Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites

Article abstract of DOI:10.1016/j.bmc.2009.01.068

Dehydroepiandrosterone (DHEA) is known as an intermediate in the synthesis of mammalian steroids and a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), but not the enzyme from plants and lower eukaryotes. G6PDH cataly

Full text of DOI:10.1016/j.bmc.2009.01.068

Bioorganic & Medicinal Chemistry 17 (2009) 2483–2489
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by
human steroids and their effects on the viability of cultured parasites
a
b
Artur T. Cordeiro a, , Otavio H. Thiemann , Paul A. M. Michels
*
a Centro de Biologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, Av. Trabalhador São Carlense 400, São Carlos, Brazil
b Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Université catholique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Dehydroepiandrosterone (DHEA) is known as an intermediate in the synthesis of mammalian steroids
and a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), but
not the enzyme from plants and lower eukaryotes. G6PDH catalyzes the first step of the pentose-phos-
phate pathway supplying cells with ribose 5-phosphate, a precursor of nucleic acid synthesis, and NADPH
for biosynthetic processes and protection against oxidative stress. In this paper we demonstrate that also
G6PDH of the protozoan parasite Trypanosoma brucei is uncompetitively inhibited by DHEA and epian-
drosterone (EA), with Ki values in the lower micromolar range. A viability assay confirmed the toxic effect
of both steroids on cultured T. brucei bloodstream form cells. Additionally, RNAi mediated reduction of
the G6PDH level in T. brucei bloodstream forms validated this enzyme as a drug target against Human
African Trypanosomiasis. Together these findings show that inhibition of G6PDH by DHEA derivatives
may lead to the development of a new class of anti-trypanosomatid compounds.
Received 20 November 2008
Revised 26 January 2009
Accepted 30 January 2009
Available online 4 February 2009
Keywords:
Dehydroepiandrosterone
Trypanosoma brucei
Glucose-6-phosphate dehydrogenase
Uncompetitive inhibition
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction
with NADPH for biosynthetic processes and protection against oxi-
dative stress. G6PDH deficiency affects 400 millions of people in
Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are
parasitic protozoans responsible for diseases that affect mankind
in tropical and subtropical countries: Human African Trypanosomi-
asis or sleeping sickness, Chagas disease and leishmaniasis, respec-
tively. Control of such diseases relies mainly on chemotherapy, but
current drugs are highly inadequate. Recently, most efforts have
been directed to the development of drugs that act specifically on
validated metabolic targets. Several enzymes of carbohydrate
metabolism, participating in glycolysis1 and the pentose-phosphate
pathway2 (PPP), have been perceived as promising targets for drug
design strategies. The use of RNA interference (RNAi) in T. brucei has
become the preferable approach to identify essential proteins for
growth and survival of the parasites. Expression of double-stranded
RNA to induce RNAi against hexokinase, phosphofructokinase,
pyruvate kinase, phosphoglycerate mutase and enolase was used
to evaluate the importance of these enzymes for the glycolytic flux
and growth of cultured pathogenic bloodstream-stage forms of the
trypanosome.3
the world, it is the most common human enzyme pathology. Most
G6PDH-deficient individuals are asymptomatic and unaware of
their status. The illness generally manifests itself as acute haemol-
ysis, trigged by oxidative stress conditions.4 G6PDH deleted
embryonic stem cells can be cultured in usual Dulbecco’s Modified
Eagle’s medium, although with an impaired cloning efficiency; that
can be overcome by reducing the oxygen pressure of the medium.5
In such cells, the NADPH production is maintained by cytosolic iso-
citrate dehydrogenase and malic enzyme; G6PDH is overexpressed
in response to oxidative agents.6 In trypanosomatids, isocitrate
dehydrogenase is a mitochondrial enzyme and in T. cruzi and T.
brucei, but not Leishmania species, it is also predicted to occur in-
side the peroxisome-like organelles called glycosomes.7 Therefore,
trypanosomatids rely on malic enzyme8 and both dehydrogenases
of the PPP (G6PDH and 6-phospho-gluconolactone dehydrogenase,
6PGDH) for cytosolic NADPH production.
The possibility of using the PPP of T. brucei as a drug target is
supported by studies on 6PGDH. Differences between sheep and
T. brucei 6PGDHs structures9 permitted the design of selective
inhibitors with trypanocidal activity.10 It is noteworthy that
6PGDH inhibition leads to accumulation of 6-phospho-gluconate,
which is a competitive inhibitor of glucose-6-phosphate isomerase
in glycolysis.11
Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the
first step of the PPP, converting glucose 6-phosphate (G6P) into
6-phospho-gluconolactone and reducing NADP+ to NADPH. The
oxidative branch of the PPP produces ribose 5-phosphate (R5P)
which is a precursor for nucleic acid synthesis and supplies the cell
Dehydroepiandrosterone (DHEA) is an intermediate in the syn-
thesis of mammalian steroid hormones such as testosterone and
estradiol. It is produced in the adrenal cortex and reaches maximal
* Corresponding author. Tel.: +55 16 33739868; fax: +55 16 33739881.
E-mail address: arturcor@gmail.com (A.T. Cordeiro).
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.068

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A. T. Cordeiro et al. / Bioorg. Med. Chem. 17 (2009) 2483–2489
values in the circulation between the ages of 20 and 30 years;
thereafter, the serum DHEA level decreases markedly.12 Its admin-
istration to elderly people has been associated to ameliorating the
signs of the aging process and some derivatives have been synthe-
sized aiming the development of new drugs against cancer, diabe-
tes, obesity and auto-immune diseases.13 DHEA affects mammalian
cell proliferation, but the precise mechanism by which it does so is
unclear. The enzymes G6PDH of the PPP and 3-hydroxy-3-methyl
glutarylCoA reductase (HMGR) of the mevalonate pathway are
inhibited by DHEA. HMGR catalyzes the rate-limiting step in sterol
biosynthesis and its product, mevalonate, is a precursor of farnesyl
diphosphate, necessary for protein farnesylation reactions occur-
ring in many cellular processes such as signal transduction.14
One observation in favor of G6PDH as the main target is that cell
growth suppression by DHEA was partially prevented by addition
of ribonucleosides, but not mevalonolactone.15 Probably, the
growth inhibition effect of DHEA might not be attributed to a sin-
gle mechanism, but G6PDH inhibition seems to be an important
contributing factor, either by reduction of 5-carbon sugars for the
synthesis of ribonucleosides and deoxyribonucleosides or by
depletion of NADPH, the reductive agent for lipid and cholesterol
biosynthesis and reactive oxygen species (ROS) detoxification.16
DHEA is an uncompetitive inhibitor of mammalian G6PDH, but
not the enzyme from plants and lower eukaryotes.17,18 Neverthe-
less, DHEA derivatives were effective against parasitic infections
by Plasmodium falciparum,19 Cryptosporidium parvum,20 Schisto-
soma mansoni,21 and T. cruzi.22 In such cases, it was suggested that
DHEA acts by stimulating the host immune response. In the case of
parasitic infections by Taenia crassiceps23 and Entamoeba histolyti-
ca,24 DHEA presented a parasiticidal effect not related to the im-
mune response. The anti-parasitic properties of DHEA are
remarkable, but more information is needed to clarify its mecha-
nism of action.
Figure 1. (A) Cumulative growth curve of T. brucei bloodstream forms transfected
with a G6PDH RNAi construct. In the absence of RNAi induction, both wild-type (s)
and transfected (h) cells grow at equal rates. In the presence of the RNAi-inducer
tetracycline, wild-type cells (d) grow at normal rates, while transfected cells (j)
die after 48 h. (B) Western blot analysis of the G6PDH levels in T. brucei bloodstream
form cells transfected with a RNAi construct. Samples were obtained at 24 h
intervals from cultures grown in the absence (ꢁ) and presence (+) of tetracycline.
Triosephosphate isomerase (TIM) was used as a sample loading control.
TbG6PDH exerts control on the growth or viability of the blood-
stream-form trypanosomes. The possibility that levels of other, un-
known essential proteins are affected by off-target effects and
contribute to the observed growth phenotype cannot be excluded
but is considered unlikely. The G6PDH sequence is unique; no
other genes with stretches of DNA highly similar to that of the
G6PDH gene were detected in the T. brucei genome database.
In the work presented here, we (1) evaluate the effect of deplet-
ing G6PDH transcript levels in T. brucei bloodstream form by RNAi;
(2) determine kinetic constants for recombinant T. brucei and Leish-
mania mexicana G6PDH (Tb and LmG6PDH), demonstrating that
both enzymes form ternary complexes with their substrates G6P
and NADP+; (3) measure DHEA and EA inhibition constants against
TbG6PDH and (4) assay the effect of DHEA and EA on growth and
viability of cultured T. brucei and L. mexicana. All together, the re-
sults indicate that DHEA (or EA) might be explored as a template
molecule for the development of new anti-trypanosomatid com-
pounds that act specifically against the parasites’ G6PDH.
2.2. T. brucei and L. mexicana G6PDH kinetic mechanism
N-terminally His-tagged recombinant Tb- and LmG6PDH were
expressed in Escherichia coli BL21 transformed with recombinant
pET28 plasmids containing either of the parasites genes, and puri-
fied to homogeneity by a single nickel affinity chromatographic
step. Approximately 1 and 3 mg of essentially pure Tb and
LmG6PDH were recovered per liter of E. coli culture. Enzyme kinet-
ics analyses were performed for both His-tagged recombinant
G6PDHs. Reaction velocities, expressed as nmoles of NADPH pro-
duced per second, were obtained by varying the G6P concentration
at different concentrations of NADP+ (Fig. 2). The convergent pat-
terns of Lineweaver-Burk lines observed for both Tb and LmG6PDH
2. Results
2.1. Conditional depletion of G6PDH levels by RNAi
are consistent with a bi-bi ordered reaction mechanism.25 Linear fit
To genetically validate G6PDH as a drug target in bloodstream-
form T. brucei, a cell line was created by transfection with a con-
struct enabling the production of double-stranded RNA corre-
app-g6p
to secondary plots (1=V
vs 1/[NADP+] and (Km/Vmax app-g6p vs
)
max
1/[NADP+]) allowed to calculate kinetic constants using Cleland’s
velocity equation:
sponding to the G6PDH gene from
a tetracycline-inducible
promoter, and so to specifically decrease the level of the enzyme
by RNAi. A reduction in the growth rate of bloodstream-form cells
was observed between 24 and 48 h after tetracycline addition to
the culture. After 48 h, TbG6PDH depleted cells began to die.
Non-induced transfected trypanosomes presented a similar growth
rate as wild-type cells cultured in the presence or absence of tetra-
cycline (Fig. 1A). No decrease of intracellular TbG6PDH levels was
observed after 24 h of RNAi induction, but at 48 h, the level of
TbG6PDH in cellular extract was considerably reduced, almost
undetectable by western-blot analysis (Fig. 1B). The reduction of
the growth rate of the culture thus correlates with the beginning
of the RNAi induced decrease of the protein level, indicating that
m
¼ V1½Aꢀ½Bꢀ=ðKiaKb þ Kb½Aꢀ þ Ka½Bꢀ þ ½Aꢀ½BꢀÞ;
where Ka and Kb are the Michaelis constants for substrates A (G6P)
and B (NADP+), respectively; and V1 and Kia are the maximum veloc-
ity and the dissociation constant for substrate A, respectively. The
kinetic constants derived for Tb and LmG6PDH are presented in Ta-
ble 1.
2.3. Inhibition of G6PDH by DHEA and EA
DHEA and EA are uncompetitive inhibitors with respect to both
substrates, G6P and NADP+, of TbG6PDH, but not in the case of

A. T. Cordeiro et al. / Bioorg. Med. Chem. 17 (2009) 2483–2489
2485
Figure 2. Reaction velocity, Lineweaver-Burk and secondary plots for Tb (A) and LmG6PDH (B). Velocity (V) is expressed in nmoles of NADPH produced per second. In velocity
and Lineweaver-Burk plots, each curve was obtained at a fixed concentration of NADP+ (as indicated aside of the plots) while the G6P concentration was varied (at values of
1250, 625, 312.5, 156.25, 78.12 and 39.06 lM).
(Fig. 3A and B). Due to solubility limitation, the highest EA and
Table 1
T. brucei (Tb), and L. mexicana (Lm) G6PDH kinetic constants for an ordered bi-bi
reaction mechanism
DHEA concentrations achieved in LmG6PDH inhibition assays were
50 and 100 lM, respectively. At these concentrations, the com-
pounds had no significant effect on LmG6PDH, resulting in Linewe-
aver-Burk lines that superpose to those obtained in the inhibitor-
free assay (Fig. 3C). EA and DHEA Ki values were calculated for each
of the TbG6PDH substrates separately (Table 2).
G6PDH
KG6P
M)
KNADP
M)
V1
kcat
KiG6P
M)
(l
(l
(nmoles of
(sꢁ1
)
(
l
NADPH sꢁ1
)
Tb
Lm
57.8 2.4
74.5 3.0
9.4 0.4
12.1 0.5
36.2 1.5
31.2 1.2
16.4 0.6
22.2 0.9
47.6 1.9
86.4 3.5
2.4. T. brucei and L. mexicana viability assays
LmG6PDH. The uncompetitive inhibition is characterized by pro-
portional changes in apparent Km and Vmax, which result in parallel
Lineweaver-Burk lines for different inhibitor concentrations
The reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt
(MTS) to formazan was used as a measure of viable cells grown

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A. T. Cordeiro et al. / Bioorg. Med. Chem. 17 (2009) 2483–2489
Figure 3. Dehydroepiandrosterone (DHEA) and Epiandrosterone (EA) inhibition of T. brucei (A and B) and L. mexicana G6PDH (C). Inhibitors were assayed against each
substrate in separate experiments. First, G6P was varied from 312.5 to 19.5
to 1.95
l
M with NADP+ fixed at a saturating concentration (250 M), then NADP+ was varied from 31.25
l
l
M while G6P was kept at a saturating concentration (1.25 mM). The parallel lines in the Lineweaver-Burk plots confirm that DHEA is an uncompetitive inhibitor of
TbG6PDH.
3. Discussion
Table 2
Comparison of inhibition constants of Dehydroepiandrosterone (DHEA) and Epian-
drosterone (EA) between T. brucei (TbG6PDH) and human (HsG6PDH) glucose-6-
phosphate dehydrogenase
3.1. T. brucei G6PDH is a valid drug target
The conditional RNAi-dependent G6PDH depletion in transfec-
ted T. brucei bloodstream cells causes a reduction in growth rate
followed by culture death after 48 h (Fig. 1). This result strongly
suggests that TbG6PDH is an essential enzyme for proliferation of
T. brucei bloodstream forms and a valid drug target against Human
African Trypanosomiasis.
TbG6PDH
NADP
HsG6PDHa
G6P
G6P
NADP
K0i DHEA
(
M)
l
M)
1.70 0.1
1.10 0.1
8.9 0.3
3.4 0.3
6.2 0.5
3.0 0.1
K0i EA
(
l
0.49 0.03
0.46 0.03
Values extracted from Gordon et al., 1995.18
a
NADPH, more than R5P, is pointed as the most important end
product of the oxidative branch of the PPP to sustain mammalian
embryonic stem cells cloning efficiency.5 In G6PDH deficient cells,
R5P might be provided by the successive action of transaldolase
and transketolase on fructose 6-phosphate, an intermediate of gly-
colysis. In trypanosomatids, the first seven glycolytic steps occur
inside a peroxisome-like organelle denominated glycosome; other
pathways like the PPP, b-oxidation of fatty acids, purine salvage,
and biosynthetic pathways for pyrimidines, ether-lipids and squa-
lenes are also at least partially localized inside glycosomes.26 In T.
brucei procyclic forms, G6PDH is found in both glycosomes and
cytosol;27 this dual subcellular localization is evidence that NADPH
is required inside glycosomes too for the biosynthetic processes
in the presence of different concentrations of DHEA and EA. LD50
was defined as the inhibitor concentration that reduces the num-
EA
50
ber of viable cells by 50%. The LD50 of EA (LD ) and DHEA
(LDD50HEA) for T. brucei bloodstream forms were 24.5 0.7 and
43.8
2 lM, respectively. The EA and DHEA toxicity data for T. bru-
cei fitted a dose–response curve with R2 = 0.995 and 0.984, respec-
tively (Fig. 4A). L. mexicana was not affected by the presence of EA
and DHEA in the culture medium (Fig. 4B). At concentrations supe-
rior to 96 lM, crystalline precipitates were observed in the L. mex-
icana assays hampering the measurements at higher
concentrations.

A. T. Cordeiro et al. / Bioorg. Med. Chem. 17 (2009) 2483–2489
2487
3.3. DHEA and EA are toxic to T. brucei, but not to L. mexicana
The observation that DHEA and EA are toxic for in vitro cultures
of T. brucei bloodstream forms but not L. mexicana early-metacyclic
promastigotes is in agreement with the notion that they arrest par-
asite growth by specifically inhibiting G6PDH. Our results clearly
show that EA and DHEA are potent inhibitors of only TbG6PDH,
but not of LmG6PDH and they also confirm that both steroids cause
death of trypanosomes, but do not affect Leishmania growth.
Although off-target effects, like HMGR inhibition, remain to be ex-
cluded, our data suggest that EA and DHEA toxicity for trypano-
somes is most likely a consequence of G6PDH inhibition. If other
targets than G6PDH exist in T. brucei bloodstream form, their
homologues in L. mexicana must also be unsusceptible to DHEA,
otherwise we would expect a L. mexicana culture to be killed once
challenged with DHEA or EA. It may be possible that DHEA also
interferes with other vital cellular processes; however, the inhibi-
tion of G6PDH certainly impairs T. brucei bloodstream forms prolif-
eration, as observed by RNAi experiments targeted against
TbG6PDH.
It is also worth noting that in T. brucei bloodstream form, differ-
ently from other trypanosomatids, cholesterol is the predominant
sterol and it is scavenged from host serum lipoproteins; Leishmania
species synthesize predominantly ergosterol-related molecules,
although in its amastigote form it can also acquire cholesterol from
the host macrophage.34 The capability of surviving with a variable
profile of sterol may assist the parasites to overcome an inhibition
of sterol biosynthesis and it imposes additional challenges to the
development of specific inhibitors for such pathway enzymes.
Figure 4. T. brucei bloodstream forms (A) and L. mexicana early-metacyclic
promastigotes (B) viability at different concentrations of DHEA and EA. Dose–
response curves for DHEA and EA are presented in the T. brucei plot.
located in them, and also because a cascade of detoxification reac-
tions is initiated in these organelles with the reduction of trypano-
4. Conclusion
thione by
a NADPH-dependent trypanothione reductase, an
Contrary to the general concept that DHEA and its derivatives
are specific inhibitors of mammalian G6PDHs, we showed here
that DHEA and EA are also potent inhibitors of the G6PDH of the
protozoan T. brucei. The protective effect of DHEA derivatives on
different parasitic infections has previously been attributed to
the enhancement of the host immune response. However, we dem-
onstrated that DHEA derivatives may also be cytotoxic against hu-
man parasites such as T. brucei bloodstream forms. Inhibition of
parasite G6PDH, if not lethal by interrupting an essential glu-
cose-catabolizing process, may at least decrease the parasite’s tol-
erance to the oxidative burst mediated by host defensive cells.
Based on the presented results, DHEA derivatives may thus be ex-
plored as a novel class of anti-trypanosomal agents due to their
inhibitory effect on the G6PDH. In order to be considered effica-
cious anti-trypanosomal molecules, selectivity against the parasite
enzyme and minimal androgenic effect are desired properties for
DHEA derivatives to be developed. We are currently conducing
crystallization trials to determine the structure of TbG6PDH in
complex with EA. Information about such a complex will help us
in reaching the desired selective anti-trypanosomal properties for
DHEA derivatives.
enzyme that also has been shown to have a dual localization.28
3.2. Inhibition of T. brucei G6PDH by human steroids
EA and DHEA are potent inhibitors of TbG6PDH, with K0i values
in the low micromolar range. An initial study on the interaction of
G6PDH with human steroids detected an inhibitory effect on puri-
fied human enzyme and the enzyme in rat crude tissue extracts but
not in spinach extracts or on partially purified yeast enzyme.17
Based on these findings it was suggested that only mammalian
G6PDHs were inhibited by DHEA derivatives.18 However, we found
that TbG6PDH is also inhibited by DHEA and EA, and that the par-
asite’s enzyme has even a higher affinity (lower Ki) than does
HsG6PDH. Comparison of DHEA and EA effects on Tb and HsG6PDH
(Table 2) shows that inhibition constants for the protozoan enzyme
are approximately 6-fold lower than the values obtained for the
human enzyme. To our knowledge, TbG6PDH is the first non-mam-
malian enzyme for which uncompetitive inhibition by human ste-
roids has been demonstrated. Uncompetitive inhibition is rarely
observed in known cell metabolism. The specific interaction of an
uncompetitive inhibitor with an enzyme–substrate ternary com-
plex can increase metabolic intermediates to toxic concentrations
inside cells.29 The formation of a ternary complex is essential for
the binding of uncompetitive inhibitors. Here, we demonstrate that
Tb and LmG6PDH follow the ordered bi-bi reaction mechanism, in
which both substrates bind the enzyme, forming a ternary com-
plex, before the chemical reaction occurs. At this stage no informa-
tion is available about the localization of the steroid binding site in
the trypanosomatid G6PDH. Crystallographic structures for NADP-
dependent G6PDHs are known for only two species: human30,31
and the bacterium Leuconostoc mesenteroides.32,33 The latter is
not inhibited by DHEA and the structure of the human enzyme
did not reveal a possible binding site to DHEA.
5. Experimental
5.1. RNA interference
Conditional depletion of TbG6PDH by interfering with its expres-
sion was performed similarly as in the previously reported RNAi ex-
periments for T. brucei glycolytic enzymes.3 To induce RNAi in T.
brucei bloodstream forms, a construct was prepared for the tetracy-
cline-inducible synthesis of a double-stranded RNA hairpin mole-
cule. The construct consists of two short, complementary
DNA fragments of the TbG6PDH gene (Tb10.70.5200, 1677 bp), one

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A. T. Cordeiro et al. / Bioorg. Med. Chem. 17 (2009) 2483–2489
somewhat longer than the other, that were inserted side by side but
in opposing directions into vector pHD677,35 and so allowing the
synthesis of a transcript with a stem-loop structure. The primers
used in PCR were Z1, 50-AAAGCTTATTCGTGGTACTTCGAC-30 (HindIII
site underlined); Z2, 50-AGGGCCCGCTGCTGGTAAATATCC-30 (ApaI
site underlined); Z3, 50-AGGATCCATTCGTGGTACTTCGAC-30 (BamHI
site underlined) and Z4, 50-AGGGCCCTCATCAACAAACTTTTG-30
(ApaI site underlined). The sense-loop (521 bp) and anti-sense
(494 bp) fragmentswereamplifiedfromthe30-terminalgeneregion,
using primer pairs Z1/Z2 and Z3/Z4, respectively. The PCR products
have been cloned first in pCR2.1-TOPO, sequenced and subse-
quently simultaneously ligated into pHD677 between the HindIII
and BamHI restriction sites. The recombinant plasmids have been
screened by double digestion with HindIII and BamHI (insert
1029 bp) and by a single digestion with ApaI (approximately 7 kb).
Prior to transfection of T. brucei bloodstream forms, the
pHD677-G6PDH construct was linearized by NotI digestion to per-
mit its homologous recombination into a transcriptionally silent
region of the genome, the spacer of the ribosomal RNA gene repeat.
For transfection, 1 ꢂ 107 T. brucei bloodstream cells, grown in HMI-
9 medium supplemented with 10% Fetal Bovine Serum (FBS), were
harvested by centrifugation, washed twice in Cytomix solution and
resuspended in 1 ml of the same solution. 0.4 ml of Cytomix solu-
tion containing T. brucei bloodstream cells was incubated with
Lm or TbG6PDH in the mix solution was chosen in order to allow
the measurement of constant NADPH formation rates for a mini-
mum of 60 s. In wells from each plate’s line the reaction occurs
at different G6P (1250, 625, 312.5, 156.25, 78.12, 39.06, 19.53
and 9.77 l
M) and a unique NADP+ concentration. The procedure
was repeated for the different NADP+ concentrations. From the
program GraphPad Prism v.5.0 (GraphPad), a non-linear fit module
was used to calculate Kampp-g6p and Vapp-g6p for different NADP+ con-
max
centrations, while a linear regression module transformed the data
into a Lineweaver-Burk plot and calculated the x- and y-intercept
app-g6p
max
values in secondary plots. In ‘1=V
versus 1/NADP+’ secondary
plots, the y-intercept (when x = 0) and x-intercept (when y = 0) cor-
app-g6p
respond to 1/V1 and ꢁ1/KNADP, respectively; and in ‘(Km/Vmax
)
versus 1/NADP+’ secondary plots, the y-intercept (when x = 0) and
x-intercept (when y = 0) correspond to KG6P/V1 and ꢁKG6P
/
(KNADPꢃKiG6P), respectively. The parameters V1, KG6P, KNADP and KiG6P
are the kinetic constants from Cleland’s24 ordered bi-bi reaction
equation.
5.4. Tb and LmG6PDH inhibition by DHEA and EA
20-fold DHEA and EA stock solutions were prepared in 50%
DMSO. In the final reaction volume, the DMSO concentration was
reduced to 2.5%. TbG6PDH was assayed in the presence of concen-
10
electric discharge from a Genetronics BTX ECM630 electroporator
set to 1250 V, 25 , and 50 F to obtain a pulse with a voltage of
800 V and a time constant of 275 s. After the electric discharge
lg of the NotI linearized DNA construct and submitted to an
trations of EA varying between 3.125 and 0.39
tween 20 and 2.5 M of DHEA. LmG6PDH was assayed against 50
and 100 M of EA and DHEA, respectively. A Biomek3000 robot
lM and DHEA be-
l
X
l
l
l
was used to pipette assay solutions into 96 well plates and NADPH
formation rates were recorded in SpectraMax Plus384 (Molecular
Devices). The final reaction volume was 0.2 ml per well. Lm and
TbG6PDH inhibition were assayed separately for G6P and NADP+.
cells were transferred to 12 ml of HMI-9 medium (10% FBS) and
incubated at 37 °C (5% CO2). After 24 h, hygromycin was added to
the medium and the culture split in a 24 wells microtiter plate.
Antibiotic resistant cells were detectable after 7–10 days.
For G6P as varying substrate, 5
loaded into wells of four plate’s columns, each plate line received
a different G6P concentration varying from 1250 to 9.77 M. The
ll of G6P stock solutions were
5.2. Tb and LmG6PDH expression and purification
l
reaction was initiated by addition of a mix solution containing
the enzyme (2 nM), inhibitor and NADP+ (0.25 mM) in RB reaction
The cloning of the TbG6PDH gene in the pET28 expression vec-
tor has been described elsewhere.36 The TbG6PDH gene was used
as a probe for identifying the LmG6PDH gene in a genomic library
of L. mexicana strain NHOM/B2/84/BEL46.37 The LmG6PDH gene
was subcloned in the pET28a vector between the NdeI and BamHI
restriction sites. Lm and TbG6PDH were overexpressed in E. coli
BL21(DE3) transformed with the recombinant pET28 constructs
and incubated in ZYM-5052 autoinduction medium for 48 h at
25 °C. Cells were harvested by centrifugation at 4000g during
20 min and disrupted in a French Press cell at 13,000 psi. His-
tagged Lm and TbG6PDH were purified to homogeneity by metal
affinity chromatography on columns containing 2 ml HisLink resin
(Promega) following the manufacturer’s recommended procedure.
Lm and TbG6PDH were eluted in 50 mM tricine pH 8, 0.5 M NaCl
and 0.4 M imidazole.
buffer. For NADP+ as varying substrate, 5 l of NADP+ stock solu-
l
tions were loaded into wells of four plate’s columns, each plate line
received a different NADP+ solution. The final NADP+ concentration
varied from 250 to 1.95 lM. The reaction was initiated by addition
of a mix solution containing the enzyme (2 nM), inhibitor and G6P
(1.25 mM) in RB reaction buffer. Linear absorbance rates were re-
corded for 80 s. Using GraphPad Prism v.5, data were adjusted by
non-linear regression to the uncompetitive inhibition equation.
5.5. T. brucei and L. mexicana viability assay
4 ꢂ 105 cells of T. brucei (427 strain) bloodstream forms in the
initial exponential growth phase were incubated in HMI-9 medium
supplemented with 10% FBS, 1% DMSO and DHEA or EA at concen-
trations of 4, 6, 8, 12, 16, 24, 32, 48, 64 and 96 lM for 48 h at 37 °C
5.3. Measurements of steady-state kinetic parameters
and under 5% CO2. For the L. mexicana toxicity assay, 2x106 early-
metacyclic promastigotes38 were incubated in M119 medium, sup-
plemented with 10% FBS, 2% DMSO and DHEA or EA at concentra-
In order to determine the reaction mechanism of Tb and
LmG6PDHs, the rates of NADPH formation were measured at
340 nm in a SpectraMax Plus384 spectrophotometer (Molecular De-
vices), using a 96 well titer plate and a final reaction volume of
0.2 ml per well. All pipetting steps were performed automatically
tions of 8, 12, 16, 24, 32, 48, 64, 96, 128, 196
Next, 80
l of MTS (5% PMS) from the CellTiter96Ò AQueous One
Solution Cell Proliferation Assay (Promega) was added to 400
of each T. brucei and L. mexicana culture; the suspensions were dis-
tributed over a 96 well plate (100 l per well) and incubated at
lM for 48 h at 25 °C.
l
ll
in a Biomek3000 robot (Beckman Coulter). Initially, 5 ll of eight
l
different G6P stock solutions were loaded into the wells of the first
four columns. The reaction was started simultaneously in all wells
of each column by addition of a mix solution containing the en-
zyme (2.2 nM of LmG6PDH or 1.4 nM TbG6PDH) and NADP+ (200,
100, 50, 25 or 12.5 lM) in RB reaction buffer (50 mM triethanola-
mine pH 7.6, 2.5 mM MgCl2). The robot took less then 20 s to load
37 °C (5% CO2) for an additional 3 h. Soluble formazan, produced
by viable cells by reduction of MTS, was measured at 590 nm with
a SpectraMax Plus384 spectrophotometer (Molecular Devices).
Data obtained by three independent experiments were normalized
and fitted to a dose–response curve using GraphPad Prism v.5
(GraphPad).
solutions into the four columns of the plate. The concentration of

A. T. Cordeiro et al. / Bioorg. Med. Chem. 17 (2009) 2483–2489
2489
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13. Schwartz, A. G.; Pashko, L. L. Ageing Res. Rev. 2004, 3, 171.
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A.C. acknowledges a current postdoctoral fellowship from the
Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP,
contract no. 2007/02663-6) and a previous ‘Unicore’ postdoctoral
fellowship from the de Duve Institute. This research was supported
by grants from the Interuniversity Attraction Poles-Belgian Federal
Office for Scientific, Technical and Cultural Affairs (contract no. P5-
29, to P.M.), the European Commission (INCO-DEV programme,
contract no. ICA4-CT- 2001-10075, to P.M.), the Center for Struc-
tural Molecular Biotechnology (CEPID-FAPESP contract no. 98 /
14138-2, to O.T.), and the São Paulo State Bioprospecting Network
(FAPESP contract no. 05/51966-6, to O.T).
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