enzyme
intermediate and the l-γ-glutamyl enzyme intermedi-
ate differ by only 2.5-fold, suggesting that there may be
a minor difference in the acceptor site or a slight differ-
ence in the orientation of the acyl bond depending on
the isomer of glutamate that is bound.
Modification of the benzyl ring, structure activity
studies
In order to determine whether modification of the
phenyl ring remote to the benzosulfonamide ring has
an effect on the behaviour of Compound 11, a series of
structural analogs of Compound 11 were synthesized
© 2012 Informa UK, Ltd.

484 S. Wickham et al.
with modifications to this portion of the inhibitor. Kinetic
parameters for modifications of the benzyl ring of nitro
compounds are shown in Table 3. A 3- to 6-fold activation
of the Vmax for hydrolysis (the ratio of V∞ to Vo in Table 3)
was observed with an activation constant, Kact, of 22–54
µM for Compounds 13 thru 19. ese activators may
alter the conformation of the enzyme or be oriented dif-
ferently than OU749, allowing greater access of water to
the acyl bond.
In the transpeptidation reaction, the nitro-containing
derivatives of Compound 11 behave in a manner that is
both qualitatively and quantitatively similar to the par-
ent compound, OU749 (Table 3). Addition of a chlorine
group at the Y position resulted in an inhibitor (Table 3,
Compound 16) with a Kii of 61.9 µM in the transpepti-
dation reaction. An OCH3 at the Y position increased
the l-GpNA Kii in the transpeptidation reaction less
than 2-fold and increased the glygly Kis more than
2-fold (Table 3, Compound 11) when compared with
Compound 16. No substitution on the benzyl ring
(Table 3, Compound 13) increased the l-GpNA Kii by
almost 2-fold compared to Compound 16, while only
slightly increasing the glygly Kis. Addition of a methyl
group at the Y position yielded results similar to having no
substitution (Table 3, Compound 17). Addition of any of
a series of chemical groups at the Z position reduced the
strength of this family of inhibitors in the transpeptidation
reaction by 2-fold, while slightly increasing the strength of
the compound as a competitive inhibitor of the acceptor,
glygly (Table 3, Compounds 15 and 18). Addition of an
NO2 at the Y position (Table 3, Compound 14) more than
doubled the Kii and Kis relative to Compound 16 in the
transpeptidation reaction. ere is very little difference
in the behaviour of any of these compounds in the trans-
peptidation reaction with the exception of Compound
19 and Compound 20, which exhibited time dependent
inhibition and, together with acivicin, will be discussed
below.
Acivicin and acivicin-like inhibition
Acivicin is an analog of γ-glutamyl donor substrates
[GpNA, GSH, etc.] and exhibits reversible competitive
inhibition against these substrates with a very slow rate
of release27. e time course for production of pNA in the
presence of increasing concentrations of acivicin is shown
in Figure 6. e time course for hydrolysis of 0.287mM
d-GpNA in the absence of acivicin is linear for at least 30
minutes (Figure 6A). In the presence of increasing con-
centrations of acivicin, the initial rate is unchanged, but
the rate decreases with increasing time, with the most pro-
nounced decrease seen at the highest acivicin concentra-
tion (Figure 6A). A fit of the time courses to the equation
for a first-order process gives an observed rate constant,
kobs, that is a linear function of acivicin concentration.
Figure 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 (■),ꢀ125ꢀµMꢀ(•)ꢀCompoundꢀ11ꢀ(A).ꢀVelocityꢀvsꢀCompoundꢀ11ꢀ
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 40mM glygly (C) or
varying glygly with 3mM l-GpNA (D) in the presence of 0 (◆), 15.2 µM (▼), 31.25 µM (▲), 62.5 µM (■),ꢀ125ꢀµMꢀ(•)ꢀCompoundꢀ11.ꢀDataꢀshownꢀ
are average triplicate values S.D.
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis and transpeptidation reactions 485
is linear dependence is expected for an irreversible
inhibitor, where kobs is the pseudo-first order net on-rate
constant for modification of enzyme by acivicin times the
concentration of acivicin. e slope of the line depicted in
the inset of Figure 6 thus describes the second order rate
constant, kon, as 0.171mM−1min−1. Since acivicin is com-
petitive versus d-GpNA, kobs is equal to the pseudo-first
order rate constant divided by (1 + [d-GpNA]/KD-GpNA).
Given a KD-GpNA of 0.16mM (Table 1), the kon, corrected for
the presence of d-GpNA, is 0.48mM−1min−1.
with glygly, and the results were qualitatively the same
(data not shown). In the transpeptidation reaction with
l-GpNA, the time courses were curvilinear even in the
absence of acivicin. Unlike d-GpNA, which can only act
as a donor to form the γ-glutamyl enzyme intermediate
(F-form), l-GpNA can also act as an acceptor18,21,26. As a
result, a complete analysis was only carried out for the
hydrolysis reaction using d-GpNA.
Increasing the d-GpNA concentration to saturation
eliminated the development of inhibition by acivicin
over the same time course as would be expected for
competitive inhibition against d-GpNA (Figure 6B).
Kinetic analysis of acivicin was also carried out for
the transpeptidation reaction using l-GpNA or l-GpNA
Table 3. Kinetic parameters for activators of the D-GpNA hydrolysis reaction and Inhibition of the L-GpNA transpeptidation reaction.
Hydrolysis D-GpNA
Inhibition L-GpNA
a
Vo
V∞
KAct
Fold
Transpeptidation Transpeptidation
Compound # Substitution
(mM/min/nM)
1.59 0.06
1.27 0.01
1.30 0.01
1.59 0.14
1.71 0.09
2.62 0.79
1.15 0.01
1.53 0.09
—
(mM/min/nM)
(µM)
activation
l-GpNA Kii (µM)
glygly Kis (µM)
59.6 3.7
81.2 8.8
43.8 7.7
95.7 7.6
44.4 3.4
56.9 8.3
34.3 0.9
31.8 0.7
10.3 0.5
13
14
15
11b
16
17
18
19
20c
None
Y=NO2
Z=Cl
9.09 0.55
5.08 0.41
5.86 0.23
8.63 1.21
7.70 0.46
11.25 4.27
5.08 0.20
5.39 0.28
—
54.4 3.0
51.9 0.8
38.6 0.3
37.1 5.6
31.4 2.2
25.3 7.6
25.2 0.2
22.2 2.0
—
5.72 0.32
4.00 0.11
4.51 0.05
5.43 2.04
4.50 0.63
4.29 2.20
4.42 0.64
3.52 0.60
—
111.5 5.5
132.5 5.5
119.1 7.1
Y=OCH3
Y=Cl
92.8 4.3
61.9 2.4
Y=CH3
Z=CH3
Y=F
112.5 5.2
121.5 8.8
Time Dependent
Time Dependent
Y=Cl
Z=Cl
aVo =maximum velocity at zero activator; V∞ =Vo(KID/KIN) in eq. 2, maximum rate at infinite activator; Kact, activation constant or
concentration that gives ½(V∞ + Vo), KID in eq. 2; fold activation=V∞/ Vo.
bNormalization of all values to Compound 11 yielded values similar to Compound 11.
cCompound 20 competitively inhibited the hydrolysis reaction with a Kii with d-GpNA of 78.2 1.9 µM.
Figure 6. Time courses for the hydrolysis of d-GpNA in the presence of acivicin. e release of pNA from 0.287 mM d-GpNA (A) or 2mM
d-GpNA (B) was monitored over time in the presence of acivicin (concentrations of acivicin noted in A). e reactions were conducted at
pH 7.4. e increase in the Km of d-GpNA with increasing concentrations of acivicin is shown (insert 6A).
© 2012 Informa UK, Ltd.

486 S. Wickham et al.
Figure 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 (■),ꢀ125ꢀµMꢀ(•)ꢀCompoundꢀ20ꢀ(A).ꢀDoubleꢀreciprocalꢀplotꢀofꢀtheꢀinitialꢀvelocityꢀofꢀ
the transpeptidation reaction with varying concentrations of glygly with 3mM l-GpNA (B) in the presence of 0 (○), 15.2 µM (◆), 31.25 µM
(▼), 62.5 µM (▲), 125 µM (■),ꢀ250ꢀµMꢀ(•)ꢀCompoundꢀ20.ꢀDataꢀshownꢀareꢀaverageꢀtriplicateꢀvaluesꢀ±ꢀS.D.ꢀForꢀmanyꢀdataꢀpointsꢀtheꢀS.D.ꢀisꢀ
smaller than the symbol.
Time-dependent inhibition of the transpeptidation reac-
tion was observed for the monofluoro- (Compound 19)
and dichloro- (Compound 20) substituted compounds
shown in Table 3, suggesting that they also exhibit
either irreversible or slow-onset inhibition. A complete
analysis of this phenomenon is outside the scope of the
present studies.
OU749 binding to the F-form of the enzyme. Despite the
fact that l-GpNA has the capacity to act as an acceptor in
the transpeptidation reaction, its maximum turnover rate
was only about three-times higher than that measured for
d-GpNA in the absence of glygly (Table 1). us, although
l-GpNA can act as an acceptor, it is not very effective. e
second order rate constant measured with l-GpNA is
similar to the rate constant with d-GpNA (8 min−1 nM−1
and 13 min−1 nM−1, respectively), indicating that forma-
tion of the γ-glutamyl-enzyme intermediate (F-form) is
independent of the stereochemistry of the γ-glutamyl
moiety on the nitroanilide substrate. To develop effective
inhibitors for therapeutic use, each compound must be
evaluated in the physiologic hydrolysis reaction.
We began our comparative analyses by characterizing
the substructural elements of OU749 that contribute to
its efficacy as an inhibitor. e terminal benzosulfon-
amide moiety of OU749, SBS, was evaluated for activity as
an inhibitor (Figure 4A and C). SBS is an uncompetitive
inhibitor versus d- and l-GpNA and a competitive inhibi-
torversusglyglyinthel-GpNAreaction. Weexpandedour
analysis to a series of OU749 analogs with modifications
on the benzosulfonamide ring. As with SBS, Compounds
2–10 were more potent inhibitors of the transpeptida-
tion reaction than the hydrolysis reaction (Table 2).
However, compared to OU749, all of these structural ana-
logs bound with either equal (Compounds 2–4) or lower
(Compounds 5–10) affinity to the d-γ-glutamyl enzyme.
As observed for glygly, analogs of OU749 with a strong
electron-withdrawing group (–NO2 or −CF3) para to the
sulfonamide group activate the hydrolysis reaction with
d-GpNA (Compounds 11 and 12, Table 2). Both analogs
inhibit the reaction with l-GpNA and exhibit an affinity
very similar to that of Compounds 2–10 in Table 2. us,
as was observed for glygly in the absence of inhibitors,
Compounds 11 and 12 activate the hydrolysis reaction.
is suggests that like glygly, they bind to the accep-
tor site of the E~γ-d-glutamyl intermediate (F-form),
and in doing so, they may alter the conformation of the
All of the nitro-containing OU749 analogs accelerated
the hydrolysis reaction except Compound 20 (Table 3).
Compound 20 has chlorines both para (Y) and meta
(Z) on the benzyl ring and a NO2 at the para position on
the benzosulfonamide ring. e inhibition mechanism
of Compound 20 is unique among the OU749 analogs,
because it is competitive with the γ-glutamyl substrate
in the hydrolysis reaction, yielding a Kii with d-GpNA
of 78 µM (Figure 7A). In addition to being competitive
with the γ-glutamyl substrate, Compound 20 is also
competitive with the acceptor glygly in the transpeptida-
tion reaction (Figure 7B), with a Kis of 10 µM (Table 3).
However, we were unable to establish a Kii with l-GpNA
in the transpeptidation reaction, because Compound 20
exhibits slow-onset binding similar to acivicin. us, the
transpeptidation reaction is too complex for accurate
analysis and establishment of inhibition constants for
this compound.
Discussion
In this analysis, new structural analogs of OU749 were
evaluated for their inhibitory activity in both the hydroly-
sis and transpeptidation reactions. All of the new analogs
were weaker inhibitors of the hydrolysis reaction relative
to OU749, but some were more potent inhibitors than
OU749 in the transpeptidation reaction. In addition to
structure-activity information, the divergent results pro-
vide insights into the reaction catalyzed by GGT. In both
the hydrolysis and transpeptidation reactions, OU749
is an uncompetitive inhibitor of the donor γ-glutamyl
substrate. e data provided herein are consistent with
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis and transpeptidation reactions 487
intermediate in a manner that allows a more facile attack
by water on the ester carbonyl. e SO2 of the benzosul-
fonamide group may interact with residues in or near the
active site.
It is difficult at best to model the results obtained in
this study on a molecular level. e crystal structure for
human GGT has not yet been determined. Homology
models based on the crystal structures of bacterial GGT
All of the OU749 analogs containing a nitroxide
group (Compounds 11, 13–20), except Compound 20,
accelerated the hydrolysis reaction. However, these
same analogs inhibited the transpeptidation reaction
with an affinity (Kii) very similar to that of Compounds
2–10 (Table 2). A series of analogs in which the meth-
oxyphenyl ring of Compound 11 was modified (Table 3)
elicited no major effect on the inhibition of the trans-
peptidation reaction, with the exception of p-F or
p-,m-dichloro analogs (Compounds 19 and 20), which
exhibited a distinct mode of inhibition similar to that
observed for acivicin.
lack the resolution necessary for molecular modeling33–35
.
Mutational analysis have indicated that the active nucleo-
phile r-381, Arg-107 and Asp-423 interact with the
α-amino and the α-carboxylate of glutamate29–30,36. Two
adjacent serines (Ser-451 and Ser-452) have also been
proposed to stabilize the rate-limiting transition state
of the catalytic reaction29. Identification of amino acids
involved in the binding of the acceptor has been more
elusive, and there is little solid structural work available
to delineate the “acceptor site” of GGT from any organ-
ism. All of the inhibitors tested, with the exception of
acivicin, target the acceptor site of human GGT. Although
one could speculate as to the residues or sites with which
different inhibitors interact, no definitive statements can
be made until a reasonable structure is obtained with the
acceptor site occupied.
A schematic representation of the hydrolysis and
transpeptidation reaction in the presence of a high affin-
ity competitive inhibitor (acivicin) and an uncompetitive
inhibitor (i.e. OU749 and analogs, I) is presented in Figure
8. In the hydrolysis reaction (Figure 8A), d-GpNA binds to
GGT, and the γ-glutamyl moiety is cleaved, releasing pNA.
e d-γ-glutamyl group is retained by GGT establishing
the F-form. Water can then hydrolyze the acyl bond of the
F-form, releasing d-glutamate and free enzyme. Acivicin
can inhibit GGT by binding the free enzyme, competing
with the γ-glutamyl substrate, thus inhibiting both the
hydrolysis and transpeptidation reactions equally. After
establishment of the F-form of GGT in the transpeptida-
tion reaction with l-GpNA (Figure 8B), the acyl bond can
be hydrolyzed by water, or attacked by an acceptor (i.e.
glygly or l-GpNA). l-glutamate, l-γ-glutamyl-glygly, or
l-γ-glutamyl-l-GpNA are then released, and the enzyme
returns to the free enzyme state. OU749 and its analogs
are uncompetitive, in that they bind the F-form of the
enzyme, competing with the acceptor. erefore, OU749
and its analogs only inhibit the second half of the reac-
tion of GGT: the attack of the acyl bond by either water
or an acceptor.
Acivicin is an analog of the γ-glutamyl donor sub-
strate27–30. It exhibits time-dependent inhibition at low,
but not at high, concentrations of l-GpNA (Figure 6). e
second order rate constant for binding acivicin, kon, is
0.48mM−1 min−1. is is the same type of inhibition of the
transpeptidation reaction as was observed for Compounds
19 and 20, indicating that they also exhibit either irrevers-
ible or time-dependent inhibition. Of further interest is
the fact that the p-, m-dichloro analog (Compound 20) is
the only compound in this series of analogs that does not
accelerate the hydrolysis reaction but, rather, serves as a
competitive inhibitor of the d-GpNA substrate. is (p-,
m-dichloro analog) is the only OU749 analog that inhibits a
GGT reaction by competing with the γ-glutamyl substrate.
To fully understand the affect of the OU749 analogs
in the hydrolysis and transpeptidation reactions, the
reactions had to be independently analyzed with both
donor substrates, d- and l-GpNA. In the absence of the
acceptor glygly, the second order rate kinetics are similar
for both d- and l-GpNA in the GGT hydrolysis reaction,
indicating the stereochemistry of the γ-glutamyl group of
the substrate does not affect the binding and cleavage of
the substrate in the hydrolysis reaction. However, when
the acceptor glygly is present in the transpeptidation
reaction, with l-GpNA serving as the donor substrate, the
second order rate kinetics increase by 15-fold. is dra-
matic increase suggests three possibilities (i) the bind-
ing of glygly increases the rate of hydrolysis, (ii) glygly
could be binding to another site, perhaps an affector site,
instead of to the acceptor site that increases the turnover
rate, or (iii) glygly could be binding free enzyme, forming
an E-glygly complex, prior to l-GpNA binding. e third
possibility would imply a sequential mechanism instead
of a ping-pong mechanism. In a ping-pong mechanism,
the donor substrate’s second order rate constant (V/K)
will not change in the absence or presence of an accep-
tor molecule. A sequential mechanism can also yield the
classic ping-pong plots, if the Km for glygly is greater than
the Ki of glygly for the E-glygly complex. is is only pos-
sible when the glygly concentration is varied around the
Km of glygly, as was carried out in these studies and previ-
Expression of GGT on the surface of the cell initiates
the cleavage of extracellular GSH, thereby releasing
cysteine and providing an additional source of cysteine
for intracellular GSH synthesis5,37. GSH and free cysteine
within the cell are potent reducing agents that protect
the cell against oxidative stress and detoxify electro-
philic metabolites. Conjugation of many chemotherapy
drugs to GSH result in their inactivation, and the con-
jugates are then exported from the cell38. GSH has been
shown to be increased in various cancers including
breast39, lung40, bone marrow41, ovarian42, head and
neck, and laryngeal43. Increased intracellular GSH has
also been shown to contribute to the inhibition of apop-
tosis by inducing Bcl-2 and inducing resistance to anti-
hormonal therapy44,45. Studies have shown that, in mice,
ous studies of the GGT transpeptidation reaction14,31,32
.
© 2012 Informa UK, Ltd.

488 S. Wickham et al.
Figure 8. Proposed overall kinetic mechanism of inhibition and activation of GGT. Mechanism of hydrolysis of d-GpNA (A). Enzyme
(E) is γ-glutamylated by d-GpNA with release of pNA to give a form of the enzyme (F) that can transfer the γ-glutamyl moiety to water.
d-GpNA is not capable of acting as an acceptor. e ester bond can be hydrolyzed with rate constant khyd, 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 γ-glutamylated by GpNA with release of pNA to give a form of the enzyme (F) that can transfer the γ-glutamyl
moiety to an acceptor (l-GpNA and/or glygly). e ester bond can be hydrolyzed with rate constant khyd, 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.
GGT-positive tumours are more resistant to treatment
References
with cisplatin than GGT-negative tumours6. In Phase I
1. Hanigan MH. γ-Glutamyl transpeptidase, a glutathionase: its
expression and function in carcinogenesis. Chem Biol Interact
1998;111–112:333–342.
clinical trials, acivicin was found to be neurotoxic and
cannot be used clinically as an inhibitor of GGT10–13
.
2. Hanigan MH, Frierson HF Jr. Immunohistochemical detection of
γ-glutamyl transpeptidase in normal human tissue. J Histochem
Cytochem 1996;44:1101–1108.
3. Lieberman MW, Wiseman AL, Shi ZZ, Carter BZ, Barrios R,
Ou CN et al. Growth retardation and cysteine deficiency in
γ-glutamyl transpeptidase-deficient mice. Proc Natl Acad Sci USA
1996;93:7923–7926.
4. Hanigan MH, Frierson HF Jr, Swanson PE, De Young BR. Altered
expression of γ-glutamyl transpeptidase in human tumors. Hum
Pathol 1999;30:300–305.
5. Ruoso P, Hedley DW. Inhibition of γ-glutamyl transpeptidase
activity decreases intracellular cysteine levels in cervical
carcinoma. Cancer Chemother Pharmacol 2004;54:49–56.
6. Hanigan MH, Gallagher BC, Townsend DM, Gabarra V. γ-glutamyl
transpeptidase accelerates tumor growth and increases the
resistance of tumors to cisplatin in vivo. Carcinogenesis 1999;
20:553–559.
erefore, less toxic inhibitors need to be developed. A
complete understanding of the mechanism of inhibi-
tion of GGT is crucial to optimize GGT inhibitors for
clinical use. In the current study, the stereochemistry of
the donor substrates provided insight into the heteroge-
neity of the acceptor site, and the potential interaction
of the OU749 analogs within the acceptor site. ese
studies also emphasize the need to study the mecha-
nism of inhibition of GGT in both the hydrolysis and
transpeptidation reactions. e hydrolysis reaction is
the physiologic reaction, and inhibitors of GGT need to
be thoroughly analyzed and optimized in this reaction.
Declaration of interest
7. Hanigan MH, Pitot HC. γ-glutamyl transpeptidase–its role in
hepatocarcinogenesis. Carcinogenesis 1985;6:165–172.
8. LowryMH,McAllisterBP,JeanJC,BrownLA,HugheyRP,Cruikshank
WW et al. Lung lining fluid glutathione attenuates IL-13-induced
asthma. Am J Respir Cell Mol Biol 2008;38:509–516.
is work was supported, in whole or in part, by National
Institutes of Health Grant RO1CA57530 (M.H.H.) and the
Grayce B. Kerr endowment to the University of Oklahoma
to support the research (to P.F.C.).
Journal of Enzyme Inhibition and Medicinal Chemistry

Hydrolysis and transpeptidation reactions 489
9. Ahluwalia GS, Grem JL, Hao Z, Cooney DA. Metabolism and
action of amino acid analog anti-cancer agents. Pharmacol er
1990;46:243–271.
29. Ikeda Y, Fujii J, Anderson ME, Taniguchi N, Meister A. Involvement
of Ser-451 and Ser-452 in the catalysis of human γ-glutamyl
transpeptidase. J Biol Chem 1995;270:22223–22228.
10. Weiss GR, McGovren JP, Schade D, Kufe DW. Phase
I
and
30. Castonguay R, Halim D, Morin M, Furtos A, Lherbet C,
Bonneil E et al. Kinetic characterization and identification of
the acylation and glycosylation sites of recombinant human
γ-glutamyltranspeptidase. Biochemistry 2007;46:12253–12262.
31. ompson GA, Meister A. Modulation of the hydrolysis, transfer,
and glutaminase activities of γ-glutamyl transpeptidase by maleate
bound at the cysteinylglycine binding site of the enzyme. J Biol
Chem 1979;254:2956–2960.
32. Tate SS, Meister A. Serine-borate complex as a transition-state
inhibitor of γ-glutamyl transpeptidase. Proc Natl Acad Sci USA
1978;75:4806–4809.
33. Okada T, Suzuki H, Wada K, Kumagai H, Fukuyama K. Crystal
structures of γ-glutamyltranspeptidase from Escherichia coli, a key
enzyme in glutathione metabolism, and its reaction intermediate.
Proc Natl Acad Sci USA 2006;103:6471–6476.
pharmacological study of acivicin by 24-hour continuous infusion.
Cancer Res 1982;42:3892–3895.
11. Earhart RH, Koeller JM, Davis TE, Borden EC, McGovren JP, Davis
HLetal.PhaseItrialandpharmacokineticsofacivicinadministered
by 72-hour infusion. Cancer Treat Rep 1983;67:683–692.
12. Fleishman G, Yap HY, Murphy WK, Bodey G. Phase II trial of
acivicin in advanced metastatic breast cancer. Cancer Treat Rep
1983;67:843–844.
13. Taylor S, Belt RJ, Joseph U, Haas CD, Hoogstraten B. Phase I
evaluation of AT-125 single dose every three weeks. Invest New
Drugs 1984;2:311–314.
14. King JB, West MB, Cook PF, Hanigan MH. A novel, species-specific
class of uncompetitive inhibitors of γ-glutamyl transpeptidase.
J Biol Chem 2009;284:9059–9065.
15. Tate SS, Meister A. Interaction of γ-glutamyl transpeptidase
with amino acids, dipeptides, and derivatives and analogs of
glutathione. J Biol Chem 1974;249:7593–7602.
16. Tate SS, Meister A. Stimulation of the hydrolytic activity
and decrease of the transpeptidase activity of γ-glutamyl
transpeptidase by maleate; identity of a rat kidney maleate-
stimulated glutaminase and γ-glutamyl transpeptidase. Proc Natl
Acad Sci USA 1974;71:3329–3333.
34. Wada K, Irie M, Suzuki H, Fukuyama K. Crystal structure of the
halotolerant γ-glutamyltranspeptidase from Bacillus subtilis
in complex with glutamate reveals a unique architecture of the
solvent-exposed catalytic pocket. FEBS J 2010;277:1000–1009.
35. Morrow AL, Williams K, Sand A, Boanca G, Barycki JJ.
Characterization of Helicobacter pylori γ-glutamyltranspeptidase
reveals the molecular basis for substrate specificity and a critical
role for the tyrosine 433-containing loop in catalysis. Biochemistry
2007;46:13407–13414.
17. Castonguay R, Lherbet C, Keillor JW. Kinetic studies of rat kidney
γ-glutamyltranspeptidase deacylation reveal
a
general base-
36. Ikeda Y, Fujii J, Taniguchi N, Meister A. Human γ-glutamyl
transpeptidase mutants involving conserved aspartate residues
and the unique cysteine residue of the light subunit. J Biol Chem
1995;270:12471–12475.
37. Hanigan MH, Ricketts WA. Extracellular glutathione is a source
of cysteine for cells that express γ-glutamyl transpeptidase.
Biochemistry 1993;32:6302–6306.
catalyzed mechanism. Biochemistry 2003;42:11504–11513.
18. Keillor JW, Castonguay R, Lherbet C. γ-glutamyl transpeptidase
substrate specificity and catalytic mechanism. Meth Enzymol
2005;401:449–467.
19. Han L, Hiratake J, Kamiyama A, Sakata K. Design, synthesis,
and evaluation of γ-phosphono diester analogues of glutamate
as highly potent inhibitors and active site probes of γ-glutamyl
transpeptidase. Biochemistry 2007;46:1432–1447.
38. Estrela JM, Ortega A, Obrador E. Glutathione in cancer biology
and therapy. Crit Rev Clin Lab Sci 2006;43:143–181.
20. Lherbet C, Keillor JW. Probing the stereochemistry of the active site
of γ-glutamyl transpeptidase using sulfur derivatives of l-glutamic
acid. Org Biomol Chem 2004;2:238–245.
39. Perry RR, Mazetta JA, Levin M, Barranco SC. Glutathione levels
and variability in breast tumors and normal tissue. Cancer
1993;72:783–787.
21. ompson GA, Meister A. Hydrolysis and transfer reactions
catalyzed by γ-glutamyl transpeptidase; evidence for separate
substrate sites and for high affinity of L-cystine. Biochem Biophys
Res Commun 1976;71:32–36.
22. Elce JS, Broxmeyer B. γ-Glutamyltransferase of rat kidney.
Simultaneous assay of the hydrolysis and transfer reactions with
(glutamate-14C)glutathione. Biochem J 1976;153:223–232.
23. CurthoysNP,HugheyRP.Characterizationandphysiologicalfunction
of rat renal γ-glutamyltranspeptidase. Enzyme 1979;24:383–403.
40. Oberli-Schrämmli AE, Joncourt F, Stadler M, Altermatt HJ,
Buser K, Ris HB et al. Parallel assessment of glutathione-based
detoxifying enzymes, O6-alkylguanine-DNA alkyltransferase and
P-glycoproteinasindicatorsofdrugresistanceintumorandnormal
lung of patients with lung cancer. Int J Cancer 1994;59:629–636.
41. Joncourt F, Oberli-Schrämmli AE, Stadler M, Buser K, Franscini
L, Fey MF et al. Patterns of drug resistance parameters in adult
leukemia. Leuk Lymphoma 1995;17:101–109.
42. Raderer M, Scheithauer W. Clinical trials of agents that reverse
24. Allison RD, Meister A. Evidence that transpeptidation is
a
multidrug resistance.
3553–3563.
43. Mulder TP, Manni JJ, Roelofs HM, Peters WH, Wiersma A.
Glutathione S-transferases and glutathione in human head and
neck cancer. Carcinogenesis 1995;16:619–624.
A literature review. Cancer 1993;72:
significant function of γ-glutamyl transpeptidase. J Biol Chem
1981;256:2988–2992.
25. Cook PF, Cleland WW. Enzyme Kinetics and Mechanism. Garland
Science, London, New York, 2007.
26. Allison RD. γ-Glutamyl transpeptidase: kinetics and mechanism.
Meth Enzymol 1985;113:419–437.
44. Mena S, Benlloch M, Ortega A, Carretero J, Obrador E, Asensi M
et al. Bcl-2 and glutathione depletion sensitizes B16 melanoma
to combination therapy and eliminates metastatic disease. Clin
Cancer Res 2007;13:2658–2666.
45. Rudin CM, Yang Z, Schumaker LM, VanderWeele DJ, Newkirk K,
Egorin MJ et al. Inhibition of glutathione synthesis reverses
Bcl-2-mediated cisplatin resistance. Cancer Res 2003;63:312–318.
27. Stole E, Smith TK, Manning JM, Meister A. Interaction of γ-glutamyl
transpeptidase with acivicin. J Biol Chem 1994;269:21435–21439.
28. Smith TK, Ikeda Y, Fujii J, Taniguchi N, Meister A. Different sites
of acivicin binding and inactivation of γ-glutamyl transpeptidases.
Proc Natl Acad Sci USA 1995;92:2360–2364.
© 2012 Informa UK, Ltd.