Multi-omic Characterization of the Mode of Action of a Potent New Antimalarial Compound, JPC-3210, Against Plasmodium falciparum

Abstract

The increasing incidence of antimalarial drug resistance to the first-line artemisinin combination therapies underpins an urgent need for new antimalarial drugs, ideally with a novel mode of action. The recently developed 2-aminomethylphenol, JPC-3210, (MMV 892646) is an erythrocytic schizonticide with potent in vitro antimalarial activity against multidrug-resistant Plasmodium falciparum lines, low cytotoxicity, potent in vivo efficacy against murine malaria, and favorable preclinical pharmacokinetics including a lengthy plasma elimination half-life. To investigate the impact of JPC-3210 on biochemical pathways within P. falciparum-infected red blood cells, we have applied a “multi-omics” workflow based on high resolution orbitrap mass spectrometry combined with biochemical approaches. Metabolomics, peptidomics and hemoglobin fractionation analyses revealed a perturbation in hemoglobin metabolism following JPC-3210 exposure. The metabolomics data demonstrated a specific depletion of short hemoglobin-derived peptides, peptidomics analysis revealed a depletion of longer hemoglobin-derived peptides, and the hemoglobin fractionation assay demonstrated decreases in hemoglobin, heme and hemozoin levels. To further elucidate the mechanism responsible for inhibition of hemoglobin metabolism, we used in vitro β-hematin polymerization assays and showed JPC-3210 to be an intermediate inhibitor of β-hematin polymerization, about 10-fold less potent then the quinoline antimalarials, such as chloroquine and mefloquine. Further, quantitative proteomics analysis showed that JPC-3210 treatment results in a distinct proteomic signature compared with other known antimalarials. While JPC-3210 clustered closely with mefloquine in the metabolomics and proteomics analyses, a key differentiating signature for JPC-3210 was the significant enrichment of parasite proteins involved in regulation of translation. These studies revealed that the mode of action for JPC-3210 involves inhibition of the hemoglobin digestion pathway and elevation of regulators of protein translation. Importantly, JPC-3210 demonstrated rapid parasite killing kinetics compared with other quinolones, suggesting that JPC-3210 warrants further investigation as a potentially long acting partner drug for malaria treatment.

System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion

“…The alternative hypothesis that these secondary pathways are non-specific responses in dying parasites is also possible. However, different drug-specific biochemical responses were reported in metabolomics, proteomics and peptidomics studies of other antimalarials, including the 4-aminoquinolines that target the digestive vacuole via a different mechanism, and even when parasites were exposed to drugs for extended durations [ 45 – 47 , 79 , 80 ]. Combined with reports showing that peroxide antimalarials target proteins in both the phospholipid and pyrimidine biosynthesis pathways [ 19 , 20 , 27 ] and colocalise with neutral lipids within iRBCs [ 70 , 71 ], the drug-specific biochemical responses of parasites detected in metabolomics studies suggests that the secondary metabolic alterations observed here are likely ozonide-specific and related to the pleiotropic effect of peroxides on parasite metabolism.…”

“…The Hb fractionation assay was adapted from [ 35 , 80 ]. Briefly, 3D7 trophozoite-stage parasites were incubated with DHA, OZ277, OZ439, E64d or a DMSO control for either 1 or 3 h. Following incubation, Hb, haem and haemozoin species were separated and measured using the Hb fractionation assay [ 35 , 80 ], and smears were made using Giemsa stain to check for parasite viability and digestive vacuole morphology by light microscopy. For the Hb fractionation assay, the samples were normalised via a paired analysis to the DMSO control and graphed as their fold change vs DMSO ± SD.…”

“…The Hb fractionation assay was adapted from [35,80]. Briefly, 3D7 trophozoite-stage parasites were incubated with DHA, OZ277, OZ439, E64d or a DMSO control for either 1 or 3 h.…”

Identification of an inhibitory pocket in falcilysin provides a new avenue for malaria drug development

“…CQ treatment leads to marked accumulation of undigested hemoglobin levels 56 , long-chain hemoglobin-derived peptides 60 , and di-/tri-peptides 61 , indicating disruption in the hemoglobin digestion cascade. In the DV, FLN is likely to be present in a hemozoin formation complex along with HDP, falcipains, and plasmepsins 22 .…”

Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

“…Proteomics LC-MS/MS data was acquired using Dionex Ultimate® 3000 RSLCnano system coupled to Q Exactive HF mass spectrometer (Thermo Scientific) carried out as described previously 78 , with minor modifications. MS2 data was collected in data independent mode (DIA) with a 33-fixed window setup of 18 m/z effective precursor isolation over the m/z range of 375-975 Da.…”