MK-7602: a potent multi-stage dual-targeting antimalarial

Abstract

Background: The global burden of malaria remains substantial, and increasing parasite resistance to current antimalarials necessitates the development of drugs with unique mechanisms of action. This study aimed to develop and characterise a new antimalarial compound targeting Plasmodium aspartic proteases.

Methods: We conducted high-throughput screening, medicinal chemistry optimisation, and extensive in vitro and in vivo testing to develop and evaluate MK-7602, a dual inhibitor of plasmepsins IX and X.

Findings: MK-7602, a clinical candidate, acts as a dual sub-nanomolar inhibitor of plasmepsins IX and X in multiple Plasmodium species. It exhibits favourable pharmacokinetic properties and a promising safety profile. MK-7602 demonstrates activity against liver and blood life-cycle stages of the parasite and blocks transmission to mosquitoes. Importantly, it shows a high barrier to resistance development and lacks cross-resistance with Plasmodium falciparum strains resistant to other antimalarials. MK-7602 effectively inhibits both wild-type parasites and those with increased plasmepsin expression, highlighting its potential to overcome existing resistance mechanisms.

Interpretation: MK-7602 represents a new class of antimalarial for treating uncomplicated malaria with a new mechanism of action and the potential to address drug-resistant malaria. Clinical evaluation of MK-7602's activity against P. falciparum is ongoing.

Funding: This work was funded by The Wellcome Trust (109662/Z/15/Z, 202749/Z/16/Z, 219658/Z/19/Z), NHMRC (GNT1176955, GNT637406, GNT1173049), the Human Frontiers Science Program (LT0001/2022-L, JMD), Drakensberg Trust, the Victorian State Government Operational Infrastructure Support grant, and the Australian Government NHMRC IRIISS. JPo was supported by the NIH/NIAID (R01AI173171, R01AI175134 and R61AI187100) and the Pasteur International Unit PvESMEE.

Keywords: Antimalarial drug; Aspartic proteases; Falciparum; Malaria; Plasmepsin; Vivax.

Fig. 1

MK-7602 is a potent clinical candidate that inhibits P. falciparum development in the blood stage. (A) Chemical structure of WM4, WM382 and MK-7602. (B) Growth inhibitory curves and EC₅₀ values for WM4 (n = 6), WM382 (n = 10), MK-7602 (n = 10) and Chloroquine (CQ, n = 9) against P. falciparum. Data are mean ± SD. (C) Kinetics of enzyme inhibition data for MK-7602 and control compounds against P. falciparum (Pf), P. vivax (Pv) and P. knowlesi (Pk) plasmepsins V, IX and X, and other human aspartic proteases: beta-site amyloid precursor protein cleaving enzyme (BACE1), cathepsin D (CatD) and renin. (D) Parasitaemia in synchronous blood stage parasites taken at 8-h intervals in the absence or presence of MK-7602 or WM382, across one life cycle. Data are mean ± SD of triplicate values from one biological replicate. (E) Parasite developmental stage in Giemsa-stained thin blood smears of untreated, MK-7602 and WM382-treated parasites across one life cycle. Size bar is the same for all panels as shown in the bottom right. (F) Quantification of P. falciparum egress for MK-7602 and WM382-treated parasites using an exported PEXEL Nanoluc parasite line. Data are mean of triplicate values ± SEM, n = 3. (G) Still photographs of purified schizonts (left panel DMSO only control and right panel incubated with MK-7602). Examples of released merozoites are indicated by red arrows in the left panel when no MK-7602 was present. Size bar is shown at bottom right of each panel. (H) Quantification of parasite invasion for MK-7602 (2 nM) and WM382 (5 nM)-treated parasites. Compound 1 (1 μM) served as negative control. Data are mean ± SD of 3 replicate invasion samples. (I) Parasite reduction ratio (PRR) curves for MK-7602, WM382 and other clinically available antimalarials. PYR: pyrimethamine, PCT: parasite clearance time. Solid lines represent compounds run concurrently in the assay; dotted lines represent historical data. Data are means of quadruplicate values normalised to log (viable parasite +1) ± SD. (J)Ex vivo P. vivax field isolate invasion inhibition in response to MK-7602 and WM382. Data are mean ± SD from P. vivax (Pv) isolated from 5 patients. DF, drug-free.

Fig. 2

MK-7602 is a dual inhibitor of plasmepsins IX and X. (A) Differential soluble protein abundance analysis of parasite lysate treated with MK-7602 or WM382 relative to DMSO control after thermal challenge. Non-significant (ns) proteins are plotted in grey, significantly stabilised proteins in red. Hit selection cut-offs of 0.73 log₂ fold-change and p < 0.01 are indicated on the graph with dashed lines. Relative abundance of soluble PMIX, PMX and PMI at 50–60 °C and 62–72 °C in treated and untreated parasite extracts is shown in the right panel. (B) SERA5 (left) and ASP (right) processing is inhibited by MK-7602 and WM382 when compared to drug-untreated control samples. Shown is the full gel uncropped for these panels. (C) A representative Surface Plasmon Resonance sensorgram from three independent experiments of MK-7602 with P. vivax (Pv) PMX and P. falciparum (Pf) PMIX and corresponding kinetic parameters determined from the concentration gradients shown. It was not possible to determine accurate off-rates in the time scale of the experiment—see Supplementary Materials and methods for details.

Fig. 3

Structural comparisons between the PvPMX_WM382 and PvPMX_MK-7602 complexes. (A) Simplified deconstructed cartoon showing a view from the front of the catalytic cleft of PvPMX highlighting those residues which form hydrogen bonds with WM382 (PDB ID: 7TBD). Hydrogen bonds are shown as yellow dashed lines. (B) Side view obtained by a 90° anticlockwise rotation of (A) about the vertical axis. (C) Simplified deconstructed cartoon showing a view from the front of the catalytic cleft of PvPMX, highlighting those residues that hydrogen bonds with MK-7602 (PDB ID:8W10). Hydrogen bonds are shown as red dashed lines. (D) A side view obtained by a 90° anticlockwise rotation of (C) about the vertical axis. (E) Cartoon with overlaid surface representations highlighting the relative location of WM382 within the catalytic cleft of PvPMX and the residues involved in hydrogen bonding. Hydrogen bonds are represented by yellow dashed lines (F) Side view of (E) obtained by a 90° anticlockwise rotation about the vertical axis. (G) View of the catalytic cleft of PvPMX from the front. MK-7602 and those residues involved in hydrogen bonding are shown in cyan. Hydrogen bonds are represented by red dashed lines. (H) Side view of (G) obtained by a 90° anticlockwise rotation about the vertical axis. (I) Summary of the PvPMX residues involved in inhibitor interaction distances <4 Å. Comparison of the residues interacting with each inhibitor in the structures for PvPMX_WM382 (left) and PvPMX_MK-7602 (right). Orange shaded residues represent those that interact with both inhibitors. Those coloured in red are also active site aspartic acids. # Residues involved with conserved hydrogen bonds (HBs) with MK-7602. # # Residues involved with unique hydrogen bonds with MK-7602. (J) Surface representation for PvPMX showing a front view of MK-7602 associated with the catalytic cleft. The distribution for those residues common to MK-7602 and WM382 interactions with PvPMX are coloured orange. The position of the S′ and S pockets are labelled. (K) A side view of (J) obtained by a 90° clockwise rotation about the vertical axis.

Fig. 4

MK-7602 suppresses P. berghei infection. (A) Effect of MK-7602 (3–300 mpk bid, i,e. twice daily, oral administration for 4 days) on P. berghei parasites in mice following infection, compared with 10 mpk chloroquine (CQ, once daily, oral administration for 4 days), n = 3–6. (B) Plasma concentration of MK-7602 at 12 h post first (dose 1) and last (dose 8) drug administration determined by LC-MS/MS. (C) Effect of MK-7602 (10–200 mpk twice daily, oral administration for 4 days) on actively replicating P. berghei parasites in mice (treatment started on day 3 when parasitaemia reached ∼1%), compared with 10 mpk chloroquine (CQ, once daily, oral administration for 4 days), n = 3. (D) Concentration of MK-7602 at 12 h post first and last drug administration (dose 1 and 8, respectively) in plasma determined by LC-MS/MS.

Fig. 5

MK-7602 prevents liver to blood infection of P. berghei in mice, and inhibits transmission of P. falciparum and P. berghei to mosquitoes. (A) Mice were infected with 4 × 10⁴PbmCherryLuci sporozoites intravenously (i.v.), then dosed orally twice with MK-7602 (100–500 mpk) at 36 and 48 hours post-infection (hpi). Parasite burden was monitored by In Vivo Imaging System (IVIS) at 52, 55 and 65 hpi, parasitaemia was monitored by Fluorescence Activated Cell Sorting (FACS) and microscopy from 65 hpi to 30 days pi. (B) Bioluminescent images showing peak liver infection (52 hpi), liver egress (55 hpi), and blood infection (65 hpi) in MK-7602 treated and untreated mice. (C) Quantification of liver parasite egress by bioluminescence at 52 and 55 hpi by photon flux (p/s: photons per second). Data are mean ± SD analysed by Mann–Whitney test. (D) Percentage loss of liver bioluminescence (parasite egress) in mice between 52 and 55 hpi. (E) Whole-body luminescence (flux) shows the first wave of blood infection in mice at 65 hpi. (F) Blood parasitaemia in mice (mCherry by FACS) at 65 hpi. (G) Time to patent blood infection for mice infected i.v. with sporozoites. All data in (D–F) are mean ± SD analysed by one-way ANOVA with Holm-Šídák multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. LOD: limit of detection. n = 5–35 mice per group. (H) Percentage of gametocytes at each stage in synchronised NF54 iGP2 parasites after treatment with MK-7602 or WM382. Data are mean ± SD, n = 3 and are compared to untreated control samples. (I) Percentage of exflagellation events in synchronised NF54 iGP2 parasites after treatment with MK-7602 or WM382 from stage I, II, III or IV gametocytes. Data are mean ± SD, n = 3 and are compared to untreated control samples. (J) Number of oocysts in mosquito midguts following treatment of P. falciparum NF54 parasites with MK-7602 (10–500 nM) or WM382 (50 nM) as control. Unt. control: drug-untreated control. Data are mean ± SD, n = 3. (K) Number of oocysts in mosquito midguts following feeding on female Swiss mice infected with P. berghei, treated with MK-7602 (3–100 mpk) or WM382 (20 mpk) as control. Unt. control: drug-untreated control. Data are mean ± SD, n = 2 mice per dose group. All data in (I–K) are mean ± SD analysed by one-way ANOVA test. ∗∗∗p < 0.001. ns, not significant.

Fig. 6

MK-7602 has a high barrier to resistance. (A) Selection of MK-7602-resistant parasites took over 15 months. Top: Copy number in 5 kilobase (kb) genomic windows estimated from whole genome sequencing of MK-7602-resistant parasites. Bottom: 30 kb and 60 kb regions (1 kb genomic windows) for chromosomes 8 and 14, showing amplification of regions encompassing genes encoding PMX and PMIX, respectively. (B) Fold-increase in EC₅₀ (left) and copy number (right) in wild-type (open bars), MK-7602-resistant (filled bars) and reversion parasite line (striped bars). Summary data are below, showing mean ± SD of EC₅₀ values, n = 3. (C) Minimum Inoculum of Resistance (MIR) for MK-7602 and WM382 against reference drugs Atovaquone and Genaplacide (KAF156). (D) Cross resistance of MK-7602 (teal), WM382 (magenta) and WM4 (purple) against 52 barcoded resistant parasite lines by Antimalarial Resistome Barcode sequencing (AReBar) assay. KAE609 (Cipargamin, PfATP4 inhibitor, black) and no-drug treatment (white) served as positive controls. Data are mean ± SD, triplicate values. (E) EC₅₀ determination for different P. falciparum lines (Parasite) to Chloroquine, Atovaquone, Mefloquine, Cipargamin (KAE609), WM4, WM382 and MK-7602. Data are mean ± SD of EC₅₀ values in triplicate samples (n = 2), except 3D7-WM4.1 (n = 1) which was also measured in triplicate.