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ACS Chemical Biology
ACS Chem Biol. 2013 September 20; 8(9): 1981–1987.
Published online 2013 July 8. doi:  10.1021/cb400411x
PMCID: PMC3780468

A Novel Allosteric Inhibitor of the Uridine Diphosphate N-Acetylglucosamine Pyrophosphorylase from Trypanosoma brucei


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Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final reaction in the biosynthesis of UDP-GlcNAc, an essential metabolite in many organisms including Trypanosoma brucei, the etiological agent of Human African Trypanosomiasis. High-throughput screening of recombinant T. brucei UAP identified a UTP-competitive inhibitor with selectivity over the human counterpart despite the high level of conservation of active site residues. Biophysical characterization of the UAP enzyme kinetics revealed that the human and trypanosome enzymes both display a strictly ordered bi–bi mechanism, but with the order of substrate binding reversed. Structural characterization of the T. brucei UAP–inhibitor complex revealed that the inhibitor binds at an allosteric site absent in the human homologue that prevents the conformational rearrangement required to bind UTP. The identification of a selective inhibitory allosteric binding site in the parasite enzyme has therapeutic potential.

The sugar nucleotide uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is an important and ubiquitous metabolite that is used in eukaryotes as the source of N-acetlyglucosamine in the biosynthesis of N-linked and O-linked glycans and the source of glucosamine in glycosylphosphatidylinositol anchors. In addition, UDP-GlcNAc is required for the formation of lipopolysaccharide and peptidoglycans used in bacterial cell wall biosynthesis and the formation of chitin for fungal cell wall biosynthesis. The enzyme UDP-GlcNAc pyrophosphorylase (UAP) is responsible for a key transformation in the biosynthesis of UDP-GlcNAc by catalyzing the reversible reaction between UTP and glucosamine-1-phosphate (Glc-1-P) forming UDP-GlcNAc and inorganic pyrophosphate (PPi) (Scheme 1). The enzyme represents a bottleneck between different glycoconjugate biosynthetic pathways that has the potential to be exploited as a therapeutic target, provided that species-specific inhibitors can be found.

Scheme 1
Enzyme-Catalyzed Formation of UDP-GlcNAc

Trypanosoma brucei is a protozoan parasite transmitted by the bite of an infected tsetse fly (Glossina spp.) and is the etiological agent of Human African Trypanosomiasis (HAT, also known as African sleeping sickness). The disease is responsible for 10,000 recorded deaths per annum in sub-Saharan Africa, although due to poor surveillance the true number is estimated to be much higher.1 Current treatments are expensive, toxic, and difficult to deliver, leaving an urgent unmet need for improved therapeutic agents.2 The parasite has a digenetic lifecycle between a mammalian host and insect vector and produces a complex array of glycoconjugates, some of which are essential for its infectivity and virulence. Several enzymes involved in the biosynthesis of glycosylphosphatidylinositol anchors35 and sugar nucleotide biosynthesis610 have been shown to be essential in bloodstream form T. brucei by genetic validation.

T. brucei UAP (TbUAP) has been genetically validated in bloodstream form parasites as essential both in vitro and in vivo and has been proposed as a potential therapeutic target, although selective inhibition of the parasite UAP would be a therapeutic requirement.8 Despite the moderate level of overall sequence similarity between TbUAP and its human counterpart (31% sequence identity, 50% sequence similarity), only two of the 15 identified substrate-interacting residues in human UAP (HsUAP) are different,8,11 and there are no known inhibitors of UAP. In this work we set out to discover novel species-specific inhibitors of TbUAP through high-throughput screening of the recombinant enzyme. Through biophysical and structural characterization, we reveal that the trypanosome and human UAP differ in the order of sequential substrate binding and that a primary hit compound is a species-specific UTP-competitive allosteric inhibitor of TbUAP.

Results and Discussion

Identification of Novel TbUAP Inhibitors

Recombinant T. brucei UAP (TbUAP) was screened against a diverse library of 63,362 molecules using a discontinuous coupled colorimetric assay that monitors phosphate generation (Scheme 1).8 The initial 73 hit compounds that showed >25% inhibition at 30 μM (0.12% hit rate) were triaged by removing compounds that displayed activity against the E. coli pyrophosphatase coupling enzyme. The 12 remaining compounds were all confirmed as TbUAP inhibitors by direct monitoring of their effects on conversion of the substrate (UTP) to product (UDP-GlcNAc) by HPLC. Their IC50 values were determined using the coupled assay. Commercially available analogues of the two most potent compounds, 1 and 2 (Scheme 2, IC50 = 37 ± 4 and 49 ± 4 μM, respectively), were identified by substructure searching, and 30 analogues were purchased and assayed for activity. None of the compounds showed improved potency over that of the parent compounds, with the relatively low potencies limiting the derivation of structure–activity relationships.

Scheme 2
TbUAP Inhibitors Identified by High-Throughput Screening

TbUAP1 Binds Its Substrates in a Different Order from HsUAP1

To examine the enzyme reaction mechanism and explore the mode of action of the most potent inhibitor 1, a series of surface plasmon resonance (SPR) experiments were employed (Figure (Figure11 and and2,2, Table 1). The reaction mechanism of UAP requires that both UTP and GlcNAc-1-P bind to the enzyme, but it was unknown if the sequential binding is random or strictly ordered. We examined the binding of the two substrates to TbUAP and the closest Human homologue UAP-AGX1 (HsUAP) by SPR (Figure (Figure1, Table1, Table 1). We found that TbUAP binds UTP alone with a KD of 83.1 ± 0.5 μM (Km = 26 μM, Stokes et al.8) but does not bind GlcNAc-1-P alone (Km = 39 μM, Stokes et al.8). In contrast, HsUAP does not bind UTP alone (Km = 53 μM, Peneff et al.11), and although it does show significant binding to GlcNAc-1-P, it was not possible to calculate an affinity due to complex binding kinetics. These data reveal that substrate binding to UAPs is strictly ordered but that, surprisingly, the order of binding is reversed between the two species. To our knowledge this is the first example of species-specificity in sequentially ordered bi–bi mechanisms and raises the intriguing possibility that UTP-competitive inhibitors may confer species specificity.

Figure 1
Surface plasmon resonance sensorgrams of TbUAP and HsUAP binding to substrates. (A) TbUAP binds UTP with a KD of 83.1 ± 0.5 μM, UTP was injected in duplicates at concentrations from 3.9 to 500 μM. The inset shows an equilibrium ...
Figure 2
Mode of inhibition by compound 1. (A) TbUAP binds 1 with a KD of 2.58 ± 0.07 μM. (B) HsUAP does not bind 1. (C) The presence of 100 μM Glc-NAc-1-P does not significantly affect the binding of 1 to TbUAP (KD of 2.35 ± 0.03 ...
Table 1
Surface Plasmon Resonance Parametersa

Compound 1 Is a Competitive Inhibitor of TbUAP1

To investigate the mode of inhibition of 1, we examined its binding to TbUAP and HsUAP by SPR (Figure (Figure22 and Table 1) and found that it was bound by TbUAP with KD = 2.58 ± 0.07 μM, but that HsUAP displayed no significant binding (Figure (Figure2A,B).2A,B). The binding of 1 to TbUAP was competitive with UTP, with the KD shifting to KD = 9.30 ± 0.1 μM (IC50 = 37 ± 4 μM) in the presence of 500 μM of UTP, while the presence of 100 μM of GlcNAc-1-P did not significantly affect binding with a KD = 2.35 ± 0.03 μM (Figure (Figure2D,C).2D,C). The selectivity observed by SPR was confirmed by testing the activity of 1 against TbUAP and HsUAP in both the coupled assay and HPLC assay, showing consistent inhibition of TbUAP but no significant inhibition of HsUAP (Figure (Figure2E-,F).2E-,F). The coupled assay was used to confirm that inhibition by 1 was competitive with UTP with an apparent Ki of 60 μM (Figure (Figure2G),2G), while the mixed mode of inhibition observed with GlcNAc-1-P did not allow an apparent Ki to be calculated (Figure (Figure22H).

Compound 1 Binds TbUAP in a Unique Allosteric Site

To gain further insight into the binding interactions with TbUAP, cocrystallization with various ligands was attempted. We were unable to obtain suitable diffraction quality crystals in the presence of substrates or product, but cocrystallization with 1 alone resulted in crystals that diffracted to high resolution. The bound complex was refined against synchrotron diffraction data to 1.75 Å (Table 3), revealing clear density for the inhibitor at a site distinct from the active site (Figure (Figure3A).3A). This represents the first UAP structure from T. brucei or indeed any protist. In common with other eukaryotic UAP structures,11,12 the TbUAP structure consists of a central pyrophosphorylase domain of eight β-strands sandwiched by eight α-helices in a Rossmann fold,13 which contains the active site, flanked by an N-terminal domain containing the N-terminus (residues 1–62) and additional β-sheets from the central domain (residues 209–231 and 377–396), and a short C-terminal domain. Strikingly, the inhibitor binding site is located away from but facing the active site in a deep hydrophobic cleft formed by the central and C-terminal domain (Figure (Figure3A,B) where3A,B) where it is able to form hydrogen bonds between the amide group of indolin-2-one and the Gly44 carboxyl group at a distance of 2.8 Å and between the carboxyl group of indolin-2-one with the amide group of Asp46 at a distance of 2.8 Å, as well as a number of hydrophobic interactions (Figure (Figure3C,D).3C,D). In the published structures of Candida albicans UAP (CaUAP) there is distinct movement in the N-terminal domain between the apo-form and the GlcNAc-1-P or UDP-GlcNAc bound forms,12 consistent with an induced-fit movement that closes the entrance to the binding site upon substrate binding. The inhibitor makes contact with residues on the opposite face of a glycine-rich loop that moves to make contact with the uridine of bound UDP-GlcNAc (Figure (Figure3B),3B), and TbUAP adopts a conformation that most closely resembles the apo-CaUAP structure (2YQC, RMSD 2.2 Å) and least resembles the CaUAP structure with UDP-GlcNAc bound (2YQJ, RMSD 2.7 Å). Thus, 1 appears to act as an allosteric competitive inhibitor of UTP by stabilizing the N-terminal domain and uridine-binding loop in a conformation that prevents the binding of UTP yet does not occupy the UTP binding site itself. Allosteric regulation of TbUAP activity is consistent with reports that Giardia lamblia UAP activity is altered in vivo by the allosteric binding of the metabolite glucosamine-6-phosphate, although in that case binding caused an increase in activity.14

Figure 3
Structure of TbUAP in complex with 1. (A) Overview of the TbUAP-1 structure (PDB ID 4bqh), which is composed of an N-terminal domain (beige), a central domain (green) containing the active site (nucleotide binding motif, red), and a C-terminal domain ...

Compound 1 Structure–Activity Relationships

The conformation of the allosteric site is such that only the (R)-enantiomer of 1 can bind, and it is likely that the kinked shape of the molecule is crucial for its shape-complimentarity to the pocket. The benzo[1,3]dioxole moiety is deeply buried, making close contact with Ala397 and Gly232 at the bottom of the cleft (Figure (Figure3C,D).3C,D). Consistent with this binding mode, the commercial structural analogue of 1 that lacks the benzo[1,3]dioxole moiety does not inhibit TbUAP1, and even replacement of the bridging methylene with ethylene is not tolerated (Table 2). The indolin-2-one sits at the top of the cleft, with the unsubstituted edge exposed to solvent and the methyl and bromide substituents on making contact with Ala239, Met370, Lys371, and Ala367 (Figure (Figure3C,D).3C,D). Removal of the bromine reduces potency >6-fold, and removal of both the bromine and methyl groups reduces potency ≥10-fold (Table 2). The observed SAR for the commercial analogues is consistent with the contacts observed in the crystal structure.

Table 2
Structures of Selected Commercial Analogues of 1a

Allosteric Binding Site Is Unique to TbUAP1

Comparison of the TbUAP-1 structure with the structure of HsUAP11 revealed that the central catalytic domains are structurally similar (RMSD 1.4 Å), but that the flanking N-terminal and C-terminal domains occupy different positions (maximum Cα atom shift is 9.5 Å, Figure Figure3E).3E). The inhibitor binding cleft formed by the central and C-terminal domain is wider (10.3 Å versus 7.7 Å) and shallower (10.9 Å versus 17.4 Å) due to both significant movement of the α-helices and nonconservative substitutions. Critically, the substitution of Gly232 in TbUAP with Asp221 in HsUAP blocks the benzo[1,3]dioxole binding site, and the substitution of Ala239 in TbUAP with Arg228 in HsUAP blocks the entrance to the cleft by forming a salt bridge with Glu44 (Figure (Figure3F).3F). The structural data thus explain the observed selectivity of 1.

Compound 1 Inhibits Growth in T. brucei Cultures

We determined that 1 has an EC50 of 30 μM against cultured T. brucei (data not shown), a surprisingly small drop-off in potency compared to the IC50 of 30 μM recorded against the recombinant T. brucei enzyme. To assess the mode of action of 1, we determined its potency against a TbUAP conditional null mutant (TbUAP-cKO) cell line in culture. The conditional null mutant, where both allelic copies of TbUAP are replaced by drug resistance cassettes, expresses TbUAP from an ectopic copy under the control of tetracycline.8 The TbUAP-cKO cell line is viable in the presence of tetracycline (permissive conditions), where expression of TbUAP occurs, although the cellular levels of UDP-GlcNAc are reduced (16 pmol/1 × 107 cells) compared to the wild type (80 pmol/1 × 107 cells) due to a reduced level of TbUAP expression. The EC50 was not significantly changed between the wild-type and TbUAP-cKO cell line, suggesting that the cytotoxicity of the compound is not driven by inhibition of TbUAP. To assess whether 1 was able to inhibit TbUAP in T. brucei cells, we treated wild-type cells with 100 μM 1 (3 × IC50) or DMSO for 3 h and measured the intracellular levels of sugar nucleotides by LC–MS/MS.15,16 No significant difference in the level of sugar nucleotides was observed between the treated and untreated samples. Taken together, these data suggest that the observed cytotoxicity of 1 against cultured T. brucei is due to an off-target effect and not through the inhibition of TbUAP.

Concluding Remarks

We have discovered a novel UTP-competitive inhibitor of T. brucei UAP that displays good selectivity for the parasite enzyme over the human homologue due to binding at a previously unidentified allosteric binding site. While the current inhibitor is of modest potency and the in vivo parasite toxicity is likely to be due to off-target effects, the structural data will facilitate the design and synthesis of more potent compounds that may have therapeutic potential. A potential drawback to the targeting of an allosteric site rather than the active site is that resistance may occur more readily due to lack of selective pressure to maintain interactions with the enzyme substrate. However, as the binding site is formed by a hinge region between two domains that undergo induced-fit movement during the catalytic cycle, such substitutions may not be tolerated.

Our studies have revealed that the UAP mechanism is strictly sequentially ordered, but that the order of substrate binding is reversed between the parasite and human enzyme. As the parasite UAP strictly binds UTP first, it follows that UTP-competitive inhibitors may show selectivity for the parasite enzyme over the human enzyme. Traditional sequence- and structure-based drug discovery approaches did not predict that species specificity would be readily achievable due to the high level of conservation of active site residues, highlighting the importance of biophysical studies in target evaluation.


Cloning, Expression, and Purification of HsUAP

Homo sapiens UAP-AX1 (HsUAP, NP_003106) was amplified by PCR from cDNA (OriGeneTechnologies) using the primers 5′-GGAATTCCATATGAACATTAATGACCTC-3′ (NdeI site underlined) and 5′-CGCGGATCCCTCGAGTCAAATACCA-3′ (BamHI site underlined) and inserted into pET15b-pp (a modified pET15b with the thrombin site replaced with PreScission protease) using the NdeI and BamHI RE sites to give the plasmid pET15b-pp-HsUAP-AX1. Recombinant HsUAP-His6 was expressed in BL21 (DE3) E. coli from the vector pET15b-pp-HsUAP-AX1 and purified in a single step using Ni2+ affinity chromatography using the same condition as reported for TbUAP-His6.8 The identity of recombinant HsUAP-AX1 was confirmed by tryptic mass finger printing (Mascot score 1814, 88% sequence coverage). Purified recombinant UAPs were stored in 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 10% glycerol at −80 °C prior to use.

Expression of TbUAP for Activity Assays

Recombinant TbUAP-His6 for activity assay was cloned from T. brucei, expressed from the plasmid pET15b-pp-TbUAP in Escherichia coli BL21 (DE3), and purified in a single step using Ni2+ affinity chromatography as reported previously.8 For crystallization trials, the TbUAP gene was cut from the pET15b-PP-TbUAP1 plasmid and cloned into a BamHI digested pGEX-6P-1 vector (GE Healthcare). The resulting plasmid, pGEX-6P-1-TbUAP, encodes a glutathione-S-transferase (GST) fusion TbUAP separated by a PreScission protease cleavage site.

High-Throughput Screening of TbUAP

The TbUAP high-throughput screen was performed using a Dundee Drug Discovery Unit in-house diverse compound collection of 63,362 molecules17 against a discontinuous coupled colorimetric assay. The assay was performed at RT in 384-well plates in a final reaction volume of 50 μL in reaction buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 2% v/v glycerol, 1 mM dithiothreitol, 0.1 mg mL–1 bovine serum albumin, 1 unit mL–1E. coli pyrophosphatase) supplemented with 30 μM UTP, 100 μM GlcNAc-1-P, 0.5 nM recombinant TbUAP, and 30 μM test compound with a final concentration 1% DMSO. Test compounds in 0.5 μL of DMSO were transferred to the plates prior to the addition of recombinant TbUAP in 24.5 μL of reaction buffer. The reaction was initiated by the addition of the substrates UTP and GlcNAc-1-P in 25 μL of reaction buffer and allowed to proceed for 8 min before termination by the addition of 50 μL of Biomol Green (0.03% malachite green, 0.2% w/v ammonium molybdate, 0.5% Triton X-100 in 0.7 M HCl). The signal was allowed to develop for a minimum of 30 min before the absorbance of each well was read at 650 nm. The assay gave a robust average Z′ of 0.8 ± 0.1, with an average coefficient of variance of 1–3% and signal/background of 2.5 ± 0.3 based on the inclusion of high (uninhibited) and low (no enzyme) control wells in each of the 183 assay plates.

Compounds with ≥25% inhibition in the screen (100, 0.16% hit rate) were cherry picked and confirmed by retesting, with a 73% confirmation rate (Supplementary Table S1). Confirmed hits were tested for potency against the pyrophosphatase by modifying the discontinuous coupled colorimetric assay to include 5 mM inorganic pyrophosphate, and compounds showing >15% difference between the pyrophosphatase and coupled assay (Supplementary Table S2) were considered TbUAP hits (12, 0.02% hit rate). The TbUAP hits were repurchased, and 10-point inhibitor IC50 curves were determined using the discontinuous coupled colorimetric assay and fitting the dose–response curve to a four-parameter fit in ActivityBase XE (IDBS).

High pH Anion Exchange Chromatography

The inhibition of TbUAP and HsUAP was measured using high pH anion exchange chromatography (HPAEC) to follow the conversion of UTP to UDP-GlcNAc by TbUAP. The reaction buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 2% v/v glycerol, 1 mM dithiothreitol, 0.1 mg mL–1 bovine serum albumin, 1% DMSO) was supplemented with 25 μM UTP, 40 μM GlcNAc-1-P, and 25 ng TbUAP or 75 ng HsUAP. The reaction (100 μL) was incubated at 30 °C for 30 min with or without inhibitor, quenched by the addition of 10 μL of 0.1 M NaOH, and then subjected to HPAEC chromatography on a CarboPac PA-1 column (Dionex) using conditions adapted from Tomiya et al.18 The eluent was monitored at 260 nm, and peaks were assigned by comparison to commercial standards. The IC50 value was calculated using a four-parameter fit of eight-point potency curves derived from three independent experiments.

Competition Studies

The kinetic parameters for TbUAP were determined in the presence of different concentrations of substrates and inhibitor using the discontinuous coupled colorimetric assay described above. The reaction was performed either at fixed concentration of 40 μM GlcNAc-1-P and 5–640 μM of UTP in the presence of 0–300 μM 1, or at a fixed concentration of 25 μM UTP and 4–500 μM GlcNAc-1-P in the presence of 0–300 μM 1, and the data were fitted to the Michaelis–Menten equation and displayed as a double reciprocal plot. The calculated apparent Km(UTP)app in the presence of a range of concentrations of 1 was used to calculate Ki by plotting Km(UTP)app against [I] to solve the equation Kmapp = (Km/Ki)[I] + Km.

Surface Plasmon Resonance

Recombinant TbUAP and HsUAP were chemically biotinylated and captured on a streptavidin surface of a Biacore T100 instrument (GE-Healthcare) at densities ~6,000–7,000 RU. To stabilize captured proteins over time all experiments were run at 4 °C. Ligands were injected over captured proteins at flow rate 30 μL min–1 in running buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween 20, 1 mM DTT, 3% DMSO), with each compound injected in duplicates in concentration series adjusted specifically for each ligand; UTP was injected at 2-fold concentration series (3.9–500 μM), GlcNAc-1-P at 3-fold concentration series (9 nM to 20 μM), and 1 at 3-fold concentration series (69 nM to 500 μM). Association was measured for 30 s and dissociation for 30–300 s depending on the off-rate. For competition studies, 500 μM UTP or 100 μM Glc-NAc-1-P was added to the running buffer. All data were double referenced for blank injections of buffer and biotin-blocked Streptavidin surface. Scrubber 2 (BioLogic Software) was used to process and analyze the data.

Expression and Purification of TbUAP for Crystallography

Recombinant TbUAP-GST was expressed from the plasmid pGEX-6P-1-TbUAP in E. coli BL21 (DE3) pLysS. Cells were grown in LB at 37 °C to an OD600 of 0.8 and cooled to RT, and protein expression was induced with 250 μM isopropyl-β-d-thiogalactopyranoside for 20 h. Cells were harvested by centrifugation at 3500 × g at 4 °C for 30 min, resuspended in buffer A (25 mM Tris pH 7.5, 150 mM NaCl) in the presence of 10 mg mL–1 DNase, a protease inhibitor cocktail (Roche) and 0.5 mg mL–1 lysozyme), lysed on a EmulsiFlex-C3 homogenizer at 20 kpsi (Avestin), and centrifuged at 40,000 × g for 30 min. The supernatant was incubated with prewashed glutathione sepharose beads (GE Healthcare) at 4 °C on a rotating platform for 2 h, and the beads were isolated by centrifugation at 1000 × g for 3 min and washed with buffer A four times. TbUAP was cleaved from the GST tag by treatment with PreScission protease in the same buffer at 4 °C on a rotating platform for 18 h, and the released protein was further purified on a Superdex75 gel filtration column (2.6 cm × 60 cm) (Amersham Biosciences) with 1.0 mL min–1 buffer A. The fractions were verified by SDS-PAGE, pooled, and concentrated to 15 mg mL–1 using a 10-kDa cutoff Vivaspin concentrator (GE Healthcare).

Crystallization, Data Collection, and Structure Determination

Crystallization was conducted using the sitting-drop vapor diffusion method at RT, where each drop contained 0.5 μL of TbUAP1 solution (15 mg mL–1 in buffer A) with an equal volume of the mother liquor. To obtain the TbUAP1-1 complex, the protein was incubated with 0.495 M compound at 4 °C for 30 min before setting up crystal trays. The complex crystallized after 4–5 days in the space group C2221 from a mother liquor containing 25% PEG3350, 0.2 M (NH4)2SO4, 0.1 M Bis-Tris pH 5.5. Crystals were cryo-protected in this solution supplemented with 15% glycerol. X-ray data were collected at the I-24 (microfocus) beamline of the Diamond (U.K.) synchrotron and processed with HKL2000.19 The phase problem was solved by the automated molecular replacement pipeline BALBES;20 REFMAC21 was used for further refinement and iterated with model building using COOT.22 Detailed crystallographic parameters are given in Table 3. The model for ligands was not included until their conformations were fully defined by unbiased |Fo| – |Fc|, [var phi]calc electron density maps. Ligand structures and topologies were generated by PRODRG.23 Images were generated with PyMol24 and LigPlot+.25 The final structure coordinates and structure factors are available in the PDB (4bqh and r4bqhsf, respectively).

Table 3
Details of Diffraction Data Collection and Structure Refinement for TbUAP1 + 1a

Trypanosoma brucei Growth Inhibition

The potency of 1 against cultured T. brucei was determined using a standard 3-day Alamar blue assay as described previously.26 Assays were conducted using the Lister 427 single marker cell line27 or a TbUAP conditional null mutant8 grown in HMI9-T.28 The EC50 values were calculated from 8-point potency curves in triplicate.

Sugar Nucleotide Analysis

T. brucei Lister 427 single marker cells grown in HMI9-T28 were treated with 100 μM of 1 in 0.1% DMSO or a 0.1% DMSO control for 3 h. Cells were harvested by centrifugation, the intracellular sugar nucleotides were extracted, and their levels were quantified by LC–MS/MS analysis as described previously.15,16


This work was funded by the Wellcome Trust (Programme Grant 085622 and 077705, and Strategic Award 083481). This work was supported by an MRC Program Grant G0900138. D.M.F.vA. is supported by a Wellcome Trust Senior Research Fellowship (WT087590MA).

Supporting Information Available

Further details of the 73 initial hits (Table S1) and the 30 commercially available compounds (Table S2). This material is available free of charge via the Internet at

Accession Codes

The TbUAP-1 structure coordinates (4bqh) and structure factors (r4bqhsf) are available in the PDB.


The authors declare no competing financial interest.

Supplementary Material


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