First small molecular inhibitors of T. brucei dolicholphosphate mannose synthase (DPMS), a validated drug target in African sleeping sickness

Article abstract of DOI:10.1016/j.bmcl.2009.01.083

Drug-like molecules with activity against Trypanosoma brucei are urgently required as potential therapeutics for the treatment of African sleeping sickness. Starting from known inhibitors of other glycosyltransferases, we have developed the first small molecular inhibitors of dolicholphosphate mannose synthase (DPMS), a mannosyltransferase critically involved in glycoconjugate biosynthesis in T. brucei. We show that these DPMS inhibitors prevent the biosynthesis of glycosylphosphatidylinositol (GPI) anchors, and possess trypanocidal activity against live trypanosomes.

Full text of DOI:10.1016/j.bmcl.2009.01.083

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1749–1752
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
First small molecular inhibitors of T. brucei dolicholphosphate mannose
synthase (DPMS), a validated drug target in African sleeping sickness
Terry K. Smith a, Benjamin L. Young b, Helen Denton a, David L. Hughes b, Gerd K. Wagner b,
*
a Centre for Biomolecular Sciences, The North Haugh, The University, St. Andrews, Scotland, UK
b Centre for Carbohydrate Chemistry, School of Chemical Sciences and Pharmacy, University of East Anglia, Earlham Road, Norwich NR4 7TJ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Drug-like molecules with activity against Trypanosoma brucei are urgently required as potential thera-
peutics for the treatment of African sleeping sickness. Starting from known inhibitors of other glycosyl-
transferases, we have developed the first small molecular inhibitors of dolicholphosphate mannose
synthase (DPMS), a mannosyltransferase critically involved in glycoconjugate biosynthesis in T. brucei.
We show that these DPMS inhibitors prevent the biosynthesis of glycosylphosphatidylinositol (GPI)
anchors, and possess trypanocidal activity against live trypanosomes.
Received 11 December 2008
Revised 22 January 2009
Accepted 23 January 2009
Available online 30 January 2009
Keywords:
African sleeping sickness
Trypanosoma
Ó 2009 Elsevier Ltd. All rights reserved.
Dolicholphosphate mannose synthase
Enzyme inhibitors
African sleeping sickness, also known as Human African Try-
panosomiasis (HAT), is an infectious disease caused by the proto-
zoan parasite Trypanosoma brucei (T. brucei). The parasite is
transmitted by the bite of an infected tsetse fly, multiplies within
the bloodstream of the mammalian host, and eventually invades
the central nervous system. If untreated, HAT is invariably lethal.
Despite becoming close to eradication by the late 1960s,1 the dis-
ease has made a dramatic re-emergence within the last half cen-
tury, with prevalence in parts of Africa now as high as they were
in the 1920s. In 2002 alone, around 48,000 deaths were reported.2
The WHO presently records around 17,500 new cases per year,3
with a cumulative rate of 50,000–70,000 cases3 and potentially
over 60 million people at risk.2
as a physical diffusion barrier for components of the innate im-
mune system.10 The VSG coat undergoes constant antigenic varia-
tion,11,12 as the parasite switches between ꢀ1000 immunologically
The situation is further exacerbated by the growing resistance
to established drug treatments for HAT, which have long been con-
sidered unsatisfactory.2 Serious side effects kill between 4% and
10% of patients who receive the arsenical melarsoprol,4 introduced
in 1949, and the drug fails to cure between 10% and 30% of pa-
tients.5 The only relatively modern anti-HAT drug, the ornithine
decarboxylase inhibitor eflornithine, is expensive, difficult to
administer, and only effective against T. brucei gambiense.6 Thus,
HAT has been described as one of the most neglected diseases of
mankind,7 and novel therapeutic approaches to HAT are urgently
needed.2
The T. brucei parasite is covered in a dense cell-surface coat of
ꢀ5 million variant surface glycoprotein (VSG) dimers8,9 which acts
Figure 1. The central role of DPMS for the biosynthesis of GPI anchors in T. brucei.
VSG: variant surface glycoprotein; GPI: glycosylphosphatidyl inositol; GDP-Man:
guanosine diphosphate mannose; Dol-P-Man: dolicholphosphate mannose; DPMS:
dolicholphosphate mannose synthase.
* Corresponding author. Tel.: +44 (0)1603 593889; fax: +44 (0)1603 592003.
E-mail address: g.wagner@uea.ac.uk (G.K. Wagner).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.083

1750
T. K. Smith et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1749–1752
distinct VSG genes,13 thus staying one step ahead of the host im-
mune system. All VSG variants are linked to the trypanosomal plas-
ma membrane via glycosylphosphatidylinositol (GPI) anchors
(Fig. 1). Genetic14–16 and chemical17,18 studies show that GPI an-
chor biosynthesis is essential for viability of the bloodstream form
of T. brucei, thus validating it as a drug target against HAT.19 Small
molecular inhibitors of enzymes involved in GPI anchor biosynthe-
sis therefore hold great promise as novel anti-trypanosomal
agents.
GPI anchor biosynthesis in T. brucei involves three mann-
osyltransferases (MTs) on the luminal face of the endoplasmic
reticulum, which catalyze the assembly of the trimannoside core
structure (Fig. 1).20,21 All three of these MTs use dolicholphosphate
mannose (Dol-P-Man) as their donor substrate, whose biosynthesis
from dolicholphosphate (Dol-P) and the sugar-nucleotide GDP-
mannose (GDP-Man) is catalyzed by another mannosyltransferase,
dolicholphosphate mannose synthase (DPMS). All T. brucei VSG
variants also contain at least one N-glycan, which requires another
four Dol-P-Man-dependant mannosyltransferases for the forma-
tion of its lipid-linked oligosaccharide precursor.22–24 Conse-
quently, T. brucei is doubly dependant upon Dol-P-Man for the
synthesis of mature, N-glycosylated and GPI-anchored VSGs, and
this double dependency makes DPMS an excellent target for inhi-
bition of VSG biosynthesis. Recently, T. brucei DPMS has also been
validated genetically as a drug target.25
The target rhodanine-3-acetic acid derivatives 2a–j (Scheme 1,
Table 1) were prepared by Knoevenagel condensation of rhoda-
nine-3-acetic acid 1 and substituted benzaldehydes 3a–j. To sim-
plify the preparation and isolation of the target compounds, we
explored different solvents and catalytic bases for this reaction,
including DMF/sodium acetate, toluene/piperidine and ethanol/
piperidine.28,32 In our hands, the ethanol/piperidine system was
the most practical one, with short reaction times and straightfor-
ward product isolation. Under these conditions, all 5-benzylidene
rhodanine-3-acetic acid derivatives precipitated from the ethanolic
solution upon cooling to room temperature, and could be collected
by simple filtration.33 Thus, all target compounds (Table 1) were
obtained as yellow or yellow-orange solids in generally good
yields. Remarkably, this procedure was also applicable to benzal-
dehydes containing a free phenolic hydroxyl group (e.g., 2f, 2g).
This was particularly important as all attempts to prepare these
analogs by debenzylation of the corresponding benzyloxy deriva-
tives (e.g., 2b, 2c) had failed.
Fortuitously, our synthetic protocol provided benzylidene thia-
zolidinone 2b in crystalline form which allowed the unambiguous
determination of its three-dimensional structure (Fig. 2).34 This is
of particular interest, as in previous reports of structurally related
thiazolidinones as glycosyltransferase inhibitors there had been
ambiguity about the configuration of the exocyclic double bond,
despite its obvious importance for structure–activity relation-
Despite its promise as a therapeutic target, no inhibitors for T.
brucei DPMS have been reported to date. The rational design of
such inhibitors is complicated by the absence of a crystal structure
for T. brucei DPMS at present. In search of a suitable lead structure
for the development of DPMS inhibitors, we noticed striking struc-
tural similarities among small molecular inhibitors for other glyco-
Table 1
Biological activity of thiazolidinones 2a–j (Scheme 1)
Compound R1
R2
R3
Residual DPMS
activity (%)a
Trypanocidal activity
b
(ED50
)
syltransferases26–28
and
sugar-nucleotide-dependent
glycoprocessing enzymes.29–31 Several such inhibitors contain a
rhodanine (2-thioxothiazolidin-4-one) scaffold, and derivatives of
rhodanine-3-acetic acid 1 (Scheme 1) have been reported as inhib-
itors of the E. coli glycosyltransferase MurG26,27 and the C. albicans
protein mannosyltransferase 1 (PMT1).28 It has been suggested
that the thiazolidinone ring can act as a mimic of the pyrophos-
phate group,26,29–31 and that this mimicry may explain the inhibi-
tory activity of thiazolidinone derivatives towards sugar-
nucleotide-dependent enzymes. As DPMS is dependent on the su-
gar-nucleotide donor GDP-mannose, we reasoned that the thiazo-
lidinone scaffold may also represent a good starting point for the
development of DPMS inhibitors.
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
OH
H
H
H
H
H
H
H
BnO
H
42 ( 5)
232 ( 12)
338 ( 31)
96 ( 5)
492 ( 24)
107 ( 12)
427 ( 19)
345 ( 23)
244 ( 17)
398 ( 19)
>1000
BnO 10 ( 2)
23 ( 3)
H
BnO BnO 20 ( 4)
H
H
OH
H
H
OH
H
CN
Cl
23 ( 8)
70 ( 4)
90 ( 5)
73 ( 6)
86 ( 5)
H
H
2j
CCH 94 ( 7)
a
Residual DPMS activity in the presence of 1 mM inhibitor, relative to control
experiments without inhibitor (activity = 100 3%).
b
All ED50 values are in
lM against cultured bloodstream T. brucei. All experi-
ments were carried out at least in duplicate.
Herein, we describe the successful application of this strategy.
We have prepared a small library of 5-benzylidene rhodanine-3-
acetic acid analogs of the general structure 2, and report herein
their inhibitory activity against T. brucei DPMS and GPI anchor bio-
synthesis as well as their trypanocidal activity against live
trypanosomes.
S
S
N
HO2C
O
O
1
H
3a-j
27-78% yield
S
R1
R3
R2
S
HO2C
N
R3
R2
O
2a-j
R1
Scheme 1. Synthesis of the target 5-benzylidene rhodanine-3-acetic acid deriva-
tives 2a–j. Reagents and conditions: NH4OAc, DMF, 80 °C, 3 h (2a) or EtOH,
piperidine, 80 °C, 3–6 h (2b–j). For substituents R1–R3 see Table 1.
Figure 2. The crystal structure of the representative 5-benzylidene thiazolidinone
2b, in complex with one molecule of ethanol, shows the exocyclic double bond
(C2@C20) in the Z configuration.

T. K. Smith et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1749–1752
1751
Figure 3. Differential effects of 5-benzylidene thiazolidinones 2a–j on DPMS and GPI anchor biosynthesis (M1: Man
a
1-4GlcNH2-PI; M2: Man
a
1-6Man
a
1-4GlcNH2-PI; M3:
Man 1-2Man 1-6Man 1-4GlcNH2-PI; aM3: Man 1-2Man 1-6Man 1-2Man
a
a
a
a
a
a
1-4GlcNH2-(acyl)PI; A0: ethanolamine-P-Man
a
a
1-6Man 1-4GlcNH2-PI).
a
ships.26,27 In our structure, the rhodanine acetic acid molecule 2b is
essentially planar, with only the carboxy group out of plane and
coordinating to one molecule of ethanol. The exocyclic double
bond exists in the Z configuration, which for arylidene rhodanines
has been reported as the thermodynamically stable
configuration.35,36
In an initial biological screen, all target compounds were tested
for inhibition of recombinant T. brucei DPMS in E. coli membranes
(Table 1).37 At 1 mM, several thiazolidinone derivatives signifi-
cantly inhibited T. brucei DPMS. A large benzyloxy substituent in
position R2 and/or R3 appears to be advantageous for DPMS inhibi-
tion (2b–d), while a small polar substituent is less well tolerated in
these positions (2f and 2g), as are rigid R3 substituents (e.g., nitrile
2h, acetylene 2j). Intriguingly, however, a polar substituent is ben-
eficial for inhibitory activity when placed at the 2-position (R1),
and the 2-hydroxy regioisomer 2e is among the most potent DPMS
inhibitors in this series.
Next, compounds 2a–j were tested for their trypanocidal activ-
ity against cultured bloodstream form T. brucei. 38 Importantly, the
in vitro activity of several thiazolidinones against DPMS did trans-
late into in vivo trypanocidal activity against live trypanosomes. In
particular, the 3-benzyloxy-substituted analog 2c and the 2-hydro-
xy regioisomer 2e showed trypanocidal activity with ED50 values in
the medium micromolar range. However, only moderate activity
was observed for some of the other potent DPMS inhibitors, nota-
bly the benzyloxy-substituted analogs 2b and 2d. The limited cel-
lular activity of these DPMS inhibitors may be due to various
factors, including limited cell penetration, which are currently
being investigated.
the formation of mannosylated GPI intermediates. Thus, the strong
inhibitory effect of 2b and 2e on GPI anchor biosynthesis in the
cell-free system may be due to inhibition of one or more GPI bio-
synthetic enzymes early in the pathway, for example, the GlcNAc
transferase, de-N-acetylase or the first mannosyltransferase
(MT1). Importantly, all of these enzymes represent therapeutic tar-
gets in their own right, and their inhibition may contribute to the
trypanocidal activity of 2b and 2e.
In summary, we have identified the first small molecular
inhibitors of trypanosomal DPMS. We demonstrate that DPMS
inhibitors compromise trypanosomal GPI anchor biosynthesis in
a cell-free system, and that inhibition of DPMS is a promising
strategy for the development of anti-trypanosomal agents. Work
on the optimization of these initial lead compounds with regard
to their DPMS inhibitory potency as well as cellular activity is
ongoing. Thiazolidinones 2b, 2d and 2e in particular are promis-
ing candidates for further development because of their respec-
tive activities against trypanosomal DPMS and GPI anchor
biosynthesis.
Acknowledgments
We thank the EPSRC National Mass Spectrometry Service,
Swansea, and Mr. Thomas Pesnot (UEA) for the recording of spec-
tra, and the 2008 MPharm mini-project students in the Wagner
group for technical assistance. T.K.S. is supported by a Wellcome
Trust Senior Research Fellowship (067441).
References and notes
In order to assess the parasite/host selectivity of these thiazolid-
inones, analogs 2a–f and 2j were tested at two concentrations (0.1
and 1.0 mM final) for their cytotoxicity against HeLa cells.38 Pleas-
ingly, none of these thiazolidinones showed any cytotoxic effect,
with the exception of 2b and 2j (20% inhibition at 1.0 mM, com-
pared to DMSO-only control), suggesting low mammalian cytotox-
icity (ED50 > 1.0 mM) and a promising margin of selectivity for T.
brucei.
In order to gain further insight into the mode of action of these
novel DPMS inhibitors, we investigated the effect of the thiazolid-
inones on GPI anchor biosynthesis in a T. brucei cell-free-system
(Fig. 3).39 This assay monitors the DPMS-catalyzed formation of
Dol-P-Man (lane 1) as well as the downstream formation of man-
nosylated GPI intermediates (lane 2). As expected, the potent
DPMS inhibitor 2d abolished the formation of Dol-P-Man almost
completely, and significantly reduced the formation of down-
stream GPI intermediates. A similar effect was observed for the
analog 2f (Fig. 3).
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37. Full experimental details will be published elsewhere. Briefly, E. coli
membranes containing recombinantly expressed, full-length (membrane-
bound) T. brucei DPMS (70 ng/ml total protein) were added to a reaction mix
(60 ml) of Na–Hepes (50 mM, pH 7.4), KCl (25 mM), MgCl2 (5 mM), MnCl2
(5 mM), dehydrofarnesol-phosphate (1 mg), GDP-[3H]Man (0.25 mCi/1 mM),
and inhibitor (1 mM), all in the presence of 0.1% n-octyl-glucopyranoside.
The reaction was incubated at 30 °C for 10 min, quenched, and lipids
extracted by the addition of CHCl3:MeOH (1:1). The resulting supernatant
was dried and partitioned between butan-1-ol and water, a portion butan-1-
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stored at ꢁ80 °C until needed. Per assay, 1 ꢂ 107 cell equivalents were used.
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pH 7.4, 25 mM KCl, 5 mM MgCl2, 100 mM tosyl-
(TLCK), and 1
g/ml leupeptin), resuspended by sonication in 20 ml of 2ꢂ
incorporation buffer (2 times concentrated wash buffer containing 10 mM
MnCl2, 1 mg/ml tunicamycin and 1 mM dithiothreitol) and added to
L-lysine chloromethyl ketone
l
a
33. Representative procedure for 2b (all thiazolidinones 2a–j were characterized
reaction tube containing an equal volume of GDP-[3H]Man (0.3 mCi per 107
cell equivalents), 1 mM UDP-GlcNAc and thiazolidinone inhibitor candidate
(in DMSO, final concentration 1 mM). The cell-free system was then
sonicated briefly, incubated at 30 °C for 1 h, and the reaction stopped by
the addition of 267 ml of CHCl3:MeOH (1:1). Glycolipid products were
recovered by extraction into CHCl3:MeOH:H2O (10:10:3), evaporated to
dryness, partitioned between butan-1-ol and water, and analyzed by HPTLC
(CHCl3:MeOH:NH4OAc (1 M):conc. NH3:H2O 180:140:9:9:23). Dried HPTLC
plates were sprayed with En3HanceTM, and radiolabeled components were
visualized by fluorography at ꢁ80 °C using Kodak Biomax MS films with an
intensifying screen.
by NMR and HRMS):
A mixture of rhodanine-3-acetic acid 1 (0.51 g,
2.61 mmol), 4-benzyloxybenzaldehyde 3b (0.60 g, 2.83 mmol) and a drop of
piperidine in absolute ethanol (25 ml) was heated to reflux for 2 h. Upon
cooling of the reaction to room temperature, a precipitate formed which was
collected by filtration, washed with cold ethanol, and dried under vacuum to
provide 0.78 g (76%) of 2b as yellow crystals. Melting point: 202–205°. dH
(400 MHz, d6-DMSO) 4.72 ppm (s, 3H), 5.21 (s, 3H), 7.20–7.22 (m, 2H), 7.34–
7.50 (m, 5H), 7.64–7.66 (m, 2H), 7.86 (s, 1H). dC (75.5 MHz, d6-DMSO) 45.0,
69.7, 116.1, 118.8, 125.7, 128.0, 128.3, 128.7, 133.3, 134.3, 136.6, 161.1, 166.7,
167.6, 193.4. m/z (ESI) 384.0370 [MꢁH]ꢁ, C10H14O4NS2 requires 384.0365.