Structure of Plasmodium vivax N-myristoyltransferase with inhibitor IMP-1088: exploring an NMT inhibitor for antimalarial therapy

This article is part of a focused issue on empowering education through structural genomics.

Plasmodium vivax, a significant contributor to global malaria cases, poses an escalating health burden on a substantial portion of the world's population. The increasing spread of P. vivax because of climate change underscores the development of new and rational drug-discovery approaches. The Seattle Structural Genomics Center for Infectious Diseases is taking a structure-based approach by investigating essential enzymes such as N-myristoyltransferase (NMT). P. vivax N-myristoyltransferase (PvNMT) is a promising target for the development of novel malaria treatments unlike current drugs, which target only the erythrocytic stages of the parasite. Here, the 1.8 Å resolution ternary structure of PvNMT in complex with myristoyl-CoA and IMP-1088, a validated NMT inhibitor, is reported. IMP-1088 is a validated nonpeptidic inhibitor and a ternary complex structure with human NMT has previously been reported. IMP-1088 binds similarly to PvNMT as to human NMT.

1. Introduction

Plasmodium vivax is responsible for the most widespread form of malaria and approximately 2.5 billion people, or over one-third of the world's population, are at risk of P. vivax infection (Battle et al., 2019). In humans, P. vivax can enter a dormant liver phase, which allows it to survive in various climates, including tropical and temperate regions, and contributes to its extensive geographical prevalence (Battle et al., 2019). P. vivax infection significantly impacts the quality of life of infected individuals, causing cyclical episodes of fever and weakness, representing a substantial burden in endemic countries due to treatment costs and productivity loss. P. vivax can persist in human hosts as hypnozoites in the liver that can cause relapses that can extend over several months or years (Flannery et al., 2022). Curing vivax malaria requires antimalarial drugs that are effective against both the blood and liver stages. Unfortunately, the sole licensed antimalarial with P. vivax activity, primaquine, has the drawback of inducing severe hemolysis in those with glucose-6-phosphate dehydrogenase (G6PD) deficiency, representing approximately 15% of the population in P. vivax endemic regions (Douglas et al., 2023).

The Seattle Structural Genomics Center for Infectious Diseases and collaborators are investigating rational malaria therapeutics discovery targeting essential proteins (Vijayan et al., 2021).

These efforts identified P. vivax N-myristoyltransferase (PvNMT) inhibitors that overcome drug resistance (Schlott et al., 2019). PvNMT is an essential enzyme that catalyzes a post-translational modification (myristoylation) through transfer of the lipid myristate from myristoyl coenzyme A (Myr-CoA) to the N-terminal glycine residues of proteins (Selvakumar et al., 2011; Udenwobele et al., 2017; McIlhinney, 1989). PvNMT catalyzes the myristoylation of substrate proteins that modulate crucial parasite cellular processes such as membrane association, protein–protein interactions, stability, turnover and signal transduction (Schlott et al., 2021; Selvakumar et al., 2011). Examples of plasmodial proteins that are myristoylated by NMT include glideosome-associated protein 45 (GAP45), which cannot perform its erythrocyte-invasion roles unless it is myristoylated (Schlott et al., 2021). Myristoylation of erythrocyte-binding antigen 175 (EBA-175) is required for P. vivax to invade erythrocytes (Bouyssou et al., 2023). Plasmodial exported protein 1 (EXP-1) and early transcribed membrane protein 11.2 (ETMP-11.2) must be myristoylated for parasites to exit the red blood cell (Cheng et al., 2015). Consequently, PvNMT inhibition significantly affects parasite development and survival (Garcia et al., 2022; Nicolau et al., 2023; Rodríguez-Hernández et al., 2023). Plasmodial adenylate kinases 2 are liver-stage proteins that must be myristoylated (Rodríguez-Hernández et al., 2023).

NMTs have been validated as targets for multiple parasitic diseases, including trypanosomiasis and leishmaniases (Corpas-Lopez et al., 2019; Wright et al., 2014; Frearson et al., 2010; Rodríguez-Hernández et al., 2023; Harupa et al., 2020). NMTs are promising drug targets for malaria and other diseases (Priyamvada et al., 2022; Garcia et al., 2022; Goncalves et al., 2017; Javid et al., 2023; Rackham et al., 2014; Rodríguez-Hernández et al., 2023; Bolling et al., 2024; Bell et al., 2012, 2020, 2022). The first reported family of NMT inhibitors was developed through rational design strategies utilizing peptide-mimicking substrates or nonhydrolyzable Myr-CoA analogs. Subsequently, novel families of NMT inhibitors have been identified through high-throughput screening (HTS) efforts (Goncalves et al., 2017).

IMP-1088 is an effective antipicornaviral agent with selectivity and pharmacological activity against NMT (Mousnier et al., 2018; Wright et al., 2014). IMP-1088 also effectively inhibits the production of infectious rhinovirus virions by blocking the N-myristoylation of rhinovirus VP0 (Mousnier et al., 2018). Other IMP-1088 chemotypes have been developed against NMT to treat multiple diseases, with recent efforts focusing on the development of novel PvNMT inhibitors as antimalarials (Bell et al., 2012; Rodríguez-Hernández et al., 2023; Schlott et al., 2018). Here, we present the structure of IMP-1088 in complex with PvNMT. Comparing the reported structure with that of human NMT in complex with IMP-1088 (PDB entry 5mu6; Mousnier et al., 2018) offers insights into repurposing this family of compounds as antimalarials.

2. Materials and methods

2.1. Macromolecule production

A codon-optimized gene (PvNMT; UniProt A0A1G4H3M1), encoding amino acids 27–410, was synthesized by GenScript with a 3C protease-cleavable hexahistidine tag (MGSSHHHHHHSAALEVLFQGP-ORF). Plasmid DNA was transformed into chemically competent Escherichia coli BL21(DE3) cells (Table 1). The plasmid containing His-PvNMT was tested for expression, and 2 l of culture was grown using auto-induction medium (Studier, 2005) in a LEX Bio­reactor (Epiphyte Three) as described previously (Serbzhinskiy et al., 2015). The expression clone can be requested online at https://www.ssgcid.org/available-materials/expression-clones/.

Table 1
Macromolecule-production information

Source organism	Plasmodium vivax Salvador I
DNA source	Synthetic, GenScript
Cloning vector	pET-11a
Expression vector	PCR-amplified plasmid DNA
Expression host	Escherichia coli BL21(DE3)R3 Rosetta
Complete amino-acid sequence of the construct produced	MGSSHHHHHHSAALEVLFQGPDYKFWYTQPVPKINDEFNESVNEPFISDNKVEDVRKDEYKLPPGYSWYVCDVKDEKDRSEIYTLLTDNYVEDDDNIFRFNYSAEFLLWALTSPNYLKTWHIGVKYDASNKLIGFISAIPTDICIHKRTIKMAEVNFLCVHKTLRSKRLAPVLIKEITRRINLENIWQAIYTAGVYLPKPVSDARYYHRSINVKKLIEIGFSSLNSRLTMSRAIKLYRVEDTLNIKNMRLMKKKDVEGVHKLLGSYLEQFNLYAVFTKEEIAHWFLPIENVIYTYVNEENGKIKDMISFYSLPSQILGNDKYSTLNAAYSFYNVTTTATFKQLMQDAILLAKRNNFDVFNALEVMQNKSVFEDLKFGEGDGSLKYYLYNWKCASFAPAHVGIVLL

PvNMT was purified in two steps: an immobilized metal (Ni²⁺) affinity chromatography (IMAC) step and size-exclusion chromatography (SEC) on an AKTApurifier 10 (GE Healthcare, now Cytiva) using automated IMAC and SEC programs (Serbzhinskiy et al., 2015). Briefly, thawed bacterial pellets (25 g) were lysed by sonication in 200 ml lysis buffer [25 mM HEPES pH 7.0, 500 mM NaCl, 5%(v/v) glycerol, 0.5%(w/v) CHAPS, 30 mM imidazole, 10 mM MgCl₂, 1 mM TCEP and five tablets of protease-inhibitor cocktail (cOmplete Mini, EDTA-free Roche, Basel, Switzerland)]. After sonication, the crude lysate was treated with 20 µl (25 U ml⁻¹) of Benzonase by incubating and mixing at room temperature for 45 min. The lysate was clarified by centrifugation at 5000g for 1 h at 277 K using a refrigerated Sorvall centrifuge (Thermo Scientific). The clarified supernatant was then passed over a 5 ml Ni–NTA HisTrap FF column (GE Healthcare, now Cytiva) which had been pre-equilibrated with loading buffer [25 mM HEPES pH 7.0, 500 mM NaCl, 5%(v/v) glycerol, 30 mM imidazole, 1 mM TCEP, 0.025%(w/v) sodium azide]. The column was washed with 20 column volumes (CV) of loading buffer and eluted with elution buffer [25 mM HEPES pH 7.0, 500 mM NaCl, 5%(v/v) glycerol, 30 mM imidazole, 1 mM TCEP, 0.025%(w/v) sodium azide, 250 mM imidazole] over a 7 CV linear gradient. Peak fractions were pooled, concentrated to 5 ml and loaded onto a Superdex 75 26/60 column (GE Biosciences) equilibrated with running buffer (20 mM HEPES pH 7.0, 300 mM NaCl, 5% glycerol, 1 mM TCEP). PvNMT eluted from the SEC column as a single, monodisperse symmetrical peak accounting for >90% of the protein product at a molecular mass of ∼40 kDa, suggesting purification as a monomer (based on a theoretical molecular weight of 47.1 kDa). The pure peak fractions were pooled and concentrated to 13.5 mg ml⁻¹ using an Amicon purification system (Millipore). The purified protein was stored in 100 µl aliquots at 193 K and can be requested online at https://www.ssgcid.org/available-materials/ssgcid-proteins/.

2.2. Crystallization

PvNMT was crystallized using sitting-drop vapor diffusion as described in Table 2. Crystals were harvested and cryoprotected with 20% ethylene glycol before data collection.

Table 2
Crystallization

Method	Vapor diffusion, sitting drop
Plate type	Tray 101-d6, 96-well plates
Temperature (K)	290
Protein concentration (mg ml^−1)	14.88
Buffer composition of protein solution	20 mM HEPES pH 7.0, 300 mM NaCl, 5%(v/v) glycerol, 1 mM TCEP, 0.5 mM IMP-1088, 0.5 mM Myr-CoA
Composition of reservoir solution	27% PEG 3350, 200 mM ammonium sulfate, 100 mM bis-Tris pH 6.0
Volume and ratio of drop	0.4 µl, 1:1
Volume of reservoir (µl)	80

2.3. Data collection and processing

Data were collected at 100 K as detailed in Table 3. Data were integrated using XDS (Kabsch, 2010) and reduced with XSCALE (Kabsch, 2010).

Table 3
Data collection and processing

Values in parentheses are for the outer shell.

Diffraction source	Beamline 08ID-1, Canadian Light Source
Wavelength (Å)	0.97949
Temperature (K)	100
Detector	Rayonix MX-300 CCD
Space group	P212121
a, b, c (Å)	57.32, 119.13, 176.61
α, β, γ (°)	90, 90, 90
Resolution range (Å)	50–1.80 (1.85–1.80)
No. of unique reflections	112736
Completeness (%)	99.9 (99.4)
Multiplicity	7.2 (6.2)
〈I/σ(I)〉	15.21 (2.98)
Rr.i.m.	0.115 (0.562)
Overall B factor from Wilson plot (Å^2)	12.320

2.4. Structure solution and refinement

The structure was determined by molecular replacement with MOLREP from the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994; Krissinel et al., 2004; Winn et al., 2011; Agirre et al., 2023) using PDB entry 4b14 (with inhibitors and waters removed) as the search model (Yu et al., 2012). The structure was refined using Phenix (Liebschner et al., 2019). The refined structure quality was assessed using MolProbity (Williams et al., 2018). Refinement statistics are listed in Table 4. The coordinates and structure factors have been deposited with the Worldwide Protein Data Bank (wwPDB) as PDB entry 5v0w. Omit electron-density maps reveal ordered electron density for all of the ligands (Supplementary Fig. S1). The ligands and waters were also checked with the CheckMyBlob server (Kowiel et al., 2019; https://checkmyblob.bioreproducibility.org/server/).

Table 4
Structure solution and refinement

Values in parentheses are for the outer shell.

Resolution range (Å)	50.0–1.80 (1.84–1.80)
Completeness (%)	99.9
σ Cutoff	F > 1.35σ(F)
No. of reflections, working set	112717 (7250)
No. of reflections, test set	2031 (119)
Final Rcryst	0.147 (0.227)
Final Rfree	0.184 (0.292)
No. of non-H atoms
Protein	9405
Ion	60
Ligand	288
Solvent	1697
Total	11450
R.m.s. deviations
Bond lengths (Å)	0.007
Angles (°)	0.935
Average B factors (Å^2)
Protein	14.5
Ion	53.4
Ligand (IMP-1088)	15.5
Ligand (myristoyl-CoA)	17.5
Water	27.8
Ramachandran plot
Most favored (%)	97
Allowed (%)	3

3. Results and discussion

The ternary structure of HsNMT1 bound to Myr-CoA and IMP-1088 was previously reported as PDB entry 5mu6 (Mousnier et al., 2018). Our reported ternary complex of PvNMT, Myr-CoA and IMP-1088 allows structure–function comparison of host and parasite inhibition by the same non­peptidic inhibitor. The ternary complex of PvNMT, Myr-CoA and the nonpeptidic inhibitor IMP-1088 was determined at a resolution of 1.8 Å (Table 3). The asymmetric unit contains three monomers (Fig. 1a). The three almost identical monomers adopt the prototypical NMT topology (Dian et al., 2020), with a compact, spherical configuration comprising 15 α-helices and 19 β-sheets (Figs. 1a and 1b). Two monomers (chains A and B) have 385 amino-acid residues (residues 26–10) and the third (chain C) has 377 residues.

Figure 1 Ternary structure of PvNMT with Myr-CoA and the inhibitor IMP-1088. (a) There are three PvNMT monomers in the asymmetric unit: A (marine), B (gray) and C (light brown). Each has a bound Myr-CoA (magenta) and IMP-1088 inhibitor (green). (b) Superposed monomers are almost identical, with an r.m.s.d. of ∼0.10 Å on C^α atoms. Each monomer is colored in a rainbow from blue at the N-terminus to red at the C-terminus. Myr-CoA is shown as magenta sticks, while the inhibitor IMP-1088 is shown as green sticks. (c) Superposed monomers of PvNMT (PDB entry 5v0w, gray), HsNMT1 (PDB entry 5mu6, pink) and HsNMT2 (PDB entry 4c2x, cyan). Myr-CoA is shown as magenta sticks, while the inhibitor IMP-1088 is shown as green sticks. (d) Solvent-accessible surface area of PvNMT colored by sequence conservation, with red indicating identical residues. The peptide/substrate-binding and myristoyl-binding cavities are shown in black parentheses.

Each monomer has two N-terminal binding cavities: the peptide/substrate-binding cavity containing the inhibitor IMP-1088 and the myristoyl-binding cavity containing Myr-CoA (Fig. 1b). Consistent with other PvNMT structures, a central core with an internal pseudo-twofold symmetry axis formed by the N-terminal and C-terminal halves shapes the structure of the peptide-binding site (Goncalves et al., 2017; Rodríguez-Hernández et al., 2023; Rudnick et al., 1993; Spassov et al., 2023; Bolling et al., 2024). All loops that are near or interacting with both binding cavities are ordered in all three monomers, notably the ab loop, which forms a lid that embraces the inhibitor within the peptide/substrate-binding cavity (Fig. 1d).

The top 82 closest structural neighbors of the reported structure were identified by PDBeFold (https://www.ebi.ac.uk/msd-srv/ssm/) analysis (Krissinel & Henrick, 2004) using a default threshold of 70% to be PvNMT structures with various ligands. The next 63 are human NMT structures, followed by NMTs from other organisms (Supplementary Table S1). ENDScript analyses (Gouet et al., 2003; Robert & Gouet, 2014) validate the PDBeFold results and reveal well conserved amino acids across the different NMTs (Supplementary Fig. S2). Structural and primary-sequence alignment reveals significant secondary-structure similarity between human and plasmodial NMTs (Fig. 2). Superposed ribbons also show the similarity in tertiary structure of human and plasmodial NMTs (Fig. 1c). A surface diagram of PvNMT reveals that the regions with the highest similarity are near the interconnected Myr-CoA-binding and peptide-binding cavities, as shown in red in Fig. 1(d). Notably, the myristoyl-binding cavity is well conserved across NMTs (Fig. 1d). Myr-CoA binding is stabilized by a few positive charges in the mainly hydrophobic myristoyl-binding cavity (Harupa et al., 2020; Rodríguez-Hernández et al., 2023; Bolling et al., 2024). LigPlus analysis (Laskowski & Swindells, 2011; Wallace et al., 1995) shows that the amino acids interacting with Myr-CoA are almost identical in PvNMT (PDB entry 5v0w) compared with HsNMT1 (PDB entry 5mu6) and HsNMT2 (PDB entry 4c2x) (Fig. 3).

Figure 2 (a) The alignment shows residue conservation between NMTs from different organisms. Residues within the cofactor-binding pocket are shown in purple, while those in the substrate-binding pocket are shown in green. The secondary-structure alignment is based on our reported structure. (b) The residues located in the C-termini of different NMTs. The following NMTs are included in the alignment: PvNMT, P. falciparum NMT, HsNMT1, HsNMT2, Saccharomyces cerevisiae NMT, Candida albicans NMT, Aspergillus fumigatus NMT, A. flavus NMT, Leishmania major NMT, L. donovani NMT and Cryptosporidium parvum NMT.

Figure 3 Myr-CoA binding by NMTs. Conserved amino-acid residues mediate Myr-CoA binding in (a) PvNMT (PDB entry 5v0w), (b) HsNMT1 (PDB entry 5mu6) and (c) HsNMT2 (PDB entry 4c2x). This figure and other ligand-interaction figures were generated with LigPlus (https://www.ebi.ac.uk/thornton-srv/software/LigPlus/).

IMP-1088 binds to a predominantly hydrophobic peptide/substrate-binding cavity stabilized by several hydrogen bonds and salt bridges (Fig. 4a). The peptide/substrate-binding cavity is less well conserved across NMTs (Bolling et al., 2024), as indicated by the white patch in Fig. 1(d). The amino-acid residues interacting with IMP-1088 are almost identical in the PvNMT (PDB entry 5v0w) and HsNMT1 (PDB entry 5mu6) structures. Notably, the serine mediating a hydrogen bond involved in inhibitor binding is conserved, as are most residues involved in IMP-1088 binding (Figs. 4a and 4b). Nonetheless, while PvNMT interacts with IMP-1088 through a leucine residue (Leu410), HsNMT forms contacts with the compound via a glutamine residue (Gln496) (Figs. 4a and 4b).

Figure 4 Comparison of inhibitor binding by NMTs. PvNMT interacts with IMP-1088 (PDB entry 5v0w) (a) with similar amino acids as HsNMT1 (PDB entry 5mu6) (b). Inhibitor IMP-1002 interacts with fewer amino acids on PvNMT (PDB entry 6mb1) (c), as does inhibitor DDD85646 with PvNMT (PDB entry 5g1z) (d).

IMP-series inhibitors generally exhibit excellent efficacy against P. vivax (Mousnier et al., 2018). For example, IMP-1031, an analog of IMP-1088, had an IC₅₀ value of approximately 200 pM in a P. berghei liver-stage assay (Bell et al., 2012, 2020, 2022). The comparison of complex structures of PvNMT and promising IMP-series inhibitors reveals similar interactions (Fig. 4). IMP-1002, an analog of IMP-1088 discovered through a fragment-reconstruction approach based on hits from screens against PvNMT and P. falciparum NMT (Mousnier et al., 2018; Schlott et al., 2019), binds similarly to IMP-1088. Interestingly, IMP-1002 exhibits a fourfold higher potency in killing parasites than the most potent previously reported PvNMT inhibitor, DDD85646 (Wright et al., 2014). LigPlus analysis of the PvNMT structures reveals that DDD85646 (PDB entry 5g1z) interacts with fewer amino-acid residues than IMP-1002 (PDB entry 6mb1) and IMP-1088 (PDB entry 5v0w) (Figs. 4c and 4d, Table 5).

Table 5
Residues involved in ligand binding

	PDB entry 5v0w	PDB entry 5mu6	PDB entry 6mb1	PDB entry 5g1z
Hydrogen-bond contacts	Ser319	Ser405	Ser319	Ser319
Non-hydrogen-bond contacts	Val96	Val181	Val96	Val96
	Asp98	Asp183	Asp98	Glu97
	Phe103	Phe188	Phe103	Asp98
	Arg104	Arg189	Phe105	Phe103
	Phe105	Phe190	Tyr107	Phe105
	Tyr107	Tyr192	Asn161	Tyr211
	Asn161	Asn246	Thr197	Phe226
	Thr197	Thr282	Gly199	Ser319
	Gly199	Gly284	Tyr211	Leu330
	Tyr211	Tyr296	Ser319	Tyr334
	His213	His298	Tyr334	Asn365
	Ser319	Ser405	Asn365
	Tyr334	Tyr420	Ala366
	Asn365	Asn451	Leu367
	Ala366	Ala452	Leu388
	Leu367	Leu453	Leu409
	Leu388	Leu474	Leu410
	Leu409	Leu495
	Leu410	Gln496

The structure of the complex of HsNMT1 with an inhibitor peptide (GNCFSKPR) and Myr-CoA (PDB entry 8q26) was released in August 2024 (Rivière et al., 2024). This structure allows the entire peptide-binding cavity of HsNMT1 to be probed, revealing the amino-acid residues involved in peptide binding (Fig. 5). LigPlus analysis after alignment of the peptide (GNCFSKPR) inhibitor with PvNMT reveals a similar network of amino-acid interactions within well conserved substrate/peptide-binding cavities (Fig. 5). Substrate-binding specificity is ensured via the preferential binding of glycine residues by the myristoyl-binding cavity (Harupa et al., 2020).

Figure 5 Interactions in the peptide/substrate-binding cavity. (a) Interactions between HsNMT1 and a peptide inhibitor (GNCFSKPR; PDB entry 8q26). (b) The modeled superposed PvNMT structure (starting from PDB entry 5v0w with ligands removed) shows conserved interactions with the peptide inhibitor.

4. Conclusions

The ternary structure of P. vivax N-myristoyltransferase (PvNMT) with IMP-1088 and Myr-CoA is presented. Ongoing efforts to develop IMP-1088-like compounds as antimalarials include testing the inhibitory activity of IMP-1088 against PvNMT.