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African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT.
Protein N-myristoylation is a ubiquitous eukaryotic co- and post-translational modification and is required for membrane targeting and biological activity of many important proteins 1,2. The N-myristoylation reaction, i.e., the transfer of C14:0 myristic acid from myristoyl-coenzyme A (CoA) to the amino group of N-terminal glycine residues within specific sequence contexts 3, is catalysed by the enzyme myristoyl-CoA: protein N-myristoyltransferase (NMT; EC 184.108.40.206) 4. In T. brucei, NMT activity is encoded by a single gene, which has been shown to be essential for parasite growth using RNA interference 5. The effects of NMT knockdown on T. brucei are likely to be complex since more than 60 proteins are predicted to be N-myristoylated in this organism 6. Experimentally validated targets for NMT include ADP ribosylation factors (Arf) 7, ADP ribosylation-like factors (Arl) 8, a calpain-type protease (CAP5.5, TbCALP1) 9 and, in the related Leishmania major and T. cruzi parasites, hydrophilic acylated surface proteins (HASPs) 10 and flagellar calcium-binding protein 11, respectively. The predicted pleiotropic effects of NMT inhibition on trypanosome physiology make it an attractive target for therapeutic intervention. NMT has also been considered as an anti-cancer 12, fungal 13 and viral 14 target. Fungal NMT orthologues have been shown to be druggable, although broad-spectrum activity has not been achieved. Nevertheless, since there is good evidence from these programmes that selectivity versus human NMT is possible, NMT has been proposed as a target for the treatment of human African trypanosomiasis (HAT) and other parasitic diseases 15,16. There are 2 human isozymes sharing 77% identity (huNMT1 and 2) 17 of which huNMT2 is the closest human homologue to TbNMT, with overall 55% identity and 69% similarity. Based upon 31 residues that are within 5Å of DDD85646 in the active site, this rises to 83% identity and 90% similarity.
To date, no drug-like, potent inhibitors of TbNMT have been reported 18. Screening of a 62,000 diversity-based compound library 19 against TbNMT identified a number of “lead-like” hits, including a chemically tractable series with moderate potency (2 μM) based on a pyrazole sulfonamide scaffold (DDD64558). Optimisation of the screening hit, involving the design and synthesis of over 200 compounds, identified highly potent inhibitors of TbNMT with single digit nanomolar IC50 values and levels of selectivity over human NMT enzymes in the 1 to >100-fold range (Fig.1a). The relative lack of activity of the piperidine analogue (DDD85635) of DDD85602 indicated the terminal basic nitrogen of DDD85602 was crucial for activity. The series was optimised by rigidifying the flexible linker to the amine moiety of DDD85602 and by adding the chlorines observed to give an increase in activity in the unelaborated template (DDD73234). TbNMT inhibitors so obtained (e.g. DDD85646) inhibited the proliferation of bloodstream form (BSF) T. brucei in culture with the best compounds yielding EC50 values between 0.8 and 3 nM and clear windows of selectivity (>200-fold) with respect to proliferation of a prototypical mammalian cell type (MRC5). We attribute the increased selectivity at the cellular level to differences in cell biology between host and parasite, although differential cellular pharmacokinetic behaviour has not been definitively ruled out. Critically, a tight correlation (R2 = 0.875) was observed between IC50 and EC50 values for TbNMT and T. brucei proliferation, respectively, over a 10,000-fold potency range (Fig. 1b); indicating that inhibition of TbNMT was driving the observed anti-parasitic effect of these compounds. The poorer correlation for the most potent compounds (see lower left quadrant of Fig. 1b) is most likely due to the limit of the enzyme assay to provide accurate IC50 determinations for such highly active, tight-binding inhibitors (see Supplementary Information, Figure 1). As a result of its impressive potency in inhibiting both TbNMT and T. brucei proliferation in vitro, together with its promising physicochemical properties, DDD85646 was assessed for efficacy in animal models of trypanosomiasis.
DDD85646 is moderately bioavailable (ca. 20%) and demonstrates good exposure after oral dosing at 10 and 50 mg kg−1 to female NMRI mice, with the free drug level above the EC99 for T. b. brucei proliferation for over 6 and 10 h, respectively (Fig. 2a). Furthermore, this compound cured all animals in the T. b. brucei acute mouse model of HAT at a minimal oral dose of 12.5 mg kg−1 (b.i.d. for 4 days) (Fig 2b). Cure of all animals was also obtained with shorter oral dosing schedules: 100 mg kg−1 b.i.d. for one day and 25 mg kg−1 b.i.d. for 2 days. Importantly, DDD85646 also cured all animals at 50 mg kg−1 (b.i.d. for 2 days) in the more refractory, but clinically relevant T. b. rhodesiense model of HAT (see Supplementary Information, Figure 2). This reduced sensitivity in vivo is not due to reduced sensitivity to the compound in vitro (T. b. rhodesiense EC50 0.6nM), but maybe a result of the known precedent for this species to occupy privileged sites in vivo. Notably, the efficacy observed for DDD85646 was comparable with the responses observed for the clinically-used drugs pentamidine and melarsoprol in the T. b. brucei model (minimal full cure doses were 1 mg kg−1 and 0.5 mg kg−1 (IP) respectively). Furthermore, despite the minimal window of in vitro enzyme selectivity between TbNMT and mammalian (human) NMT (Fig. 1a and vide infra), this compound was well tolerated at efficacious doses.
Addition of DDD85646 resulted in rapid killing of trypanosomes both in vivo and in vitro (Fig. 3a, b). Parasite counts dropped to below detectable levels within 12 h of dosing mice at 50 mg kg−1 b.i.d. Addition of compound (50 nM) to BSF T. brucei cultures in vitro also resulted in rapid killing with numbers of motile cells reduced to below detectable levels between 24 and 48 h. The apparent differences in kinetics of death between the in vivo and in vitro systems are most likely a combination of the harsher in vivo environment for drug-damaged trypanosomes and the fact that compound exposure reached higher concentrations in vivo (up to ~1 μM), compared with 50 nM in vitro.
The trypanocidal mechanism of compound action was confirmed by subjecting T. brucei treated in vitro with 50 nM compound to live/dead FACS analysis, which showed >95% cell death within 24 h of treatment (see Supplementary Information, Figure 3). Furthermore, wash-out experiments showed that death was irreversible after 48 h of exposure to 50 nM compound (data not shown). Microscopic examination of the trypanosomes treated with DDD85646 in vivo and in vitro revealed the same abnormal morphology, i.e., the development of a large vesicular structure (Figure 3c). A scanning electron micrograph of a compound-treated trypanosome clearly shows the rounded and swollen features of this phenotype compared to control (Fig. 3d). Interestingly, rapid cell killing with a similar morphological phenotype has been observed previously following treatment with myristate analogues, such as 10-(propoxy)decanoic acid 20. This morphology closely resembles the ‘BigEye’ phenotype observed in BSF T. brucei when endocytosis is disrupted through the knockdown of clathrin heavy chain, TbRab5 21,22 or TbArf1 7 leading to expansion of the flagellar pocket. Parasites with enlarged flagellar pockets are clearly visible in the DDD85646-treated trypanosome population (Fig. 3e). Additional studies are required to fully understand the cellular effects of NMT inhibition.
Incubation of BSF T. brucei with [3H]-myristic acid results in the biosynthetic radiolabelling of myristoylated substrates, particularly the highly abundant variant surface glycoprotein (VSG) 23. Using this method, but including a detergent lysis step that activates an endogenous phospholipase C to release [3H]-myristate label from the glycosylphosphatidylinositol (GPI) anchor of VSG 24, a number of putative N-[3H]-myristoylated proteins were visualised by SDS-PAGE and fluorography (Fig. 4a, lane 2). The labelling of most of these proteins was eliminated by prior treatment with DDD85646 (Fig. 4a, lane 1). To confirm that most of the proteins labelled with [3H]-myristic acid were indeed N-myristoylated, a duplicate gel was treated with 0.2 M NaOH in methanol prior to fluorography to remove base-labile hydroxy- or thio-ester linked [3H]-myristate (Fig. 4a, lanes 3 and 4). Three faint DDD85646-insensitive bands were removed (Fig. 4a, compare lanes 1 and 2 with lanes 3 and 4); these most likely include traces of residual GPI anchored VSG at 55 kDa 24 and thioester-myristoylated GPI-PLC at 42 kDa 25. In order to assess whether DDD85646 specifically inhibited N-[3H]-myristoylation, the same cells were labelled in parallel with [35S]-methionine. Pre-treatment of parasites with DD85646 had no effect on [35S]-methionine incorporation into proteins, showing that the compound has no effect on general protein synthesis (Fig. 4a, lanes 5 and 6).
Further evidence that DDD85646 was acting on-target in the trypanosome was obtained by over-expressing TbNMT (5-fold) in a tetracycline-inducible manner; this resulted in an 8-fold reduction in DDD85646 potency against these cells (Fig. 4b).
Another independent approach was taken using one of the few known substrates of TbNMT in T. brucei, TbArf1. This protein plays a central role in endocytosis and Golgi-lysosome trafficking, where tetracycline-induced expression of a constitutively-active GTP-locked mutant (Q71L) causes rapid cell death in BSF T. brucei in an N-myristoylation-dependent manner 7. Short-term treatment (5 h) with 10 nM DDD85646 rescued these TbArf1 Q71L expressing cells from death (Fig. 4c), presumably by preventing N-myristoylation of newly produced TbArf1 Q71L protein. Although the Q71L mutant protein could not be detected by Western blotting, as reported previously 7, nonetheless the inducible expression of the related, but myristoylation blocked, G2A Q71L mutant in the presence of DDD85646 does show that its effect on cell survival was not due to interference with tetracycline-induction of these mutants.
Taken together, these data provide strong evidence that DDD85646 acts to inhibit TbNMT in BSF trypanosomes and that this is directly linked to inhibition of proliferation. These data also provide a potential link between inhibition of TbNMT and disruption of the function of the TbNMT substrate TbArf1, known to operate in protein trafficking and endocytic processes.
The Target Product Profile for a new drug to combat HAT, as defined by Drugs for Neglected Diseases initiative, requires compounds that are safe and efficacious against both the stage 1 disease, when parasites are present in the blood, lymph and interstitial fluids, and the stage 2 disease, when parasites are also present in the CNS. The DDD85646 compound does not yet meet these criteria and will have to be optimised with respect to selectivity over human NMT and for diffusion into the CNS, while retaining excellent potency against TbNMT and BSF T. brucei cells. To achieve these objectives, a detailed understanding of the interaction between DDD85646 and TbNMT is required. Characterisation of the mode of inhibition of the early hits revealed competition with respect to peptide substrate as a likely mode of inhibition for the series. Thus, a shift in IC50 from 1 to 4.3 μM for an early hit was seen when the peptide substrate concentration in the assay was increased from 0.5 to 16 μM (Fig. 5a). Surface Plasmon Resonance (SPR) studies confirmed a 1:1 binding stoichiometry of DDD85646 with TbNMT (Fig. 5b) and showed that the binding affinity of the compound was increased in the presence of myristoyl-CoA from 33 nM to 1 nM (data not shown). Accurate determination of binding affinity via SPR also revealed a potential small window of selectivity between TbNMT and human NMT with binding constants of 1 and 14 nM, respectively. Due to tight binding of this compound (see Supplementary Information, Figure 1), this selectivity was less clear in the biochemical assay which recorded 2 and 4 nM (IC50), respectively. Furthermore, this emerging selectivity is clearly driven by differing off-rates and optimisation around this parameter will not only be important in improving selectivity, but also in sustaining a high residency time of binding to TbNMT. The structure of TbNMT has not been solved to date. However, using L. major NMT (LmNMT) as a model system, the binding of DDD85646 into the peptide substrate binding site has been confirmed by X-ray crystallography (Fig. 5c, for stereo view see Supplementary Information, Figure 4). LmNMT has 74% overall sequence identity with TbNMT and 94% identity within the peptide-binding site (see Supplementary Information, Figure 5). The binding mode shows the piperazine interacting with the C-terminal carboxylate of NMT, via a tightly coordinated water molecule as opposed to a direct H-bond. The sulfonamide makes water bridged interactions with the highly conserved residue His219 and to the backbone NH group of Gly397. The geometry of the sulfonamide creates a significant bend in the structure, allowing the pyrazole to fit into a lipophilic pocket where it acts as a hydrogen bond acceptor from Ser330. The binding mode is of particular interest because all but the latter interactions are through water-mediated hydrogen bonds; a mode not readily predicted using computational techniques. Overlaying the structure of Saccharomyces cerevisiae NMT in complex with substrate peptide (PDB 1IID) 26 with the LmNMT complex shows that DDD85646 occupies the peptide binding site with the basic piperazine moiety mimicking the N-terminus of the substrate (see Supplementary Information, Figure 6).
We have presented evidence that our model TbNMT inhibitor, DDD85646, kills BSF T. brucei by acting on TbNMT in situ. The downstream consequences of TbNMT inhibition in the parasite are likely to be multiple, since the enzyme has over 60 putative substrates 6, and this no doubt explains the speed of killing and the dramatic morphological changes observed upon treatment with this compound. The emergence of the ‘BigEye’ phenotype, and the possible link to TbArf1 activity, suggests that disturbance of endocytosis is one mechanism by which TbNMT inhibitors act in BSF T. brucei. Interestingly, knockdown of TbNMT levels in BSF T. brucei by RNAi to 16% of wild-type levels, while fatal in vitro and in vivo, does not lead to the ‘BigEye’ phenotype but rather to the accumulation of vesicles close to the flagellar pocket 27.
It is notable that, despite the small window of selectivity between human NMT and TbNMT, DDD85646 shows promising selectivity at the cellular level. One might speculate that T. brucei cells are hyper-sensitive to NMT inhibition because of unique or unusual aspects of their biochemistry and/or cell biology. In this context, the extremely high endocytic rate of BSF T. brucei (some 9 times faster than fibroblasts and 2.6 times faster than macrophages), combined with the entire endocytic/exocytic process occurring in the flagellar pocket, is noteworthy 28. The parasite’s requirement for this high endocytic rate relates to its need to remove antibody from the cell surface and to recycle the protective VSG coat 29.
Finally, we may conclude that TbNMT is one of the few T. brucei proteins that have been comprehensively validated as a drug target for HAT. The TbNMT inhibitors described meet many requirements for a greatly needed new therapeutic for HAT. Further optimisation of this series towards improved CNS penetration and selectivity is currently underway. In the meantime DDD85646 will serve as an excellent chemical tool for investigation of the biology of protein N-myristoylation across a range of organisms.
This work was supported by grants from the Wellcome Trust (WT077705, WT083481, WT077503 and WT085622), Scottish Funding Council (HR04013) and by the Translational Biology Theme of SULSA. We thank the European Regional Development Fund and the Wolfson Foundation for grants that provided relevant infrastructure for this work. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. We would like to thank all members of the Drug Discovery Unit for their expert technical assistance in this study, particularly Bhavya Rao, Iain Collie and Daniel James.
Enzyme activity assays were performed in scintillation proximity format18, with minor modifications. Proliferation assays were performed using resazurin as an indicator of metabolic activity30. TbNMT was immobilised onto sensor chips (NTA) using standard immobilisation protocols and kinetics of binding of compound analysed on Biacore T-100. Details of all standard methods including compound exposure studies and efficacy studies, metabolic labelling, microscopy, and crystallography can be found in the Methods section of online versions. Chemical synthesis details can be found in supplementary materials.
Female NMRI mice (25-35 g; Harlan Laboratories; n=3 per dose group) were administered DDD85646 at 10 and 50 mg kg−1 orally. The dose solutions were prepared on the day of dosing and vehicle was 5% DMSO in deionised water or 5% DMSO, 40% PEG400 in deionised water respectively. Blood samples (10 μl) were collected from the tail vein of each animal into micronic tubes (Micronic BV) containing deionised water (20 μl) at 0.25, 0.5, 1, 2, 4, 6 and 8 h post-dose and stored at −80°C to await analysis.
Sample extraction of blood was performed by a method based on protein precipitation using acetonitrile and a structural analogue of the analyte of interest as internal standard. Blood extracts were analysed by UPLC/MS/MS using a Quattro Premier XE mass spectrometer (Waters). Calibration curves were constructed in blood to cover at least 3 orders of magnitude (i.e. 1-1000 ng ml−1).
DDD85646 was tested in vivo against Trypanosoma brucei brucei using a modification of the approach used by Thuita et al. 31. In brief, female NMRI mice (25-35 g, Harlan Laboratories; 3-5 per group) were infected intraperitoneally with 1 × 104 bloodstream forms of T. b. brucei S427 (221). These bloodstream forms come from a stock of cryopreserved stabilates containing 10% glycerol. The stabilate was suspended in phosphate-saline-glucose to obtain a trypanosome concentration of 5 × 104 ml−1. Each mouse was injected with 0.2 ml. DDD85646 was administered orally from day +3 to day +6 of the experiment and parasitaemia levels monitored up to day +30. Animals with parasitaemia > 108 ml−1 were humanely killed as previous studies had shown that animals do not survive a further 24 h 32. The day of euthanisation was recorded.
NMT assays were carried out at room temperature in 384-well white optiplates (Perkin Elmer). Each assay was performed in a 40 μl reaction volume containing 30 mM Tris, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 1.25 mM DTT, 0.1% (v/v) Triton X-100, 0.125 μM [3H]-myristoyl-coenzyme A (8 Ci mmol−1), 0.5 μM biotinylated CAP5.5, 5 nM NMT and various concentrations of test compound. The IC50 values for huNMT1 and huNMT2 were essentially identical against 80 compounds tested and, for logistical reasons, only huNMT1 was used in later studies.
Test compound (0.4 μl in DMSO) was transferred to all assay plates using a Cartesian Hummingbird (Genomics Solution) before 20 μl of enzyme was added to assay plates. The reaction was initiated with 20 μl of a substrate mix and stopped after 15 min (huNMT1 or 2) or 50 min (TbNMT) with 40 μl of a stop solution containing 0.2 M phosphoric acid, pH 4.0 and 1.5 M MgCl2 and 1 mg ml−1 PVT SPA beads (GE Healthcare). All reaction mix additions were carried out using a Thermo Scientific WellMate (Matrix). Plates were sealed and read on a TopCount NXT™ Microplate Scintillation and Luminescence Counter (Perkin Elmer).
ActivityBase from IDBS was used for data processing and analysis. All IC50 curve fitting was undertaken using XLFit version 4.2 from IDBS. A 4-parameter logistic dose response curve was utilised using XLFit 4.2 Model 205. All test compound curves had floating top and bottom and pre-fit was used for all 4 parameters.
Bloodstream T. b. brucei S427 were cultured at 37°C in modified HMI9 medium (56 μM 1-thioglycerol was substituted for 200 μM 2-mercaptoethanol) and quantified using a haemocytometer. For the live/dead assay, cells were analysed using a two colour cell viability assay (Invitrogen) as described previously 7. Routine screening of test compounds against parasites was performed in 96-well plates using a modification 30 of the cell viability assay previously described by Raz et al. 34. Cell culture plates were stamped with 1 μl of an appropriate concentration of test compound in DMSO followed by the addition of 200 μl trypanosome culture (104 cells ml−1) to each well, except for one column which received media only. MRC5 cells were cultured in Dulbecco’s Modified Eagle’s Medium, seeded at 2,000 cells per well and allowed to adhere overnight. One microlitre of test compound (10 point dilutions from 50 μM to 2 nM) was added to each well at the start of the assay. Culture plates of T. brucei and MRC5 cells were incubated at 37°C in an atmosphere of 5% CO2 for 69 h, prior to the addition of 20 μl resazurin (final concentration 50 μM). After a further 4 h incubation, fluorescence was measured (excitation 528 nm; emission 590 nm) using a BioTek flx800 plate reader.
The interaction between NMT and DDD85646 was assessed using a Biacore T100 instrument. Histidine-tagged TbNMT and huNMT1 were immobilised onto a NTA sensor chip (GE Healthcare) to levels of ~ 6000 response units (RU) using standard immobilisation protocols (GE Healthcare). Experiments were carried out at 25°C using HBS-P+ running buffer (GE Healthcare) containing 100 nM myristoyl-coenzyme A. DDD85646 binding was tested by injecting varying concentrations of inhibitor (0.05 nM to 1000 nM) at a flow rate of 30 μl min−1. Different concentrations of DDD85646 were prepared so that the final DMSO concentration was 0.1%. Kinetic data analysis was performed using Biacore T100 Evaluation Software 2.0.1 and fitted to the standard 1:1 interaction model to give results.
The plasmid vector pM2cN was a gift from David Horn and Sam Alsford (LSHTM, UK). The T. brucei NMT open reading frame was amplified from genomic DNA using suitable primers and cloned into the plasmid vector pM2cN. The resulting construct encodes the target protein with an N-terminal myc epitope tag under the control of a tetracycline-inducible T7 promoter. Mid-log phase T. brucei BSF ‘single marker’ (T7RPOL TETR NEO) S427 (subsequently termed SM) were electroporated with 10 μg of NotI-digested pM2cN-TbNMT using methods described 8. Expression of myc-tagged NMT was induced in stable cell lines by incubating parasites in 1 μg ml−1 tetracycline for 24 h. Immunoblotting 8 of parasite lysates was performed using the following primary antibodies: mouse anti-myc (Invitrogen, 1:1000 dilution), rabbit anti-TbNMT (1:500 dilution) and rabbit anti-BiP (gift from Jay Bangs, Department of Medical Microbiology and Immunology, Madison, WI, used at 1:10,000 dilution), mouse anti-alpha-tubulin, TAT1 (gift from Keith Gull, Dunn School, University of Oxford). Parental and NMT overexpressing cell lines (induced with tetracycline for 24 h) were seeded in 24-well plates at 5 × 105 ml−1 in triplicate, containing 0-100 nM of DDD85646. Motile cells were counted using a haemocytometer.
Production and transfection of the constructs pM2TbARF1-Q71L and pM2TbARF1-G2AQ71L have been described previously 7. Parental and mutant cell lines were seeded in 10 ml cultures at 5 × 105 ml−1. DDD85646 (10 nM) with or without tetracycline (1 μg ml−1) was added to cultures and flasks incubated at 37°C with 5% CO2 for 6 h. A set of cultures was set up in which tetracycline was added for 1 h before addition of DDD85646 and incubated for a further 5 h. Treated cells were then analysed using a Live/Dead two colour cell viability assay as described previously 7 and immunoblotted using anti-myc antibody 8.
T. brucei cells were harvested by centrifugation at 1,000 x g for 10 min and fixed in 1 pellet volume of 4% paraformaldehyde in 0.1 M piperazine-N,N’-bis(2-ethanesulfonic acid) for 30 min at room temperature. Fixed cells were spun at 13,000 g for 10 min, the supernatant was removed, and the pellet was resuspended in 1% aqueous osmium tetroxide (to fix and stain lipids), dehydrated, and set in Durcupan epoxy resin (Sigma). Sections were cut using a Leica Ultracut UCT system and analyzed using a Philips Tecnai 12 transmission electron microscopy instrument.
Live bloodstream form T. brucei (107) were incubated with and without 0.5 μM DDU85646 compound in 10 ml HMI-9 for 6 h at 37°C in a 5% CO2 incubator and subsequently labelled with 50 μCi ml−1 [3H]-myristic acid or 20 μCi ml−1 [35S]-methionine for 1 h at 37°C. The cells were washed in trypanosome dilution buffer and lysed in 1% TX-100 in 10 mM sodium phosphate buffer, pH 8 and incubated for 10 min at 37°C. Duplicate aliquots of lysates were run in 4-12% Nupage gels, stained with Coomassie blue and subsequently treated, or not, with 0.2 M NaOH in MeOH for 1 h at room temperature. The gels were washed and soaked in En3Hance, dried and exposed to film.
LmNMT protein was obtained from the Structural Genomics Consortium parasitic disease project (courtesy R. Hui, SGC Toronto). The protein construct consisted of 6xHis, TEV cleavage site, LmNMT 5-421 which was prepared in 10 mM HEPES pH 7.5, 500 mM NaCl at 9 mg/ml for crystallisation. A 200 mM stock solution of DDD85646 was prepared in DMSO and added to the protein solution to a final concentration 10 mM. Myristoyl CoA was also added to sample to a final concentration of 1 mM prior to crystallisation. Crystallisation was carried out using vapour diffusion methods at with 2 μl of protein:ligand complex mixed with 2 μl of reservoir solution. The reservoir solution contained 26% PEG 1500, 0.2 M NaCl, 0.1M Na Cacodylate pH 5.6. X-ray diffraction data for LmNMT + DDD85646 were measured at the European Synchrotron Facility (ESRF, Grenoble, France) at beamline ID14-1. The data were integrated and scaled using the HKL suite 35. The structure was solved by molecular replacement as implemented in MOLREP 36 using the native LmNMT coordinates (PDB 3H5Z) as a search model. The structure was refined using REFMAC5 37 and manual alteration of the model carried out using COOT 38. Ligand coordinates and topology files were created using the PRODRG server 39. Data collection and refinement statistics are presented in Supplementary Table 1.
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
The authors declare no competing financial interests.