β-Carbonyl substituted glutathione conjugates as inhibitors of O. volvulus GST2

Article abstract of DOI:10.1016/S0960-894X(00)00142-6

A series of β-carbonyl substituted glutathione conjugates were prepared and evaluated as inhibitors of OvGST2. Their specificity for the parasite derived protein was assessed through comparison with their inhibition of human πGST. Inhibition of OvGST2 has been demonstrated at low micromolar concentrations for these conjugates and selectivity for OvGST2 over human π-GST of greater than 10-fold has been achieved. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00142-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 979±981
ꢀ-Carbonyl Substituted Glutathione Conjugates as Inhibitors
of O. Volvulus GST2
a
a
b
Peter M. Brophy, Alison M. Campbell, Annamaria J. van Eldik,
b
b,
c
Paul H. Teesdale-Spittle, * Eva Liebau and Meng F. Wang
aInstitute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DA, UK
bDepartment of Chemistry, De Montfort University, The Gateway, Leicester LE1 9BH, UK
cBernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str 74, Hamburg, Germany
Received 16 November 1999; accepted 28 February 2000
AbstractÐA series of b-carbonyl substituted glutathione conjugates were prepared and evaluated as inhibitors of OvGST2. Their
speci®city for the parasite derived protein was assessed through comparison with their inhibition of human pGST. Inhibition of
OvGST2 has been demonstrated at low micromolar concentrations for these conjugates and selectivity for OvGST2 over human p-
GST of greater than 10-fold has been achieved. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
contract, the glutathione binding site is closely related to
that found in the mammalian enzymes: Notably the Tyr
115 residue is almost identically placed in both the mam-
malian and parasite enzymes. This residue is a potential
target for binding of a number of p-GST inhibitors, all
bearing a carbonyl moiety b to the point of where glu-
tathione conjugation occurs.5
The ®larial nematode parasite Onchocerca volvulus causes
onchocerciasis; a major cause of preventable blindness and
severe dermatitis in Africa. Glutathione S-transferases
(
GSTs) are the linchpin of parasitic helminth defence
,6
mechanisms, providing their major phase II detoxi®ca-
tion system and accounting for up to 4% of the total
soluble protein. Two O. volvulus GSTs have been repor-
ted to date, OvGST1 and OvGST2. We have previously
reported the isolation of puri®ed active recombinant
OvGST2. GSTs detoxify electrophilic compounds,2
including many anthelmintics and cytotoxins arising
Given the signi®cant divergence from host GSTs, we
propose that selective inhibition of parasite derived
GST enzymes is a realisable chemotherapeutic target.
This should ultimately tip the molecular balance in
favour of the host during the characteristically chronic
nematode infection and may be an important adjunct in
1
3
from the e€ector mechanism of the immune response,
through catalysis of their conjugation to glutathione.
3
e€ective immuno- and chemotherapy. Selective inhibi-
tors of nematode GST will also provide new tools to
study host±parasite interactions.
Unlike mammalian GSTs, parasitic nematode GSTs are
not well characterised. Biochemical, immunological and
primary amino acid sequence analysis fails to place
OvGST2 into any of the ®ve characterised mammalian
species independent soluble GST families (a, m, p, y and
s). Homology models indicate critical structural di€er-
ences at the active site between host and parasite
enzymes. In particular, the parasitic enzymes, whilst
strongly topologically related to the mammalian p-GST4
family, have a very open hydrophobic binding cleft.
This is in contrast to that found in the mammalian
enzymes, which have a more constricted feature. By
It is our hypothesis that suitable derivatisation of b-keto
substituted glutathione S-conjugates will engender speci-
®city along with established abolition of target enzyme
activity. As part of our ongoing investigations into the
utility of GSTs as a target for antiparasitic agents, we have
synthesised a series of b-carbonyl substituted glutathione
conjugates as inhibitors of OvGST2.
Chemistry
Enones were synthesized by the base catalysed Aldol con-
densation and the a,b -unsaturated esters were synthesized
*
7
Corresponding author. Tel.: +44-116-257-7115; fax: +44-116-257-
135; e-mail: molgraph@dmu.ac.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00142-6

9
80
P. M. Brophy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 979±981
by acid catalysed esteri®cation of maleic acid. The glu-
tathione conjugates were prepared through a base cata-
lyzed Michael-type addition. Cyclohex-2-enone and S-
comparison with 1 this might be seen as the result of
the extension of the chain on the side of the carbonyl
away from the glutathione conjugation point, or as the
result of chain shortening on the side of glutathione
(
dicarboxyethyl)glutathione (8) were obtained commer-
cially. Unexpectedly, the reaction between glutathione
and dibutyl maleate yielded the monobutyl succinate glu-
0
tathione conjugate (11) (R =H in Figure 1b). In all other
Table 1. IC50 Inhibition data for selected glutathione conjugates
cases, the diester conjugate was formed. Conditions and
reagents are shown in Figure 1.
a
IC50 Data (m M)
Compound
OvGST2
pGST
Selectivity
pGST/
OvGST2)
(
Inhibition of OvGST2 was determined using 1-chloro-
2,4-dinitrobenzene (CDNB) as the second substrate in
7
the standard assay: Reduced glutathione (1 mM), inhi-
bitor (0±1 mM) and puri®ed OvGST2 (10 mg/mL) were
combined in potassium phosphate bu€er (pH 6.5, 100
mM) and equilibrated at room temperature for 5 min.
The reaction was initiated by addition of 1-chloro-2,4-
dinitrobenzene in ethanol (1 mM). The increase in
absorbance at 340 nm due to production of S-(2,5-din-
trophenyl)glutathione was determined over 5 min at
room temperature. I50 was determined as the con-
centration of inhibitor leading to 50% decrease in the
rate of S-(2,5-dintrophenyl)glutathione production. The
inhibition of OvGST2 and human p GST are presented
as IC50 values in Table 1.
1
2
3
4.1‹1.28
8.4‹ 1.44
5.2‹ 3.0
8.1‹ 2.6
>250
1.3
1.3
220.0‹ 25.7
>1.1
4
66.0‹ 8.49
>250
>3.8
5
6
90.0‹ 26.0 217‹ 9.6
41.0‹ 7.94 155‹ 13.2
2.1
3.8
Results and Discussion
7
8
31.9‹ 5.04
19.1‹ 0.8
>250
>250
>7.8
It is likely that the GST most closely related to OvGST2
4
in a human host would be in the p-GST class. For this
reason inhibition data is shown for both OvGST2 and
human p-GST, giving a measure of selectivity of the
inhibitor for the target parasite enzyme (Table 1).
>13.9
9
>250
208‹ 29.3
>250
<0.6
>1.7
0.8
Compound 1 shows optimal OvGST2 inhibition.
Extension of the alkyl chain, such as in 2, leads to slight
increase in I50 however introduction of a branch, as
found in 4 signi®cantly increases IC50. Interestingly,
replacement of tetrahedral branched feature in 4 with
planar aryl group regains some of this lost activity.
This may be due to removal of a steric constraint or
acquisition of an aromatic±stacking interaction.
1
0
143.3‹ 12.6
48.0‹ 9.7
11
40.3‹ 3.5
With the exception of the cyclic conjugate 3, 5 shows
the highest IC50 value of the enone derived series. By
aValues shown as mean‹standard deviation (n=3).
ꢀ
3
Figure 1. Reagents and conditions: (i) aldehyde (0.4 mmol), ketone (1.1 mmol), 0 C, NaOH (10%, 10 cm ), stir 2 h; (ii) glutathione (1.6 mmol), a,b -
3
3
ꢀ
unsaturated carbonyl compound (6.4 mmol), methanol (2 cm ) NaOH (2 M, 1cm ), stir 25 C, 24 h; (iii) Maleic acid (5.00 g), concd H
ROH (30 mL), re¯ux, 16 h.
2 4
SO (1.5 mL),

P. M. Brophy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 979±981
981
conjugation. A comparison of 6 and 7 make the latter
more likely.
conjugates. Whilst unsubstituted, straight-chain conjugates
show the highest activity, substituted and branched
conjugates show the greatest selectivity. Selectivity for
OvGST2 over human p-GST of greater than 10-fold has
been achieved. Further work is underway to enhance
both activity and selectivity.
The succinate conjugate, 8 can be considered as isosteric
with 4 and 6, however it is more active than either. This
may suggest an additional electrostatic interaction is
desirable. Indeed, dimethylation of the succinyl group
whilst apparently not going beyond the steric boundaries
set by 7 leads to a compound with negligible activity. The
mono succinate ester conjugate 11 regains much of the
activity lost by the succinate diesters. In summary, b-car-
bonyl substituted glutathione conjugates can demonstrate
good OvGST2 inhibition. Extension of the chain beyond
the carbonyl group is allowed, as is the introduction of an
aryl group neighboring the glutathione conjugation
point. It is likely that a hydrogen bond donating and/or
accepting group in this position is especially favored.
Acknowledgements
The authors acknowledge the support of the UNDP/
World Bank/WHO Special Programme for Research
and in Tropical Diseases (TDR), project 980381.
References
1. Liebau, E.; Wildenburg, G.; Brophy, P. M.; Walter, R.;
Henkle-Duhrsen, K. Mol. Biochem. Parasitol. 1996, 80, 27.
Those conjugates with the requirements discussed above
and possessing a point of branching next to the point of
glutathione conjugation demonstrate the highest selec-
tivity for OvGST2 over human pGST. This in accord
with expectations of a more open active site in GSTs of
È
2. Armstrong, R. N. Chem. Res. Toxicol. 1991, 4, 131.
3. Brophy, P. M.; Pritchard, D. I. Exp. Parasitol. 1994, 79,
8
4
9.
. Liebau, E.; Eckelt, V. H. O.; Wildenburg, G.; Teesdale-
4
Spittle, P. H.; Brophy, P. M.; Walter, R. D.; Henkle-Duhrsen,
K. Biochem. J. 1997, 324, 659.
parasitic origin.
5. Droog, F. N. J.; Hooykaas, P. J. J.; Vanderzaal, B. J. Plant
Physiol. 1995, 107, 1139.
6
7
. Katusz, R. M.; Colman, R. F. Biochemistry 1991, 30, 11230.
. Mannervik, B.; Guthenberg, C. In Methods in Enzymology;
Conclusion
Inhibition of OvGST2 has been demonstrated at low
micromolar concentrations for b-carbonyl glutathione
Jakoby, W.B., Ed.; Academic: New York, 1981; Vol. 77, pp
231.