Inhibition of trypanothione reductase by substrate analogues.

Article abstract of DOI:10.1021/ol0065423

Trypanothione reductase (TR) catalyzes the NAPDH-dependent reduction of the spermidine-glutathione conjugate trypanothione, an antioxidant found in Trypanosomatid parasites. TR plays an essential role in the parasite's defense against oxidative stress and

Full text of DOI:10.1021/ol0065423

ORGANIC
LETTERS
2000
Vol. 2, No. 23
3639-3642
Inhibition of Trypanothione Reductase
by Substrate Analogues
Elizabeth A. Garrard, Emily C. Borman, Brian N. Cook, Emily J. Pike, and
David G. Alberg*
Department of Chemistry, Carleton College, Northfield, Minnesota 55057
dalberg@carleton.edu
Received September 1, 2000
ABSTRACT
Trypanothione reductase (TR) catalyzes the NAPDH-dependent reduction of the spermidine−glutathione conjugate trypanothione, an antioxidant
found in Trypanosomatid parasites. TR plays an essential role in the parasite’s defense against oxidative stress and has emerged as a prime
target for drug development. Here we report the synthesis of several trypanothione analogues and their inhibitory effects on T. cruzi TR. All
are competitive inhibitors with K values ranging from 30 to 91 µM.
i
Trypanothione reductase (TR) is an NADPH-dependent
flavoenzyme found in the parasitic protozoa Trypanosoma
and Leishmania.1 These parasites are responsible for a host
of diseases in both humans and domestic animals, including
African sleeping sickness (T. brucei) and Chagas’ disease
(T. cruzi), among others. To maintain an intracellular
reducing environment and to combat oxidative stress, try-
panosomatids rely on TR to catalyze the reduction of the
antioxidant trypanothione from its disulfide to its dithiol
form.1,2 In the parasites, the trypanothione/TR system appears
to serve in place of the related glutathione/glutathione
reductase (GR) system found in most other prokaryotes and
eukaryotes.1,3
(1) Fairlamb, A. H.; Cerami, A. Annu. ReV. Microbiol. 1992, 46, 695-
729.
(2) Fairlamb, A. H.; Blackburn, P.; Ulrich, P.; Chait, B. T.; Cerami, A.
Science 1985, 227, 1485-1487.
Both TR4 and GR5 display a high degree of sequence and
structural homology, and both catalyze their NADPH-
dependent reduction reactions by analogous mechanisms.3
Despite their similarities, however, the two reductases exhibit
almost complete specificity for their respective substrates.6
This metabolic distinction between the parasites and their
hosts, combined with the parasites dependence on TR for
growth and virulence,7 makes TR a promising target for the
(3) (a) Walsh, C.; Bradley, M.; Nadeau, K. Trends Biochem. Sci. 1991,
16, 305-309. (b) Walsh, C.; Bradely, M.; Nadeau, K. Curr. Top. Cell.
Regul. 1992, 33, 409-417.
(4) For leading references, see: (a) Bond, C. S.; Zhang, Y.; Berriman,
M.; Cunningham, M. L.; Fairlamb, A. H.; Hunter, W. N. Structure (London)
1999, 7, 81-89. (b) Borges, A.; Cummingham, M. L.; Tovar, J.; Fairlamb,
A. H. Eur. J. Biochem. 1995, 228, 745-752. (c) Leichus, B. N.; Bradley,
M.; Nadeau, K.; Walsh, C. T.; Blanchard, J. S. Biochemistry 1992, 31,
6414-6420.
(5) For leading references, see: (a) Schirmer, R. H.; Krauth-Siegel, R.
L.; Schulz, G. E. In Glutathione: Chemical, Biochemical, and Medicinal
Aspects. Part A; Dolphin, D., Poulson, R., Avramovic, O., Eds.; John Wiley
and Sons: New York, 1989; pp 553-596. (b) Ghisla, S.; Massey, V. Eur.
J. Biochem. 1989, 181, 1-17. (c) Karplus, P. A.; Schulz, G. E. J. Mol.
Biol. 1989, 210, 163-180. (d) Sweet, W. L.; Blanchard, J. S. Biochemistry
1991, 30, 8702-8709.
(6) (a) Shames, S. L.; Fairlamb, A. H.; Cerami, A.; Walsh, C. T.
Biochemistry 1986, 25, 3519-3526. (b) Krauth-Siegel, R. L.; Enders, B.;
Henderson, G. B.; Fairlamb, A. H.; Schirmer, R. H. Eur. J. Biochem. 1987,
164, 123-128.
10.1021/ol0065423 CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/27/2000

design of antiparasitic drugs.3 Accordingly, TR has attracted
the attention of a number of groups interested in inhibiting
this enzyme.8
othione’s disulfide moiety during the enzymatic reduction,
by the thiol-specific reagent iodoacetamide.6 The enzyme is
also covalently inhibited by nitrosourea drugs such as
carmustine.8a These data suggest that substrate analogues
incorporating electrophilic moieties should be potent inac-
tivators of this enzyme.8,14 We now report the synthesis of
reversible TR inhibitors 5 and 6 and the evaluation of the
inhibitory activity of these compounds against T. cruzi TR.
Unfortunately, we have so far been unable to isolate potential
epoxide inhibitor 7 because of the instability of its oxirane
ring.
Like compounds 1-4, inhibitors 5a and 6a contain
3-dimethylaminopropylamide (DMAPA, a) groups in place
of trypanothione’s spermidine moiety. Inhibitors 5b and 6b,
on the other hand, replace the DMAPA groups with two
3-propylaminopropylamide (PAPA, b) chains. While the
DMAPA group has seen regular use in trypanothione
analogues9-12,15, we wished to examine the PAPA group,
since an acyclic trypanothione analogue with two PAPA
chains in the place of the spermidine moiety displays both a
lower Km (92 vs 185 µM) and higher kcat/Km (more than
2-fold) than its analogue with two DMAPA chains.9
Inhibitors 5 and 6 were all synthesized from olefin
intermediate 16. We initially prepared 16 from Cbz-aspartic
acid R-methyl ester (8), as shown in Scheme 1.16 Notable
Early substrate specificity studies showed that TR tolerates
significant variations in the structure of its substrate. Acyclic
substrate analogues incorporating amine-bearing chains in
the place of the spermidine group are turned over by the
enzyme,9 as are analogues in which the γ-glutamyl moeity
is replaced by various groups, including a simple Cbz
moiety.10 Indeed, we routinely use substrate analogue 1,
reported by El-Waer et al.,11 as an easily accessible substrate
for TR assays.
On the basis of the structure of substrate 1, Sergheraert
and co-workers12 prepared nonreducible TR inhibitors in
which the cystine moeity of 1 is replaced successively by
djenkolic acid, lanthionine, and cystathionine (2-4, respec-
tively). Analogues 2-4 all retain sulfur atoms in the bridge
connecting the peptidic halves of the molecules. We were
interested in exploring similar analogues in which the
bridging group is composed exclusively of carbon atoms (5
and 6), with the intent of ultimately using the olefin of 5 as
a means of introducing an epoxide moiety, to provide a
potential irreversible TR inhibitor (7).
Scheme 1a
a (a) i. BH3 (76%), ii. NaOCl, TEMPO (65%); (b) Ph3PdCHCHO,
toluene, ∆; (c) 9-BBN, THF; (d) CBr4, PPh3, CH2Cl2; (e) n-BuLi,
THF, -78 °C; (f) 1:1 0.5 M HCl/THF; (g) Cbz-Cl, TEA, THF; (h)
i. 0.5 M LiOH, methanol, ii. aqueous HCl.
TR is known to be alkylated specifically at Cys-53,13 an
active site nucleophile implicated in an attack on trypan-
(7) (a) Dumas, C.; Ouellette, M.; Tovar, J.; Cunningham, M. L.; Fairlamb,
A. H.; Tamar, S.; Olivier, M.; Papadopoulou, B. EMBO J. 1997, 16, 2590-
2598. (b) Tovar, J.; Cunningham, M. L.; Smith, A. C.; Croft, S. L.; Fairlamb,
A. H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5311-5316. (c) Krieger, S.;
Schwarz, W.; Ariyanayagam, M. R.; Fairlamb, A. H.; Krauth-Siegel, R.
L.; Clayton, C. H. Mol. Microbiol. 2000, 35, 542-552.
transformations include the introduction of the trans olefin
via Wittig addition of (triphenylphosphoranylidene)acet-
aldehyde to aldehyde 9, to afford R,â-unsaturated aldehyde
(8) For reviews, see: (a) Schirmer, R. H.; Muller, J. G.; Krauth-Siegel,
R. L. Angew. Chem., Int. Ed. Engl. 1995, 34, 141-154. (b) Krauth-Siegel,
R. L.; Scho¨neck, R. FASEB J. 1995, 9, 1138-1146. (c) Krauth-Siegel, R.
L.; Coombs, G. H. Parasitol. Today 1999, 15, 404-409. (d) Austin, S. E.;
Khan, M. O. F.; Douglas, K. T. Drug Des. DiscoVery 1999, 16, 5-23.
(9) Henderson, G. B.; Fairlamb, A. H.; Ulrich, P.; Cerami, A. Biochem-
istry 1987, 26, 3023-3027.
(10) El-Waer, A. F.; Smith, K.; McKie, J. H.; Benson, T.; Fairlamb, A.
H.; Douglas, K. T. Biochim. Biophys. Acta 1993, 1203, 93-98.
3640
Org. Lett., Vol. 2, No. 23, 2000

10,17 and the stereospecific alkylation of bromide 12 by the
lithiated Scho¨llkopf dihydydropyrazine 13, to furnish 14 in
89% yield.18
Scheme 3a
We recently devised a more efficient route to 16, shown
in Scheme 2 along with the elaboration of this olefin to
Scheme 2a
a (a) 2.25 equiv 13, n-BuLi, THF, -78 °C; (b) 1:1 0.5 M HCl/
THF; (c) Cbz-Cl, TEA, THF; (d) i. 0.5 M LiOH, MeOH, ii. HCl;
(e) 2.8 equiv H-Gly-NH(CH2)3N(CH3)2, PyBrop, DIEA, CH2Cl2;
(f) 480 psi H2, Rh(PPh3)3Cl, benzene/EtOH.
inhibitors 5a and 6a. The double addition of 2 equiv of
lithiated Scho¨llkopf reagent 13 to commercially available
trans-1,4-dibromobutene gave olefin 17 in 52% yield.18
Hydrolysis of the bislactim ethers of 17, followed by
treatment of the resulting diamine with 2 equiv of Cbz-Cl,
afforded diester 15 in 65% yield for the two steps. Finally,
hydrolysis of the methyl esters provided diacid 16 in
essentially quantitative yield. PyBrop19 mediated coupling
of 2 equiv of glycine-3-dimethylaminopropylamide11 to 16
gave inhibitor 5a. In turn, hydrogenation of a portion of 5a,
in the presence of Wilkinson’s catalyst, gave saturated
inhibitor 6a in 78% yield.
a (a) CH2Cl2; (b) Fmoc-Cl, DIEA, CH2Cl2; (c) TFA, CH2Cl2;
(d) 2.5 equiv 19, PyBop, DIEA, CH2Cl2; (e) DBU, CH2Cl2; (f)
400 psi H2, Rh(PPh3)3Cl, benzene/EtOH; (g) diethylamine, CH3CN.
as its trifluoroacetate salt. PyBop20 mediated coupling of 2
equiv of amine 19 with diacid 16 afforded 20 in 96% yield.
Removal of the Fmoc groups from 20 gave unsaturated
inhibitor 5b,21 while hydrogenation of 20 in the presence of
Wilkinson’s catalyst, followed by Fmoc removal, afforded
saturated inhibitor 6b.
We envisioned preparing epoxide 7b from olefin 20
(Scheme 4). Epoxidation of this olefin with mCPBA ap-
The preparation of inhibitors 5b and 6b required the
synthesis of glycine derivative 19, shown in Scheme 3. The
addition of N-propyl-1,3-propanediamine to Boc-glycine
N-hydroxysuccinimde ester cleanly afforded the correspond-
ing secondary amide, which was treated with Fmoc-Cl to
furnish 18 in 72% yield for the two steps. Removal of the
Boc moiety under standard conditions provided amine 19
Scheme 4
(11) El-Waer, A.; Douglas, K. T.; Smith, K.; Fairlamb, A. H. Anal.
Biochem. 1991, 198, 212-216.
(12) Tromelin, A.; Moutiez, M.; Meziane-Cherif, D.; Aumercier, M.;
Tartar, A.; Sergheraert, C. Bioorg. Med. Chem. Lett. 1993, 3, 1971-1976.
(13) TR amino acid residue numbering is that of the T. cruzi enzyme.
(14) One TR-selective covalent inhibitor was recently reported; see ref
4a.
(15) Yuen, C. T.; Garforth, J.; Besheya, T.; Jaouhari, R.; McKie, J. H.;
Fairlamb, A. H.; Douglas, K. T. Amino Acids 1999, 17, 175-183.
(16) This synthesis was inspired by Jurgens’ asymmetric synthesis of a
diaminopimelic acid derivative, see: Jurgens, A. R. Tetrahedon Lett. 1992,
33, 4727-4730.
(17) The expected E-olefin geometry of 10 was confirmed by the large
vinyl coupling constant observed for this material (JCHdCH ) 15.6 Hz). No
trans isomer was observed in the NMR spectrum of this compound.
(18) Scho¨llkopf, U.; Groth, U.; Deng, C. Angew. Chem., Int. Ed. Engl.
1981, 20, 798-799.
(19) PyBrop ) bromotripyrrolidinophoshonium hexafluorophosphate.
Coste, J.; Fre´rot, E.; Jouin, P. Tetrahedron Lett. 1991, 32, 1967-1970.
peared to provide oxirane 22. However, this compound
proved to be very sensitive, and it decomposed during
purification attempts. We suspect that decomposition pro-
ceeds via intramolecular nucleophilic attack by a glycyl
(20) PyBop ) benzotriazole-1-yloxytrispyrrolidinophosphonium hexaflu-
orophosphate. Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205-208.
(21) Our low yield in this normally high-yielding Fmoc deprotection
reaction was the result of accidental loss of material during purification.
Org. Lett., Vol. 2, No. 23, 2000
3641

amide nitrogen on the epoxide ring, analogous to the well-
known tendency of aspartyl â-ester-containing peptides to
cyclize to aminosuccinimides.22 However, oxidation of 20
with dimethyldioxirane23 did cleanly provide 22 in essentially
quantitative yield, but removal of the Fmoc groups from this
compound was not possible without decomposition. We now
believe that epoxides 7a,b are unlikely to be stable to the
assay conditions, and we are currently exploring structural
modifications that would provide for more robust epoxide
compounds. One possibility would be to replace the offend-
ing glycyl amide moieties with less nucleophilic esters (i.e.
replace the glycyl residues with glycolates).
Compounds 5 and 6 were evaluated as inhibitors of
recombinant T. cruzi TR.24 TR activity was assayed using 1
as the disulfide substrate and by following the oxidation of
NADPH spectrophotometrically at 340 nm.25 The Ki values
for each inhibitor are given in Table 1.
concentrations of each ([glutathione] ) 30 µM), indicating
a decided specificity of our inhibitors for the parasite enzyme.
It appears that TR has a slight preference for the DMAPA
chain (a) over the PAPA chain (b). This finding is somewhat
surprising in light of the substrate specificity results of
Henderson, et al.,9 which would suggest otherwise, although
those results were obtained with enzyme from Crithidia
fasciculata. It is also apparent that the TR active site tolerates
the different structural geometries of the saturated versus the
unsaturated inhibitors, displaying an approximate 2-fold
preference for the saturated inhibitors. Crystallography
studies indicate that the TR active site is rigid and undergoes
little conformational change upon substrate binding.4a,26 That
TR appears to be reasonably tolerant of the geometry of the
tether linking the two peptidic halves of our inhibitors,
despite the enzyme’s apparent rigidity, is reassuring and
bodes well for the eventual success of potential epoxide
inhibitors when such compounds become available.
Acknowledgment. We thank Professor Christopher Walsh
and Kari Nadeau (Department of Biological Chemistry and
Molecular Pharmacology, Harvard Medical School) for
providing the SG5 E. coli strain containing TR expression
vector pIBITczTR. We are grateful for support from a
Camille and Henry Dreyfus Foundation Faculty Start-up
Grant and a Bristol-Meyers Squibb Company Award of the
Research Corporation. Acknowledgment is made to the
donors of the Petroleum Research Fund, administered by the
American Chemical Society, for partial support of this
research. A student stipend for E.A.G. was provided by a
grant from the Howard Hughes Medical Institute. We also
thank Salwa Salah for optimizing the yields of the first three
transformations shown in Scheme 2.
Table 1. Inhibition of T. cruzi TRa
entry
inhibitor
Ki (µM)
1
2
3
4
5a
5b
6a
6b
74 ( 6
91 ( 6
30 ( 3
48 ( 3
a Assays were run at 25 °C in 100 mM HEPES (pH 7.8), 1 mM EDTA,
and 150 µM NADPH. Ki values for each inhibitor were determined by
measuring the initial rates at three different inhibitor concentrations, ranging
from 25 to 105 µM, in the presence of five substrate concentrations, varied
from 2.5 to 36 µM. The data were fit to the competitive inhibition model
using Cleland’s COMP program.27 The Km value for assay substrate 1 was
6.7 µM.
Supporting Information Available: Experimental pro-
cedures and characterization data for all compounds and
experimental details for the enzyme assays. This material is
available free of charge via the Internet at http//pubs.acs.org.
As expected, 5 and 6 are all modest competitive inhibitors
of TR, with affinities comparable to those found for
analogues 2-4.12 We also assessed the abilities of our
compounds to inhibit yeast GR. We observed no inhibition
of GR by any of our compounds at up to 250 µM
OL0065423
(26) Bailey, S.; Smith, K.; Fairlamb, A. H.; Hunter, W. H. Eur. J.
Biochem. 1993, 213, 67-75.
(22) Bodanszky, M. Principles of Peptide Synthesis, 2nd ed.; Springer-
Verlag: Berlin, 1993; pp 187-188 and references therein.
(23) Murray, R. W. Chem. ReV. 1989, 89, 1187-1201.
(24) Sullivan, F. X.; Walsh, C. T. Mol. Biochem. Parisitol. 1991, 44,
145-148.
(25) Sullivan, F. X.; Shames, S. L.; Walsh, C. T. Biochemistry 1989,
28, 4986-4992.
(27) We also attempted to fit the data to the uncompetitive and
noncompetitive inhibition models using Cleland’s UNCOMP and NCOMP
programs. As expected, the data for each inhibitor either failed to fit these
models or gave a fit significantly worse than that obtained with the
competitive model. Cleland, W. W. Methods Enzymol. 1979, 63, 103-
138.
3642
Org. Lett., Vol. 2, No. 23, 2000