Fragment screening reveals salicylic hydroxamic acid as an inhibitor of Trypanosoma brucei GPI GlcNAc-PI de-N-acetylase

Article abstract of DOI:10.1016/j.carres.2013.12.016

The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African sleeping sickness. We screened a focused library

Full text of DOI:10.1016/j.carres.2013.12.016

Carbohydrate Research 387 (2014) 54–58
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Fragment screening reveals salicylic hydroxamic acid as an inhibitor
of Trypanosoma brucei GPI GlcNAc-PI de-N-acetylase
Michael D. Urbaniak ,à, Amy S. Capes à, Arthur Crossman, Sandra O’Neill, Stephen Thompson,
⇑
⇑
Ian H. Gilbert , Michael A. J. Ferguson
Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The zinc-metalloenzyme GlcNAc-PI de-N-acetylase is essential for the biosynthesis of mature GPI anchors
and has been genetically validated in the bloodstream form of Trypanosoma brucei, which causes African
sleeping sickness. We screened a focused library of zinc-binding fragments and identified salicylic
hydroxamic acid as a GlcNAc-PI de-N-acetylase inhibitor with high ligand efficiency. This is the first small
molecule inhibitor reported for the trypanosome GPI pathway. Investigating the structure activity rela-
tionship revealed that hydroxamic acid and 2-OH are essential for potency, and that substitution is tol-
erated at the 4- and 5-positions.
Received 5 November 2013
Received in revised form 12 December 2013
Accepted 18 December 2013
Available online 30 December 2013
Keywords:
GPI
Trypanosoma brucei
Hydroxamic acid
Inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
N-Deacetylase
1. Introduction
step in biosynthesis of GPI (Fig. 1),6 and has been genetically vali-
dated as essential in bloodstream form T. brucei.3 The use of syn-
Trypanosoma brucei is a protozoan parasite that is transmitted
by the bite of an infected tsetse fly and causes the fatal African
sleeping sickness in humans and the related wasting disease Naga-
na in cattle. Current treatments are expensive, toxic, and difficult
to administer, leaving an urgent need for improved therapeutic
agents. The parasite is able to evade the host immune response
by virtue of a dense surface coat composed of 5 ꢀ 106 variant
surface glycoprotein (VSG) dimers that prevent lysis by the innate
immune response. The coat also undergoes antigenic variation to
evade the adaptive immune response.1 The VSG dimer is attached
to the plasma membrane by a glycosylphosphatidylinositol (GPI)
anchor, and GPI anchor biosynthesis has been genetically and
chemically validated as essential for the survival of the clinically
relevant bloodstream form of the parasite.2–5
thetic substrate analogues has revealed that the human enzyme
is more fastidious than the trypanosome enzyme, enabling the de-
sign of species-specific substrate-based inhibitors.7–9 The substrate
analogue approach has shown that the phosphate, 20-NHAc and
30-OH of the substrate GlcNAc-PI are critical for recognition by the
T. brucei GlcNAc-PI de-N-acetylase.10 In contrast, the diacylglycerol
is not directly recognised and may be efficiently replaced with an
octadecyl chain.9,11 Recently, we have shown that substrate-based
inhibitors containing the zinc binding moieties hydroxamic acid or
carboxylic acid can act as inhibitors of the T. brucei de-N-acety-
lase.12,13 These substrate-based inhibitors do not possess drug-like
physiochemical properties and contain too much stereochemical
complexity for tractable synthesis. Here, we report our efforts to
identify alternative scaffolds for GlcNAc-PI de-N-acetylase inhibi-
tors that resulted in the identification of salicylic hydroxamic acid
as an inhibitor with high ligand efficiency.
The GPI biosynthetic pathway has been extensively studied in
both T. brucei and mammalian systems, highlighting differences
in both the order of assembly and substrate specificity that can
be exploited to produce species-specific inhibitors. The GlcNAc-PI
de-N-acetylase is a zinc metalloenzyme that catalyses the second
2. Materials and methods
2.1. Materials
⇑
Corresponding authors. Tel.: +44 (0) 1382 384219.
E-mail addresses: i.h.gilbert@dundee.ac.uk (I.H. Gilbert), m.a.j.ferguson@
dundee.ac.uk (M.A.J. Ferguson).
The synthesis of D-glucosamine-a-(1-6)-D-myo-inositol-1-octa-
decyl phosphate (GlcN-IPC18) has been described previously.14 The
corresponding N-acetyl derivate (GlcNAc-IPC18) was prepared by
treatment with acetic anhydride, and the concentration of stock
These authors contributed equally to the work.
Present address: Division of Biomedical and Life Sciences, Faculty of Health and
à
Medicine, Lancaster University, Lancaster LA1 4GQ, UK.
http://dx.doi.org/10.1016/j.carres.2013.12.016
0008-6215/Ó 2013 Elsevier Ltd. All rights reserved.

M. D. Urbaniak et al. / Carbohydrate Research 387 (2014) 54–58
55
2.3. Trypanosome cell-free system assay
Trypanosome cell-free system assays, where the formation of
GPI precursors is monitored by following the incorporation of
[3H]-mannose were analysed using high-performance liquid chro-
matography and fluorography as described previously.13
2.4. Compound synthesis
2.4.1. Synthesis of 5-((tert-butoxycarbonyl)amino)-2-
hydroxybenzoic acid (9)
5-Aminosalicylic acid 8 (6.89 g, 45.0 mmol) was suspended in
water (60 mL), and TEA (12.4 mL, 90.0 mmol) was added. A solu-
tion of Boc2O (10.8 g, 90.0 mmol) in dioxane (120 mL) was added,
and the reaction was stirred at room temperature overnight. The
solvent was removed, and the solid residue suspended in water
(20 mL). HCl (3 M) was added until the greyish pink precipitate
stopped forming. The precipitate was filtered off and washed with
water. The precipitate was dissolved in boiling acetone and hot
filtered before being recrystallized twice from acetone to afford
the product (8.61 g, 76%) as a white powder, mp 278 °C. 1H NMR
(500 MHz, MeOD) d 7.93 (1H, s), 7.40 (1H, d, J = 7.6 Hz), 6.85 (1H,
d, J = 8.9 Hz), 1.51 (9H, s); 13C NMR (125 MHz, MeOD) d 173.3,
159.0, 155.7, 132.1, 128.7, 121.8, 118.2, 113.5, 80.8, 28.7; HRMS,
calcd mass for C12H16NO5 [M+H+]: 254.1023. Found: 254.1027
+
Figure 1. GlcNAc-PI de-N-acetylase catalyses the second step in the GPI biosyn-
(ꢁ1.4 ppm).
thetic pathway. The zinc-dependent metalloenzyme is inhibited by zinc chelators.
2.4.2. Synthesis of tert-butyl (3-((benzyloxy)carbamoyl)-4-
hydroxyphenyl)carbamate (10)
solutions determined by measurement of the inositol content by
selected ion-monitoring GC–MS.13 Bloodstream form Trypanosome
brucei (variant MITat1.4) was isolated and membranes (cell-free
system) prepared as described previously and stored at ꢁ80 °C.15
Recombinant GST-tagged T. brucei de-N-acetylase (GST-TbGPI12)
was expressed and purified as described previously and stored at
ꢁ80 °C.13
O-Benzylhydroxylamine hydrochloride (3.54 g, 22.2 mmol) was
suspended in CHCl3 (85 mL) and cooled to 0 °C, and then TEA
(3.21 mL, 23.1 mmol) was added. A solution of compound 9
(5.62 g, 22.2 mmol) in THF (140 mL), DMAP (169 mg, 1.12 mmol)
and DCC (5.06 g, 24.4 mmol) was added, and the reaction was
stirred for 16 h. The solvent was removed and the solids were
resuspended in EtOAc, filtered, and then washed with HCl (1 M,
50 mL), water (50 mL), ammonia (1 M, 2 ꢀ 50 mL) and filtered
through cotton wool. The solvent was removed and the crude
was dried on to silica then purified by column chromatography
(EtOAc/hexane, 0:100 to 1:1) to afford the product (3.63 g, 46%)
as a pale yellow powder, mp 102–103 °C. 1H NMR (500 MHz,
CDCl3) d 11.33 (1H, br s), 9.42 (1H, br s), 7.63 (1H, br s), 7.44–
7.41 (2H, m), 7.39–7.34 (3H, m), 7.13 (1H, dd, J = 8.9, 2.4 Hz),
6.90 (1H, d, J = 8.9 Hz), 6.45 (1H, br s), 4.97 (2H, s), 1.49 (9H, s);
13C NMR (125 MHz, CDCl3) d 157.1, 153.3, 134.9, 129.6, 129.3,
128.9, 128.7, 128.7, 126.1, 118.8, 116.0, 112.1, 80.8, 78.6, 28.3;
2.2. Mass spectrometry based activity assays
Inhibition assays were performed in 100 lL final volume, with
1% v/v DMSO with or without inhibitor. Recombinant GST-TbGPI12
(10
l
g per assay) or trypanosome cell-free system (2.5 ꢀ 106 cell
equivalents per assay) in incorporation buffer (25 mM Tris pH
8.0, 50 mM KCl, 5 mM MnCl2) was added to wells containing
500 pmol GlcNAc-IPC18 with or without the inhibitor and incu-
bated at 37 °C for 1 h. The reactions were quenched by the addition
LRMS (ES+), m/z 359.2 [M+H+]; HRMS, calcd mass for
of 200
were bound to C18 resin (50 mg Isolute cartridge), washed three
times with 200 L 5% propan-1-ol, 5 mM NH4OAc and eluted with
100 L 40% propan-1-ol, 5 mM NH4OAc. Enriched glycolipids were
analysed by liquid chromatography coupled to an electrospray
tandem mass spectrometer (LC–MS/MS). Samples (40 L) were
injected on to a 10 ꢀ 1 mm C18 column (ACE, 5 M) and eluted
lL of 5% propan-1-ol, 5 mM NH4OAc, and the glycolipids
+
C
19H23N2O5 [M+H+]: 359.1601. Found: 359.1591 (2.8 ppm).
l
l
2.4.3. Synthesis of 5-amino-N-(benzyloxy)-2-hydroxybenzamide
TFA salt (11)
l
Compound 10 (1.99 g, 5.56 mmol) was dissolved in wet TFA
(5.3 mL), and the mixture was stirred for 1 h. The TFA was removed
in vacuo; the residue was suspended in Et2O and filtered to afford
the product (1.76 g, 85%) as a tan powder, mp 169–170 °C. 1H NMR
(500 MHz, DMSO-d6) d 11.48 (1H, br s), 9.96 (2H, br s), 7.64 (1H, d,
J = 2.6 Hz), 7.47 (2H, d, J = 7 Hz), 7.42–7.36 (3H, m), 7.31 (1H, dd,
J = 8.7, 2.8 Hz), 7.03 (1H, d, J = 8.8 Hz), 4.96 (2H, s); 13C NMR
(125 MHz, DMSO-d6) d 163.4, 155.6, 135.7, 128.8, 128.29, 128.27,
126.9, 124.5, 122.7, 117.87, 115.5, 113.2, 77.0; LRMS (ES+), m/z
l
using a binary gradient of 5–80% propan-1-ol in 5 mM NH4OAc
(Dionex Ultimate 3000). The gradient consisted of 2 min 0% B,
2–4 min 0–100% B, 4–8 min 100%, 8–9 min 100–0% B, 9–10 min
0% B where buffer A consisted of 5% propan-1-ol, 5 mM NH4OAc
and buffer B 80% propan-1-ol, 5 mM NH4OAc. The glycolipids were
analysed on an electrospray triple quadrapole mass spectrometer
(Micromass Quattro Ultima) in multiple reaction monitoring mode.
The turnover of the substrate GlcNAc-IPC18 (m/z 715 > 223) to
GlcN-IPC18 (m/z 672 > 223) was used to calculate the percentage
of substrate conversion to product in a given sample.6 Inhibitor
IC50 values were calculated using a four-parameter fit of eight-
point potency curves derived from three independent experiments,
and are quoted with standard deviation.
259.1 [M+H+]; HRMS, calcd mass for C14H15N2O3 [M+H+]:
+
259.1077. Found: 259.1069 (3.3 ppm).
2.4.4. General method for the synthesis of the amide series
Compound 11 (200 mg, 0.592 mmol), DMAP (cat.) and the acyl
chloride (0.592 mmol) were dissolved in THF (2 mL) and DCE

56
M. D. Urbaniak et al. / Carbohydrate Research 387 (2014) 54–58
Figure 2. T. brucei GlcNAc-PI de-N-acetylase inhibitors. The activity of the recombinant enzyme against the synthetic substrate
D-glucosamine-a-(1-6)-D-myo-inositol-1-
octadecyl phosphate was measured using a mass spectrometry based assay (A) Fragments were screened at 1 mM in duplicate. (B) The six most potent fragments showed
dose–response. (C) For the most potent compound 1 an eight-point potency curve was determined in triplicate giving an IC50 = 63
4 lM. (D) Structures of the six most
potent fragments.
(0.5 mL). Pyridine (96
l
L, 1.184 mmol) was added, and the reaction
ꢁ1.5 kcal molꢁ1 per non-hydrogen atom.17 By way of comparison,
the previously reported glucocyclitol-phospholipid substrate ana-
logue 1R,2R-1-O-[2-C-(carboxymethyl N-hydroxyamide)-2-deoxy-b-
stirred for 24 h. The mixture was diluted with DCM (10 mL) and
water (10 mL) then stirred vigorously. The mixture was passed
through a phase separator, and then the solvent was removed.
The crude was recrystallized from MeOH/Et2O. Characterisation
of compounds 12–19 is reported in the Supplementary data.
D
-glucopyranosyl]-cyclohexanediol 2-(n-octadecylphosphate) has
an IC50 of 19 0.5 l
M and a molecular weight of 666.4,13 giving
a substantially lower ligand efficiency of only ꢁ0.13 kcal molꢁ1
per non-hydrogen atom.
2.4.5. General method for amide deprotection
The result of the fragment screen compared favourably to our
screening of 16,037 lead-like compounds from the Dundee Drug
Discovery Unit’s compound collection18 against the T. brucei de-
N-acetylase in a cell-based functional complementation assay,i
where no inhibitors were identified (data not shown). Interestingly,
compound 1 (also known as SHAM) has previously been identified as
an inhibitor of trypanosome alternative oxidase (TAO), and is toxic
in vivo to the clinically relevant bloodstream form T. brucei.19,20
A 0.05 M solution of benzyl-protected amide (12–19) and AcOH
(2 equiv) in MeOH/THF, MeOH or THF, was passed through a H2
flow reactor (1 mL/min, 30 °C, 1 atm) using a 20% Pd(OH)2 catalyst
cartridge. The solvent was removed and the product was recrystal-
lized from THF/MeOH, or washed with THF or MeOH. Characterisa-
tion of compounds 20–27 is reported in the Supplementary data.
3. Results and discussion
3.1. Fragment screening
3.2. Inhibition of the T. brucei cell-free system
The ability of 1 to act as an inhibitor of the T. brucei GPI pathway
was confirmed using the trypanosome cell-free system (Fig. 3).
Priming the cell-free system with GlcAc-IPC18 in the presence of
GDP-[3H]-mannose stimulates the production of radiolabelled
mannosylated GPI intermediates that can be separated by high
performance thin-layer chromatography and visualised by fluorog-
To discover more drug-like scaffolds for zinc-binding de-N-acet-
ylase inhibitors we screened a small focused library of zinc binding
fragments. Because we have previously shown that the enzyme
may be inhibited by substrate analogues containing hydroxamic
acid, N-hydroxyurea or carboxylic acid groups,12,13 we selected
26 commercially available fragments, which contained these zinc
binding moieties, with an average molecular weight of 220 (Sup-
plemental Table S1). We screened the inhibition of recombinant
T. brucei de-N-acetylase by the fragments using a mass spectrome-
try-based activity assay to directly monitor the conversion of the
raphy. Priming the cell-free system with GlcNAc-IPC18 (10
lM) was
prevented by incubation with >300 M of 1 (Fig. 3B), a five-fold in-
l
crease over the IC50 value against the recombinant enzyme. To ver-
ify that the decrease in potency was not due to a reduction in
potency against the endogenous enzyme compared with the re-
combinant enzyme, we repeated the mass spectrometry based
activity assay with the T. brucei cell-free system. We found that it
was necessary to reduce the amount of cell-free system by 40-fold
in the mass spectrometry-based assay compared with the
radiometric assay to achieve measurements in the linear range
for the turnover of GlcNAc-IPC18. The potency of compound 1
synthetic substrate N-acetyl-
tol-1-octadecyl phosphate (GlcNAc-IPC18) to
6)- -myo-inositol-1-octadecyl phosphate (GlcNAc-IPC18). The
D
-glucosamine-
a
-(1-6)-
D
-myo-inosi-
D-glucosamine-a-(1-
D
replacement of the diacylglycerol moiety of GlcNAc-PI with a sim-
ple C18 alkyl chain in GlcNAc-IPC18 has no effect on the substrate
recognition, and enables the use of a unique mass-transition to fol-
low the reaction.6 Initial screening of the fragments at 1 mM in
duplicate revealed six compounds with >50% inhibition
(Fig. 2A + D). However, only one compound showed >50% inhibi-
against the cell-free system with an IC50 = 66
8 lM (Fig. 3B)
was comparable to that against the recombinant enzyme.
tion at 100
had an IC50 of 63
l
M (Fig. 2B). The most potent compound 1 (Fig. 2C)
M and a molecular weight of 153, giving
4
l
i
16
The assay used the CHO MS2S cell line, which has a defective de-N-acetylase and
an impressive ligand efficiency (-RT.lnIC50/Nnon-H
)
of
atoms
expresses the GPI anchored human CD55, functionally complemented by transfection
with T. brucei de-N-acetylase. Cells were treated with trypsin, plated onto compound,
and GPI biosynthesis monitored by aCD55 ELISA at 48 h.
ꢁ0.57 kcal molꢁ1 per non-hydrogen atom, which is over one third
of the theoretical maximum affinity for organic compounds of

M. D. Urbaniak et al. / Carbohydrate Research 387 (2014) 54–58
57
Figure 3. Trypanosome GPI biosynthesis in the cell-free system. (A) The T. brucei cell-free system was incubated with GlcNAc-PI (10
l
M) in the presence of 0, 100, 300, or
1000 lM of 1 and 0.5 l
Ci of GDP-[3H]-mannose to stimulate the production of radiolabelled mannosylated GPI intermediates. Glycolipid products were extracted, separated
by high-performance thin-layer chromatography, and visualised by fluorography. DPM–dolichol-phosphate-mannose, M1–Man1GlcN-IPC18, M2–Man2GlcN-IPC18, M3–
Man3GlcN-IPC18, A0–EtNPMan3GlcN-IPC18. (B) Inhibition of the turnover of GlcNAc-IPC18 (10
lM) by the presence of 1 measured in the T. brucei cell-free system in the LC–MS/
MS assay.
Table 1
2-hydroxyl group or substitution with bromide or amine com-
pletely abrogated the inhibitory activity, whereas substitution at
the 40 position with bromine was tolerated (Table 1).
Inhibitory activity of commercially available analogues of 1
To further explore the available chemical space, a small array of
analogues of compound 1 were synthesised from the literature
compound 1121 by coupling to the 5-NH2 position with commer-
cially available acid chlorides (Scheme 1). After deprotection of
the hydroxamic acid, the majority of compounds showed
decreased inhibitory activity compared with 1 (Table 2). The two
ID
R1
R2
R4
Inhibition at 1 mMa (%)
1
2
3
4
5
6
7
NHOH
OH
OH
OH
H
H
H
H
H
H
Br
Br
97
25
2
1
most potent compounds 20 and 21 had IC50 values of 45 11
lM
NHOH
NHOH
NHOH
NHOH
NHOH
ꢁ6 14
and 69 M respectively (Supplementary Fig. 1), comparable to
6
l
Br
ꢁ4 15
the parent compound albeit with reduced ligand efficiency
(ꢁ0.30 and ꢁ0.29 kcal molꢁ1 per non-hydrogen atom respectively),
suggesting that the 50-amide substitution was tolerated but
contributed little to binding.
NH2
OH
H
ꢁ5
2
2
86
9.6 15
a
Inhibition of T. brucei de-N-acetylase (cell-free system).
The structure–activity relationship derived from the data pre-
sented here suggest that the 2-OH group of 1 is essential for activ-
ity and substitution at the 4- and 5- position is tolerated (Fig. 4).
Comparing 1 with the natural substrate GlcNAc-PI could suggest
that 1 is mimicking the GlcNAc moiety, with the 2-OH of 1 mimick-
ing the 30-OH that is essential for substrate recognition. In the
proposed reaction mechanism for the de-N-acetylase, the catalytic
base D43 assists the nucleophilic attack of a zinc-bound water mol-
ecule on the activated acetamidocarbonyl, and the subsequent
protonation of the tetrahedral intermediate promotes loss of acetic
3.3. Structure–activity relationship
Substructure searching identified commercially available
analogues of 1 that were screened for activity against in the mass
spectrometry based assay against T. brucei cell-free system.
Replacement of the hydroxamic acid with carboxylic acid
decreased the potency of inhibition, potentially due to the carbox-
ylic acid acting as a less efficient zinc chelator. Removal of the
Scheme 1. Synthesis of a small array of 50 amide derivatives of 1. Reagents and conditions: (a) TEA, Boc2O, dioxane, RT, 18 h, 76%. (b) BnONH2ꢂHCl, DCC, DMAP, TEA, CHCl3/
THF, 0 °C, 16 h to RT, 24 h, 46%. (c) TFA (wet), RT, 1 h, 85%. (d) R1-COCl, pyridine, DMAP (cat.), THF/DCE 4:1, RT, 24 h, 21–79%. (e) AcOH, H2, 20% Pd(OH)2, MeOH/THF, 0.5 mL/
min, 1 atm, 45–100%.

58
M. D. Urbaniak et al. / Carbohydrate Research 387 (2014) 54–58
Table 2
3.4. Conclusions
Inhibitory activity of novel derivatives of 1
We have identified salicylic hydroxamic acid 1 as an inhibitor of
ID
R1
Inhibition at 1 mMa (%)
99 1.6
T. brucei GlcNAc-PI de-N-acetylase with high ligand efficiency, rep-
resenting the first non-substrate analogue inhibitor of the trypano-
some GPI pathway. Although 1 shows modest potency with an IC50
20
of 63
4 l
M, it has impressive ligand efficiency of 0.57 kcal molꢁ1
21
96 0.4
per non-hydrogen atom, and thus may be a useful starting point to
develop more potent inhibitors.
Me
22
23
24
62 24
25 14
40 17
Acknowledgments
CH3(CH2)16
AcN
This work was funded by the Medical Research Council (stu-
dentship to ASC) and the Wellcome Trust (Programme Grant
085622 and 077705, and Strategic Awards 083481 and 100476).
The funders had no further role in the study design.
25
26
75 7.0
56 8.3
O
O
O
Supplementary data
27
86 11
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.12.
016.
a
Inhibition of T. brucei de-N-acetylase (cell-free system).
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Figure 4. Proposed mode of action. (A) Structure–activity relationships of 1.(B)
Components of the natural substrate GlcNAc-PI recognised by the enzme. R1 (1-
6)- -myo-inositol-1-phosphate-dimyristolglycerol. (C) The GlcNAc-PI de-N-acety-
lase contains catalytic zinc chelated by the residues H49, D52 and H157, and the
catalytic base D43. (D) Compound 1 could inhibit the enzyme through binding of
the hydroxamic acid to the catalytic zinc, replacing both the acetamidocarbonyl and
activated water.
=
a
D
acid. We propose that 1 could inhibit the enzyme through the
hydroxamic acid binding to the catalytic zinc, and replacing both
the acetamidocarbonyl and the activated water (Fig. 4).
21. Chen, H.; Boiziau, J.; Parker, F.; Mailliet, P.; Commercon, A.; Tocque, B.; Le Pecq,
J. B.; Roques, B. P.; Garbay, C. J. Med. Chem. 1994, 37, 845–859.