The eukaryotic enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase) has been characterized in a range of species from Saccharomyces cerevisiae to Homo sapiens. NMT is essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans, and the parasitic protozoa Leishmania major and Trypanosoma brucei. We have purified the Leishmania and T. brucei NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and specific peptide substrates. A number of inhibitory compounds that target NMT in fungal species have been tested against the parasite enzymes in vitro and against live parasites in vivo. Two of these compounds inhibit TbNMT with IC50 values of <1 μM and are also active against mammalian parasite stages, with ED50 (the effective dose that allows 50% cell growth) values of 16–66 μM and low toxicity to murine macrophages. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against infectious diseases including African sleeping sickness and Nagana.

INTRODUCTION

Co-translational modification of proteins by N-myristoylation is a mechanism utilized by eukaryotes to facilitate membrane attachment and protein–protein interactions [1,2]. Catalysed by the enzyme NMT (myristoyl-CoA:protein N-myristoyltransferase), this process involves the covalent attachment of myristic acid to the N-terminal glycine residue of target proteins, which include signalling cascade components and structural proteins [2]. NMT catalysis proceeds via an ordered Bi Bi reaction mechanism [3]. Initial binding of myristoyl-CoA to the enzyme causes a conformational change, leading to the formation of a peptide-binding site and generation of a ternary myristoyl-CoA:NMT:peptide complex. Following catalysis, the N-myristoylated product is released. NMTs have been characterized in a range of eukaryotes, including Saccharomyces cerevisiae [4], Plasmodium falciparum [5] and human cells [6], and shown to be essential for viability in a number of human pathogens, including the fungi Candida albicans and Cryptococcus neoformans [7], and the parasitic protozoa Leishmania major and Trypanosoma brucei [8]. Comparative sequence and biochemical analyses have demonstrated high conservation of myristoyl-CoA-binding sites in human and fungal NMTs but divergent peptide binding specificities [2]. Given these observations, peptide-based and peptidomimetic inhibitors have been developed that show selectivity against the NMTs of pathogenic fungal species as compared with human NMT [911]. These results suggest that targeting NMT could be a valid approach for the development of chemo-therapeutics against a range of infectious diseases.

Parasitic kinetoplastid protozoa, including Trypanosoma and Leishmania species, are major causes of tropical infection worldwide (see http://www.who.int/tdr/index.html) and yet only a very limited number of effective drugs are available for use in areas of endemic disease. We have previously used gene targeting and RNAi (RNA interference) to demonstrate that NMT is essential for viability in L. major and T. brucei [8], suggesting that inhibition of NMT activity in these species might be a useful strategy for a drug development programme [12]. In the present study, we further characterize the NMTs of L. major and T. brucei and test a panel of compounds developed as fungal NMT inhibitors for their specificity and sensitivity against these parasite enzymes in vitro and in vivo. Our results suggest that targeting TbNMT (T. brucei NMT) may be effective in the development of anti-trypanocidal compounds.

METHODS

PCR amplification, cloning and expression

The TbNMT gene was amplified from genomic DNA using Pfu DNA polymerase (Promega) at 58 °C annealing temperature and the primers, TbNMTfor (5′-TTATTATCATATGACTGACAAAGCATTTACG-3′) and TbNMTrev (5′-ATTAGGATCCTTAAACCATCACAAGAC-3′) based on the gene sequence TRYP10.0.001826-6. The NdeI and BamHI sites used to clone the amplified fragment into the vector pET-15b (Novagen) are underlined (as are other restriction sites below). The resulting plasmid, pNMTtb, was transformed into Escherichia coli BL21(DE3) for expression. The LmNMT (L. major NMT) gene was amplified from pNMT [8] using primers LmNMTfor (5′-ATACGGATCCTGTCTCGCAATCCATCGAACTC-3′) and LmNMTrev (5′-AATACTCGAGCTACAGCATCACCAAGGCAACCT-3′) and Pfu DNA polymerase, as above.

The amplified fragment was digested with BamHI and XhoI and cloned into the pGEX-5X-1 vector (Amersham Biosciences). The resulting plasmid, pGNMT, was transformed into E. coli BL21(DE3) for expression. The pHASPA expression plasmid has been described previously [8].

The L. major HASPA gene was amplified from pHASPA using primers HAwtfor (5′-TACACCATGGGAAGCTCTTGCACGAAGGAC-3′) or HAG2Afor (5′-TACACCATGGCAAGCTCTTGCACGAAGGAC-3′) and HArev (5′-AATAAGGATCCCTAGTTGCCGGCAGCGT-3′). The amplified fragments were digested with the restriction enzymes NcoI and BamHI and ligated into pET28 vector (Novagen). The resulting plasmids, p28HASPA and p28HASPAm, express wild-type and mutant (G2A mutation) L. major HASPA (hydrophilic acylated surface protein A) protein with a C-terminal His tag when transformed into E. coli BL21(DE3).

Expression of N-terminally His6-tagged recombinant TbNMT was induced after growth at 37 °C to A600 (absorbance) of 0.6 by addition of IPTG (isopropyl β-D-thiogalactoside) to 1 mM final concentration. After a further 4 h growth at 30 °C, the bacterial cells were lysed in ice-cold buffer A (50 mM Tris/HCl, pH 7.4, and 300 mM NaCl) and centrifuged at 14000 g for 45 min to pellet insoluble material, prior to affinity chromatography using TALON™ beads (BD Biosciences). The His6-tagged protein was eluted with imidazole (75 mM imidazole in buffer A) and dialysed extensively to 50 mM Tris/HCl (pH 7.4), prior to Resource™ Q anion-exchange chromatography (Amersham Biosciences). Following gradient elution with 0–1 M NaCl, TbNMT protein was visualized using SDS/PAGE.

Recombinant LmNMT was expressed from pGNMT by addition of IPTG to 1 mM final concentration, following bacterial growth to A600 0.7 at 37 °C. After a further 6 h growth at 18 °C, the bacterial cells were lysed in ice-cold PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4 and 1.7 mM KH2PO4, pH 7.4). After centrifugation at 14000 g for 45 min, the soluble material was added to glutathione–Sepharose 4B beads and incubated at 4 °C for 16 h. The beads were subsequently washed extensively with PBS, followed by a final wash in buffer B (50 mM Tris/HCl, pH 8.0, 1 mM CaCl2 and 100 mM NaCl). Factor Xa (Amersham Biosciences) was added to the beads (10 cleavage units per mg of fusion protein) and incubated overnight at 4 °C.

After transfer to a PD-10 column (Amersham Biosciences), the flow through was collected and buffer B-exchanged prior to Resource™ Q anion-exchange chromatography and detection by SDS/PAGE. For immunoblotting, proteins were size-separated by SDS/PAGE and transferred on to nitrocellulose membrane (Millipore), prior to probing with anti-His antibody (1:2000; Santa Cruz Biotechnology). Immune complexes were detected using ECL® (Amersham Biosciences).

Functional co-expression of recombinant TbNMT in E. coli was carried out as described previously [8].

Parasite culture, immunofluorescence microscopy and in vivo inhibition studies

PCF (procyclic form) and BSF (bloodstream form) T. brucei parasite strains were maintained as described previously [13]. Immunoblotting was used to detect NMT expression in both parasite stages: cells were harvested by centrifugation (800 g for 10 min at 4 °C), washed with ice-cold PBS, resuspended in SDS loading buffer [50 mM Tris/HCl, pH 6.8, 100 mM DTT (dithiothreitol), 2% (w/v) SDS, 0.1% Bromophenol Blue and 10% glycerol], denatured for 5 min by boiling, and total proteins were separated by SDS/PAGE (10% gel), prior to immunoblotting with the cross-reactive antibody raised against LmNMT [8].

For cell fractionation, washed BSF T. brucei cells were lysed in 0.2 M Tris/HCl (pH 8.0), 6 mM MgCl2, 1 mM EDTA and 1 mM DTT plus complete protease inhibitors (Roche) by mechanical disruption, using 300 mm glass beads (Sigma). Undisrupted cells were cleared by centrifugation at 500 g for 10 min at 4 °C. The supernatant was then subjected to centrifugation at 100000 g (Beckman TLA 100 rotor) for 1 h at 4 °C for separation of membranes from cytosolic fractions. After precipitation of the proteins with trichloroacetic acid, both fractions were resuspended in SDS loading buffer, separated by SDS/PAGE and immunoblotted.

Indirect immunofluorescence assays were performed essentially as described in [13], using the rabbit primary antibody against LmNMT (1:50 dilution) and Alexa Fluor® 633-conjugated secondary antibody (Invitrogen). Samples were co-stained with DAPI (4′,6-diamidino-2-phenylindole) and visualized by confocal microscopy using a Zeiss LSM 510 meta, with a Plan-Apochromat ×63/1.4 Oil DIC I objective lens (where DIC is differential interference contrast). Images were acquired using LSM 510 version 3.2 software (Zeiss).

To test inhibitory compounds, 2 ml of BSF T. brucei (at 5×104 cells/ml) or L. major exponential-phase promastigotes (grown as described in [8]) were grown in 24-well flat-bottom plates in the presence and absence of test compounds, over a period of 72 h. Parasite numbers were counted at regular intervals using a haemocytometer. Alamar Blue® was also used as a colorimetric indicator of viability [14] following parasite incubation with test compounds for 24 h.

The concentration of inhibitor required to reduce parasite growth rate to 50% as compared with controls [ED50 (the effective dose that allows 50% cell growth)] was determined. The cytotoxicity of test compounds for murine macrophages (RAW264 cells, maintained at 2×105 cells/ml) was also determined using Alamar Blue®.

Potential NMT inhibitory compounds were provided by Pfizer (Sandwich, Kent, U.K.); structural information is provided in Table 2. UK-370485 is a benzothiazole-based compound and has been described previously [15].

In vitro activity assays

LmNMT and TbNMT were assayed using the synthetic octapeptides GQLFTSRR and GCGGSKVK respectively. GQLFTSRR is derived from the N-terminal sequence of L. major ARL1 (ADP-ribosylation factor related protein 1) (GQLFTSLN; LmjF04.0340; [13]) but modified at positions 7 and 8 (L7R, N8R) to generate positive charge at pH 7.3. Peptide GCGGSKVK is based on the N-terminal sequence of CAP5.5 (cytoskeleton-associated protein 5.5) [16]. The assay was modified from the method of King and Sharma [17]. In brief, reactions were run in a total volume of 50 μl containing [3H]myristoyl-CoA (Amersham Biosciences) and peptide, and initiated by the addition of TbNMT or LmNMT in 30 mM Tris (pH 7.4), 0.5 mM EDTA, 0.45 mM EGTA, 4.5 mM 2-mercaptoethanol and 1% Triton X-100. After 10 min incubation at 30 °C, the reaction was terminated by spotting 10 μl on P81 phosphocellulose paper (Whatman). For calculation of the Km for the ARL1 peptide, 5 ng of purified LmNMT protein was incubated with 5 μM [3H]myristoyl-CoA and 12.8–205 μM peptide. To determine the Km for myristoyl-CoA, the peptide concentration was maintained at 103 μM, while varying the myristoyl-CoA concentration (0.38–9.16 μM). The procedure was repeated for the TbNMT enzyme using CAP5.5 peptide at 0.32–80.0 μM. For in vitro inhibition assays, the NMT enzymes were incubated with 0.2 mM of compound before reaction initiation by the addition of peptide.

The IC50 values for compounds CP-014553 and CP-030890-27 with TbNMT were determined using the activity assay described above. Different concentrations of each compound were pre-incubated with the enzyme for 10 min at 30 °C before initiating the reaction.

Maximum velocities and Km values were obtained and fitted to the appropriate kinetic equation using the GraFit package (version 4.04; Erithacus Software; http://www.erithacus.com/grafit/index.htm).

SPA (scintillation proximity assay) for NMT activity

An SPA for NMT activity has been developed for inhibitor screening in 96-well plate format. Full details of the assay have been published elsewhere [17a] and are available from P. W. B. on request. Briefly, each 100 μl reaction contained equal volumes of inhibitor (or buffer alone), NMT, 500 nM [3H]myristoyl-CoA (8 Ci/mmol; Amersham Biosciences) and 500 nM biotinylated peptide substrate. All solutions were prepared using assay buffer (30 mM Tris, 0.5 mM EGTA, 0.5 mM EDTA, 2.5 mM DTT adjusted to pH 7.4 with HCl and 0.1% Triton X-100). The reactions were run for 30 min before analysis using streptavidin SPA beads (Amersham Biosciences) and scintillation counting using a Chameleon plate reader (Hidex, Finland).

IC50 values were determined for compounds CP-014553 and CP-005240 over the concentration range 25 μM–16.4 nM, and data were analysed using the GraFit package. The values quoted are the means derived from triplicate assays.

RESULTS

Expression and localization of TbNMT

The 1341 bp open reading frame of the single copy TbNMT gene codes for TbNMT, a 50.5 kDa protein of pI 6.2 that shares >50% amino acid identity with NMT from other trypanosomatid species [8], including those amino acids implicated in catalysis, e.g. the glutamic and leucine residues that form the floor of the active site pocket [18]. Because of this high relatedness, antiserum raised against LmNMT also recognizes TbNMT [8]. Hence this reagent could be used to determine the expression pattern of the T. brucei enzyme in vector PCFs and mammalian BSFs of the parasite. As might be predicted from RNAi analysis in both parasite stages [8], immunoblotting with anti-LmNMT (Figure 1A) revealed approximately equivalent levels of the 50.5 kDa TbNMT protein in both parasite stages relative to the endoplasmic reticulum marker protein, BiP (immunoglobulin heavy-chain binding protein) (which is expressed at approximately twice the level in BSF as in PCF; [19]). No other proteins were detected by anti-LmNMT in T. brucei lysates. Thus TbNMT is constitutively expressed and, most importantly, present in those parasites that cause disease in mammals.

TbNMT is constitutively expressed and partitions between membrane and cytosolic fractions

Figure 1
TbNMT is constitutively expressed and partitions between membrane and cytosolic fractions

(A) Total parasite lysates from BSF and PCF T. brucei were size-separated and immunoblotted using anti-NMT or anti-BiP antibodies (to monitor protein loading between lanes). (B) Lysed BSF were fractionated into cytosolic (C) and membrane (M) components, prior to size separation and immunoblotting with anti-NMT or anti-VSG antibodies (for detection of the major BSF lipid-anchored surface antigen). (C) Immunofluorescence microscopy of BSF and PCF T. brucei. Cells are shown as viewed under phase contrast, visualized for fluorescence with anti-LmNMT (red) and co-stained with DAPI (blue, to reveal positions of the nucleus, n, and kinetoplast, k). Scale bar, 5 μm.

Figure 1
TbNMT is constitutively expressed and partitions between membrane and cytosolic fractions

(A) Total parasite lysates from BSF and PCF T. brucei were size-separated and immunoblotted using anti-NMT or anti-BiP antibodies (to monitor protein loading between lanes). (B) Lysed BSF were fractionated into cytosolic (C) and membrane (M) components, prior to size separation and immunoblotting with anti-NMT or anti-VSG antibodies (for detection of the major BSF lipid-anchored surface antigen). (C) Immunofluorescence microscopy of BSF and PCF T. brucei. Cells are shown as viewed under phase contrast, visualized for fluorescence with anti-LmNMT (red) and co-stained with DAPI (blue, to reveal positions of the nucleus, n, and kinetoplast, k). Scale bar, 5 μm.

The LmNMT antiserum was also used to determine the cellular localization of TbNMT. Following cell lysis and fractionation, membrane and cytosolic fractions of BSF were analysed by immunoblotting (Figure 1B). Relative to VSG (variant surface glycoprotein), the major GPI (glycosylphosphatidylinositol)-anchored surface component specific to BSFs, NMT is distributed equally between membranes and the cytosol, an observation confirmed by immunofluorescence analysis (Figure 1C) and consistent with NMT distribution in Drosophila, Arabidopsis thaliana, human cells and Leishmania [8,2022], but not in S. cerevisiae, in which NMT is predominantly cytosolic [23]. In the micrographs in Figure 1(C), TbNMT fluorescence is excluded from the nucleus in both BSF and PCF parasites but is otherwise distributed throughout the cytoplasm with a punctate appearance, suggestive of partial localization to the endomembrane system and other intracellular sites. Expression levels are also similar in both cell types, in agreement with the immunoblots in Figure 1(A).

TbNMT is active in E. coli

A previously described bacterial co-expression assay system [24] was used to demonstrate the activity of TbNMT in vivo. Plasmids expressing TbNMT and the LmNMT substrate, HASPA, were co-transformed into E. coli, prior to induction of protein expression from both plasmids and radiolabelling to monitor N-myristoylation [8]. The [3H]myristate-radiolabelled products were size-separated by SDS/PAGE, followed by Coomassie Blue staining and autoradiography. As shown in Figure 2(A), radiolabelled proteins migrating at 50.5 and 17 kDa were specifically expressed in whole-cell lysates following induction of protein expression in the co-transformed bacteria (lane 4). These proteins were identified as TbNMT and HASPA respectively by immunoblotting (results not shown). The apo-TbNMT forms a high-affinity complex with myristoyl-CoA [3], explaining the presence of the radiolabelled 50.5 kDa TbNMT. The HASPA was N-myristoylated by the plasmid-encoded TbNMT, yielding a 17 kDa labelled product. Transformation with pHASPA alone (lane 6) gave high protein expression but no detectable radiolabelled product. Thus recombinant TbNMT is active in this heterologous in vivo bacterial system and is required for the N-myristoylation of HASPA. Expression of TbNMT in the absence of substrate in this assay resulted in N-myristoylation of an endogenous 25 kDa E. coli protein (lane 2), as observed previously [8]. This protein is also N-myristoylated in the presence of the HASPA substrate (lane 4), despite the difference in their expression levels, suggesting that the bacterial protein carries an N-myristoylation motif that is favourable for TbNMT.

Recombinant TbNMT requires a Gly-2 residue to myristoylate an L. major substrate in E. coli

Figure 2
Recombinant TbNMT requires a Gly-2 residue to myristoylate an L. major substrate in E. coli

(A) pNMTtb and pHASPA were transformed into E. coli, either together or separately, and recombinant proteins (50.5 and 17 kDa respectively) were expressed following induction with IPTG (lanes 2, 4 and 6) in the presence of [3H]myristate. The proteins were size-separated by SDS/PAGE (12% gel) and visualized using Coomassie Blue (upper panel). Three radiolabelled products were detected by autoradiography: the NMT:myristoyl-CoA binary complex (*), the myristoylated HASPA protein (arrowed in A and B) and a myristoylated endogenous E. coli protein (lower panel). (B) pNMTtb and p28HASPA/p28HASPAm were transformed into E. coli as in (A). Three radiolabelled products were detected with p28HASPA (expressing wild-type protein) as in (A) (lane 4). Only two products were detected in the presence of p28HASPAm (expressing the L. major G2A mutant HASPA protein substrate, Δ), the NMT:myristoyl-CoA binary complex and the endogenous E. coli protein (lower panel).

Figure 2
Recombinant TbNMT requires a Gly-2 residue to myristoylate an L. major substrate in E. coli

(A) pNMTtb and pHASPA were transformed into E. coli, either together or separately, and recombinant proteins (50.5 and 17 kDa respectively) were expressed following induction with IPTG (lanes 2, 4 and 6) in the presence of [3H]myristate. The proteins were size-separated by SDS/PAGE (12% gel) and visualized using Coomassie Blue (upper panel). Three radiolabelled products were detected by autoradiography: the NMT:myristoyl-CoA binary complex (*), the myristoylated HASPA protein (arrowed in A and B) and a myristoylated endogenous E. coli protein (lower panel). (B) pNMTtb and p28HASPA/p28HASPAm were transformed into E. coli as in (A). Three radiolabelled products were detected with p28HASPA (expressing wild-type protein) as in (A) (lane 4). Only two products were detected in the presence of p28HASPAm (expressing the L. major G2A mutant HASPA protein substrate, Δ), the NMT:myristoyl-CoA binary complex and the endogenous E. coli protein (lower panel).

The E. coli co-expression assay was also used to demonstrate that a glycine residue in position 2 of the HASPA substrate is essential for N-myristoylation to occur. Plasmids expressing wild-type and G2A L. major HASPA proteins (p28HASPA/p28HASPAm respectively) were co-transformed together with pNMTtb and [3H]myristate added to the cultures, prior to analysis of the radiolabelled products as described above. As shown in Figure 2(B), expression of the C-terminally His-tagged proteins was not detectable by staining but the presence of these proteins was verified by immunoblotting, using anti-His antibody (results not shown). Expression of wild-type HASPA in the presence of TbNMT and [3H]myristate resulted in the expected N-myristoylation of the 17 kDa HASPA (lower panel, lane 2) but expression of the G2A HASPA protein under the same conditions yielded no detectable radiolabelled product (lower panel, lane 4). Radiolabelled TbNMT:myristoyl-CoA binary complex was detected at equivalent levels with both protein substrates. These observations correlate with in vitro experimental results demonstrating that replacement of glycine with alanine at the N-terminus removes the ability of peptides to act as substrates for either S. cerevisiae or C. albicans NMT [25,26].

Purification and kinetic analyses of recombinant T. brucei and L. major N-myristoyltransferases

The LmNMT gene was cloned into pGEX-5X, for expression of LmNMT as a C-terminal fusion with the 26 kDa Schistosoma japonicum GST (glutathione S-transferase). The resulting plasmid, pGNMT, was transformed into E. coli BL21(DE3) and expression was induced by addition of IPTG. A fusion protein of the expected size (75 kDa) was detected by immunoblotting with anti-NMT antibody (Figure 3A, left panel). This GST–LmNMT protein was purified by affinity chromatography, and NMT was cleaved from the fusion protein using Factor Xa (Figure 3A, left panel, lane 3). The digestion products containing LmNMT were buffer-exchanged prior to anion-exchange chromatography and NaCl elution. LmNMT eluted from the column at 100 mM NaCl and was detected as a single band by SDS/PAGE (Figure 3A, right panel).

Purification of L. major and T. brucei recombinant NMTs

Figure 3
Purification of L. major and T. brucei recombinant NMTs

(A) Recombinant LmNMT was expressed as a GST–NMT fusion protein and purified by affinity chromatography with GST-affinity beads, followed by Factor Xa cleavage and anion-exchange chromatography. Left panel: GST–LmNMT, detected as a 75 kDa protein in SDS/PAGE separations of whole-cell lysates (Coomassie Blue staining, upper lanes 1 and 2) and immunoblotted with anti-NMT antibody (lower lanes 1 and 2). Lane 3, LmNMT (the lower band of 50.5 kDa band) following Factor Xa cleavage. Right panel: further purification of LmNMT by anion-exchange chromatography. Lane 1, starting material; lane 2, wash; lanes 3 and 4, elution fractions, stained by Coomassie Blue (upper panel) or immunoblotted with anti-NMT antibody (lower panel). (B) TbMT was expressed as a His-tagged fusion protein and purified by affinity chromatography, followed by anion-exchange chromatography as in (A). Left panel: soluble lysate of BL21(DE3) E. coli expressing TbNMT (Coomassie Blue staining, upper lanes 1 and 2) was applied to a TALON™ column and bound protein was eluted using an imidazole step gradient (upper lanes 3–6). Immunoblotting with anti-NMT antibody is shown below. Right panel: the imidazole fractions containing TbNMT were pooled and subjected to ion-exchange chromatography with NaCl elution to yield TbNMT purified to apparent homogeneity (E). M, protein markers.

Figure 3
Purification of L. major and T. brucei recombinant NMTs

(A) Recombinant LmNMT was expressed as a GST–NMT fusion protein and purified by affinity chromatography with GST-affinity beads, followed by Factor Xa cleavage and anion-exchange chromatography. Left panel: GST–LmNMT, detected as a 75 kDa protein in SDS/PAGE separations of whole-cell lysates (Coomassie Blue staining, upper lanes 1 and 2) and immunoblotted with anti-NMT antibody (lower lanes 1 and 2). Lane 3, LmNMT (the lower band of 50.5 kDa band) following Factor Xa cleavage. Right panel: further purification of LmNMT by anion-exchange chromatography. Lane 1, starting material; lane 2, wash; lanes 3 and 4, elution fractions, stained by Coomassie Blue (upper panel) or immunoblotted with anti-NMT antibody (lower panel). (B) TbMT was expressed as a His-tagged fusion protein and purified by affinity chromatography, followed by anion-exchange chromatography as in (A). Left panel: soluble lysate of BL21(DE3) E. coli expressing TbNMT (Coomassie Blue staining, upper lanes 1 and 2) was applied to a TALON™ column and bound protein was eluted using an imidazole step gradient (upper lanes 3–6). Immunoblotting with anti-NMT antibody is shown below. Right panel: the imidazole fractions containing TbNMT were pooled and subjected to ion-exchange chromatography with NaCl elution to yield TbNMT purified to apparent homogeneity (E). M, protein markers.

The TbNMT gene was amplified from genomic DNA and cloned into pET15b, for expression as an N-terminally His6-tagged protein. Following transformation of the plasmid into E. coli BL21(DE3), a 50 kDa protein was expressed after IPTG induction and this was recognized by anti-NMT antibody when immunoblotted (Figure 3B, left panel). The TbNMT was initially purified by TALON™ affinity beads (Figure 3B, left panel), prior to buffer exchange and anion-exchange chromatography. As with LmNMT, TbNMT was eluted from the column at 100 mM NaCl and was detected as a single band following SDS/PAGE (Figure 3B, right panel) consistent with a predicted molecular mass of 50.5 kDa.

Kinetic analyses of both recombinant parasite NMTs were carried out using a modified discontinuous assay to monitor the activities of the purified enzymes [17]. The Km, Vmax, kcat and catalytic efficiency (kcat/Km) values were obtained using octapeptide substrates specific for each enzyme (see the Methods section). Purified TbNMT and LmNMT showed similar affinities for myristoyl-CoA (Km=1.78 and 1.37 μM respectively) when calculated using CAP5.5 and ARL1 peptides respectively. These values are in the same order of magnitude as those calculated for human NMT (9.3 μM) and S. cerevisiae NMT (1.4 μM), when assayed with the substrate GNAAAARR [27]. For TbNMT, the octapeptide substrate demonstrated a Km of 11.3 μM, while for LmNMT, the ARL1 substrate showed a Km of 109 μM (Table 1).

Table 1
Peptide and myristoyl-CoA kinetic parameters for LbNMT and TbNMT

Kinetic analyses of LmNMT and TbNMT were carried out using a modified discontinuous assay [17]. The Km, Vmax, kcat and catalytic efficiency (kcat/Km) values were obtained using the octapeptide substrates, ARL1 and CAP5.5, specific for LmNMT and TbNMT respectively (see the Methods section).

LmNMTTbNMT
SubstratesKm (μM)Vmax (μM/s)kcat (s−1)kcat/Km (μM−1·s−1)Km (μM)Vmax (μM/s)kcat (s−1)kcat/Km (μM−1·s−1)
ARL1 110±33 0.060±0.006 24.00 0.219 – – – – 
CAP5.5 – – – – 11.3±1.41 0.036±0.0017 18.2 1.61 
Myristoyl-CoA 1.37±0.29 0.059±0.01 23.60 17.22 1.78±0.40 0.076±0.0059 76.8 43.2 
LmNMTTbNMT
SubstratesKm (μM)Vmax (μM/s)kcat (s−1)kcat/Km (μM−1·s−1)Km (μM)Vmax (μM/s)kcat (s−1)kcat/Km (μM−1·s−1)
ARL1 110±33 0.060±0.006 24.00 0.219 – – – – 
CAP5.5 – – – – 11.3±1.41 0.036±0.0017 18.2 1.61 
Myristoyl-CoA 1.37±0.29 0.059±0.01 23.60 17.22 1.78±0.40 0.076±0.0059 76.8 43.2 

In vitro inhibition of parasite NMT activities using antifungal compounds

Using the indirect discontinuous assay, a range of compounds developed as antifungal agents were tested for their inhibitory activity (at 0.2 mM) against purified LmNMT and TbNMT (Table 2). LmNMT activity appeared relatively insensitive to those compounds tested, while more varied effects were observed upon TbNMT activity. For example, as shown in Table 2, UK-370485-01 and UK-145974 did not inhibit TbNMT activity, while CP-030890-27 and CP-014553 were highly effective inhibitors in comparison (with IC50 values of 250 and 460 nM respectively). Notably, the last two compounds were also the only ones that showed any, although weak, inhibition of LmNMT activity (Table 2).

Table 2
In vitro inhibition of LmNMT and TbNMT

Compounds originally developed as antifungal agents were tested for their inhibitory activity at 0.2 mM concentration against purified LmNMT and TbNMT (columns 3–5) in the modified discontinuous assay. Two compounds were also tested against TbNMT using the SPA (columns 6 and 7). n.d., not determined.

1234567
CompoundStructureLmNMT activity at 0.2 mM (%)TbNMT activity at 0.2 mM (%)TbNMT IC50 (nM)TbNMT (SPA) activity at 0.05 mM (%)TbNMT IC50 (nM)
UK-370485-01 
graphic
 
99 104 n.d. 77 n.d. 
UK-145974 
graphic
 
101 102 n.d. 105 n.d. 
CP-030890-27 
graphic
 
60 250±40 n.d. n.d. 
CP-014553 
graphic
 
58 460±40 770±70 
CP-005240 
graphic
 
n.d. n.d. n.d. 740±80 
1234567
CompoundStructureLmNMT activity at 0.2 mM (%)TbNMT activity at 0.2 mM (%)TbNMT IC50 (nM)TbNMT (SPA) activity at 0.05 mM (%)TbNMT IC50 (nM)
UK-370485-01 
graphic
 
99 104 n.d. 77 n.d. 
UK-145974 
graphic
 
101 102 n.d. 105 n.d. 
CP-030890-27 
graphic
 
60 250±40 n.d. n.d. 
CP-014553 
graphic
 
58 460±40 770±70 
CP-005240 
graphic
 
n.d. n.d. n.d. 740±80 

Using the SPA with TbNMT, a similar level of inhibition was obtained for compound CP-014553 (IC50 770 nM), as well as with an additional compound, CP-005240 (IC50 740 nM; Table 2).

Antifungal compounds inhibit growth of T. brucei BSFs

All anti-fungal compounds were tested for their effect on cell growth and division against cultured T. brucei BSF, insect-stage L. major and murine macrophages (RAW267). The ED50 was determined for each compound, as was the SI (selectivity index) for T. brucei only (SI is the ratio of ED50 for RAW267 macrophages to ED50 for T. brucei BSF), as shown in Table 3. None of the compounds tested was effective against insect-stage L. major, including those that showed some inhibitory activity against the LmNMT in Table 2.

Table 3
Selective growth inhibition of BSF T. brucei and L. major promastigotes

Antifungal compounds described in Table 2 were tested for their effects on growth and division of cultured L. major promastigotes, BSF T. brucei and murine macrophages (see the Methods section). The ED50 values were determined for each compound. The results shown represent the means±S.E.M. obtained from triplicate measurements in two independent experiments. The SI, describing the ratio of ED50 for murine macrophages to ED50 for the parasite, was determined for T. brucei only. NE, not effective; MΦ, macrophage.

ED50 (μM)SI
CompoundL. majorT. bruceiMurine MΦMurine MΦ/T. brucei
UK-370485-01 NE 160±21 980±43 6.1 
CP-145974 NE 650±42 400±37 0.62 
CP-030890-27 NE 66±6 >1500 >23 
CP-014553 NE 16±3 328±27 21 
ED50 (μM)SI
CompoundL. majorT. bruceiMurine MΦMurine MΦ/T. brucei
UK-370485-01 NE 160±21 980±43 6.1 
CP-145974 NE 650±42 400±37 0.62 
CP-030890-27 NE 66±6 >1500 >23 
CP-014553 NE 16±3 328±27 21 

However, each of the compounds tested showed activity against T. brucei BSF, with the most potent compounds, CP-030890-27 and CP-014553, giving ED50 values of 66 and 16 μM respectively. In addition, CP-030890-27 was remarkably non-toxic to murine macrophages (ED50>1500 μM), whereas the SI of CP-014553 was among the highest measured. Growth curves for T. brucei incubated with and without these most effective compounds (CP-030890-27 and CP-014553) are shown in Figure 4. Under these conditions, a 50 μM final concentration of compound CP-014553 was lethal within 24 h, whereas CP-030890-27 was tolerated at a higher concentration over the same time period.

Effect of anti-NMT compounds on BSF T. brucei

Figure 4
Effect of anti-NMT compounds on BSF T. brucei

Parasites (1×105) were incubated in the presence or absence of compounds CP-014553 (A) and CP-030890-27 (B) over a concentration range and viable parasite numbers counted at the time points indicated, over a 72 h period. Data points were derived from one of three independent experiments and are expressed as ±S.E.M. for three determinations.

Figure 4
Effect of anti-NMT compounds on BSF T. brucei

Parasites (1×105) were incubated in the presence or absence of compounds CP-014553 (A) and CP-030890-27 (B) over a concentration range and viable parasite numbers counted at the time points indicated, over a 72 h period. Data points were derived from one of three independent experiments and are expressed as ±S.E.M. for three determinations.

DISCUSSION

Compounds that inhibit NMT activity have been studied intensively as potential antifungal agents, with a principal focus on the S. cerevisiae and C. albicans enzymes. Both in vitro and in vivo studies, using myristate and myristoyl-CoA analogues, histidine analogues, aminobenzothiazoles, quinolines and benzofurans to target enzyme activity, have demonstrated the potential for selective inhibition of these and other fungal NMTs [28]. This work has provided the framework for investigation of NMTs from protozoan parasites, with the aim of taking a ‘piggyback’ medicinal chemistry approach to the development of urgently needed antiparasitic agents [12]. We have previously shown that NMT activity is essential for viability in Leishmania and T. brucei parasites [8], suggesting that this enzyme may also be an exploitable drug target in these organisms. In the present study, we have expressed and purified recombinant LmNMT and TbNMTs and obtained kinetic parameters for substrates and myristoyl-CoA that correlate with those recorded for fungal NMTs. While the GST tag was removed from LmNMT for this analysis, the TbNMT His tag was not removed. His-tagged proteins have been used routinely in the development of NMT inhibitors [29,30]. Based on known crystal structures, in which His tags are almost always disordered, there is little evidence to suggest that their presence interferes with enzyme activity, a conclusion supported by studies with His-tagged and non-His-tagged C. albicans NMT ([30]; P. W. Bowyer, unpublished work).

In vitro studies using four antifungal compounds have demonstrated only weak inhibition of the L. major enzyme with these reagents, which also have little effect on parasite growth in vivo (results not shown). However, two of the compounds tested (CP-030890-27 and CP-014553) are able both effectively to inhibit TbNMT and also to inhibit the growth of mammalian stages of T. brucei in culture. These active compounds are characterized by fused ring systems with particularly oxygen-rich substituents providing opportunities for hydrogen bond formation.

They were identified following high-throughput screening for activity against fungal NMTs and are known to be active against Ca. albicans NMT (Pfizer and P. W. Bowyer, unpublished work) but, to our knowledge, are unlike any NMT inhibitory structures so far reported in the literature. These observations provide ‘proof-of-concept’ for this experimental approach and suggest that the compounds may represent viable leads for future exploitation.

Protein N-myristoylation is a two-substrate reaction in which a high-affinity complex is formed between NMT and myristoylCoA in the absence of substrate peptide. Upon subsequent peptide binding, reaction occurs followed by the release of free CoA and the myristoylated peptide. In the experiments described here, the Km values for both parasite NMTs with myristoyl-CoA were in the same range as those previously recorded for both the S. cerevisiae enzyme and human NMT1 [27]. The peptides used to determine the Km values for the parasite enzymes were derived from known N-myristoylated protein substrates in these species: for LmNMT, an ARL1-related protein [13], and for TbNMT, a cytoskeletal protein of the calpain family, CAP5.5 [16]. The eight N-terminal residues of a peptide or polypeptide substrate appear to be a minimal requirement for binding to the peptide-binding site, and those substrates that have been used to determine Km values for NMTs vary greatly in their sequence and length [27]. For example, a mutated version of the N-terminal peptide of the catalytic subunit cAMP-dependent protein kinase (GNAAAARR) has been used to obtain the Km for S. cerevisiae, C. albicans and bovine NMT, yielding 30, 600 and 250 μM respectively [31]. Using substrate-specific octapeptide substrates for TbNMT and LmNMT, the Km values reported here, 11.3 and 109 μM respectively, are comparable. This suggests that the peptide-binding site in these parasitic enzymes shares similar recognition features for binding the N-terminal eight residues of peptide substrates as observed for other eukaryotic NMTs [27].

Using the previously described in vitro activity assay [17] to test the inhibitory effect of a small number of anti-NMT compounds (Pfizer), we found that none of the compounds tested was effective in targeting LmNMT enzyme activity. In contrast, two compounds from the same group were able to strongly inhibit TbNMT enzyme activity when used at 0.2 mM. The IC50 values for these reagents were 0.25 μM for CP-030890-27 and 0.46 μM for CP-014553. These compare favourably with IC50 values generated in a screen of non-peptidic and peptidomimetic compounds against Ca. albicans NMT [32,33], which were in the range of 0.2–2 μM or less. Initial data using the SPA correlated well with that achieved using the indirect discontinuous assay.

All five of the anti-NMT compounds were also evaluated for their antiparasitic effect against T. brucei BSF. Strikingly, the compounds that most effectively inhibited TbNMT in vitro were also the most toxic against bloodstream trypanosomes, with ED50 values of 66 and 16 μM (for CP-030890-27 and CP-014553 respectively). These values are comparable with those measured with other putative anti-T. brucei compounds such as the ‘first generation’ farnesyltransferase inhibitors, which have generated ED50 values of 2–50 μM [34]. More recent peptidomimetic farnesyltransferase inhibitors are 1000-fold more effective, with ED50 values of 5–20 nM [35]. Nonetheless, two lead compounds worthy of further investigation have been identified in the present study, on the basis of their inhibitory effect on TbNMT in vitro, as well as their inhibitory effect on parasite viability in vivo. Both of these show specificity for T. brucei, do not inhibit LmNMT and are non-toxic to cultured murine macrophages. These results suggest that inhibition of parasite NMTs may provide another avenue for the development of inhibitory compounds and justify the extension of this work to high-throughput screening and testing against human-infective T. brucei species and the intracellular stage of Leishmania.

We thank Tanya Parkinson, Andy Bell and colleagues at Pfizer for provision of compounds, technical advice and helpful discussions, and Jay Bangs (Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, U.S.A.) and Mark Field (Department of Pathology, University of Cambridge, Cambridge, U.K.) for antibodies. This work was funded by the Wellcome Trust (061343, programme grant to D.F.S.; 065514, studentship to P.W.B.) and the U.K. Biological and Biotechnological Scientific Research Council (studentship to C.P.).

Abbreviations

     
  • ARL1

    ADP-ribosylation factor related protein 1

  •  
  • BiP

    immunoglobulin heavy-chain binding protein

  •  
  • BSF

    bloodstream form

  •  
  • CAP5.5

    cytoskeleton-associated protein 5.5

  •  
  • DAPI

    4′,6-diamidino-2-phenylindole

  •  
  • DTT

    dithiothreitol

  •  
  • ED50

    the effective dose that allows 50% cell growth

  •  
  • GST

    glutathione S-transferase

  •  
  • HASPA

    hydrophilic acylated surface protein A

  •  
  • IPTG

    isopropyl β-D-thiogalactoside

  •  
  • NMT

    myristoyl-CoA:protein N-myristoyltransferase

  •  
  • LmNMT

    Leishmania major NMT

  •  
  • PCF

    procyclic form

  •  
  • RNAi

    RNA interference

  •  
  • SI

    selectivity index

  •  
  • SPA

    scintillation proximity assay

  •  
  • TbNMT

    Trypanosoma brucei NMT

  •  
  • VSG

    variant surface glycoprotein

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Author notes

1

Present address: Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, London SW3 6JB, U.K.