Figure 1.
Reaction catalyzed by Myristoyl CoA:protein N-myristoyltransferase (NMT).
NMT catalyzes the covalent attachment of fatty acid myristate to the N-terminal glycine of a subset of proteins. The fatty acid moiety is provided by myristoyl-CoA, which binds to the apo-enzyme first, forming a binary complex. This complex then binds the substrate protein and the reaction occurs. Subsequently, CoA is released, followed by release of the myristoylated protein product [17].
Figure 2.
Evidence for the presence of N-myristoyltransferase (NMT) in nematodes and conservation of NMT among prokaryotes and eukaryotes.
Rooted phylogenetic tree analysis of NMT and various homologs. Deduced open reading frames of NMTs were used to generate the phylogram. Branch length was generated using ClustalW (http://www.genome.jp/tools/clustalw/). The percentage of amino acid identity and similarity between B. malayi NMT and orthologs are shown.
Figure 3.
Modeling the structure of B. malayi NMT and structural comparison with Leishmania major NMT.
(A) Ribbon diagram of the predicted crystal structure of NMT from B. malayi. (B) Comparison of B. malayi NMT (tan) with L. major NMT (blue: 2wsa). An overlay of the structure of these enzymes using UCSF Chimera [26] reveals a nearly identical conformation of the binding sites for myristoyl-CoA (yellow) and inhibitor DDD85646 (magenta). The 2 small helixes (arrow) formed by an insertion of 21 amino acids in L. major NMT are replaced with a loop in B. malayi NMT.
Figure 4.
Biochemical analysis of recombinant C. elegans NMT and B. malayi NMT.
Purified recombinant B. malayi NMT and C. elegans NMT enzymes myristoylate several synthetic peptide substrates (ARL-1 ADP ribosylation factor related protein; ABL-1 tyrosine kinase and SRC-1 tyrosine kinase). No activity was detected in the absence of peptide. Enzyme activity is expressed as radioactivity in disintegrations per minute (DPM).
Figure 5.
Structures of the NMT inhibitors, DDD85646 and DDD100870.
Figure 6.
Inhibition of C. elegans (A) and B. malayi (B) NMTs using DDD85646 and DDD100870.
Enzyme mixtures were preincubated with various concentrations of each compound (0.0025–1 µM). Reactions performed in the absence of inhibitor (positive control) or peptide (negative control), were included. Background radioactivity values generated from ‘non-peptide’ control samples were subtracted from the values obtained from experimental samples. Percent activity relative to the positive control ± propagation of error (http://laffers.net/blog/2010/11/15/error-propagation-calculator/) was determined. Assays were performed in triplicate.
Figure 7.
NMT is essential in C. elegans.
(A) Phenotypic analyses were performed using the mutant strain nmt-1(tm796)/hT2[qla48]. Predicted genomic organization of CeNMT and location of the deletion are shown. Boxes and lines denote exons and introns respectively. To verify the deletion genomic DNA was prepared from a single mutant animal “−/−”, and one worm from heterozygous “+/−” and wild-type “+/+” strains using standard methods. (B) Primers were designed to flank the deletion site and the deletion was confirmed on the basis of the change in size of a PCR product. Bands of the expected sizes were obtained. (C) RNAi knockdown of NMT in C. elegans. Three C. elegans strains were used for RNAi knockdown of NMT: C. elegans wild-type and two RNAi sensitive C. elegans strains, one containing a mutation in rrf-3, and a second strain carrying mutations in both eri-1 and lin-15B. RNAi was performed by feeding worms E. coli expressing dsRNA corresponding to NMT, or pL4440 plasmid vector without CeNMT.
Figure 8.
Effect of NMT inhibitors on C. elegans growth.
Wild-type C. elegans were grown on NGM plates seeded with OP50 E. coli. Compound screening (DDD85646 and DDD100870) commenced with L4-staged worms placed into a well of a sterile 96-well micro titer plate (Falcon 3072) containing a 100 µL suspension of previously frozen HB101 E. coli bacteria in S medium. Various concentrations (25, 50 or 100 µM) of compound or DMSO (control) were then added. Plates were maintained at 20°C in a humidity chamber for 7 days. Worm growth and development was scored daily by measuring a decrease in OD600 nm resulting from consumption of E. coli, (A) and by microscopic examination of the number of F1 progeny produced and size of worms treated with DDD100870 (B). For each condition, 10 L4-stage worms were used, and the average ± standard deviation was plotted.
Figure 9.
Effect of NMT inhibitors on B. malayi in vitro.
Living B. malayi worms including adult female (A), adult male (B) and microfilariae (C) were exposed to NMT inhibitors for 7 days. Compounds DDD85646 (orange) or DDD100870 (blue) at 100 µM (circle) or 50 µM (square) or 1% DMSO (grey) were added to adult worm culture. Microfilariae (mf) were cultured in the presence of compounds at a final concentration of 100 µM (circle), 50 µM (square), 25 µM (diamond) or 12.5 µM (triangle). Experiments were performed in triplicate. The culture media were replaced each day with fresh media containing compound or DMSO (grey) at corresponding concentrations. Parasite motility was video-recorded daily and observations expressed as a percentage of the motility relative to the motility scored on day 0 of the experiment. Production of microfilaria from female worms (D) was assessed on days 1, 3 and 5. The data obtained from triplicate samples are expressed as mean ± standard deviation.
Table 1.
Listing of predicted B. malayi NMT substrates.