A Potent Kalihinol Analogue Disrupts Apicoplast Function and Vesicular Trafficking in P. falciparum Malaria

Abstract

Here we report the discovery of MED6-189, a new analogue of the kalihinol family of isocyanoterpene (ICT) natural products. MED6-189 is effective against drug-sensitive and -resistant P. falciparum strains blocking both intraerythrocytic asexual replication and sexual differentiation. This compound was also effective against P. knowlesi and P. cynomolgi. In vivo efficacy studies using a humanized mouse model of malaria confirms strong efficacy of the compound in animals with no apparent hemolytic activity or apparent toxicity. Complementary chemical biology, molecular biology, genomics and cell biological analyses revealed that MED6-189 primarily targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses in P. falciparum revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. The high potency of MED6-189 in vitro and in vivo, its broad range of efficacy, excellent therapeutic profile, and unique mode of action make it an excellent addition to the antimalarial drug pipeline.

Figure 1.. Effect of MED6-189 on P. falciparum intraerythrocytic development

A. Chemical structures of the natural product kalihinol B (left) and its analog MED6-189 (16, 17). B. SYBR Green-based dose response assays were conducted on early-ring stage parasites (6 hours post invasion). The parasites were exposed to serial dilutions of MED6-189 for 72 hours, after which parasite growth was assessed. 3D7 WT (blue), NF54 (green) and drug-resistant strains Dd2 (red), HB3 (black), W2 (grey) and D10-Acp-GFP (purple) lines (Sigmoidal, 4PL, X is concentration, n≥3, nonlinear regression, CI:95%). C. Schematic diagram of the development of P. falciparum following two consecutive erythrocytic cycles. The time points at which MED6-189 was introduced are depicted in green. D. Giemsa-stained images of synchronized 3D7 parasites that were incubated with either DMSO or MED6-189 (at its IC80 concentration). The images depict various developmental stages of the parasite’s intraerythrocytic life cycle. Bordered images represent timepoints when drug is first introduced (n=3, p<0.05) E. (%) parasitemia following exposure of 3D7 parasites to either DMSO (Control) or MED6-189 at various stages of the parasite life cycle within erythrocytes (p< 0.05, n=3, 2-way ANOVA, Tukey t-test). F. Inhibition of P. falciparum gametocyte development following MED6-189 treatment (blue) during early gametocytogenesis compared to the control (black) (p<0.05, n=3, 2-way ANOVA).

Figure 2.. MED6-189’s localization and activity in combination with other antimalarials.

A. Cellular localization of MED6-131 (Red) in D10-Acp-GFP P. falciparum transgenic parasites. Nuclei are stained with DAPI (Blue). Overlap between ACP-GFP (Green) and MED6-131 can be seen during the trophozoite and schizont stages of the cell cycle. B. Dose-dependent interactions between MED6-189 (Blue) and various antimalarials with known mechanisms of action (Red). The figures show logarithmic growth of parasites (Y-axis) as a function of drug concentrations for MED6-189 (M), Fosmidomycin (F), Chloroquine (C) or Atovaquone (A) (X-axis). The regression line represents a nonlinear regression (Variable slope with four parameters), with significant differences considered if p<0.05. Activity correlations between each compound and MED6-189 were analyzed using Pearson correlation (r) using GraphPad Prism 9 (GraphPad Software, Inc.), n=3 (See table S2, fig. S3). C. Rescue of 3D7 parasites exposed to DMSO (black), Fosmidomycin (Red), MED6-189 (Blue), along with several other known antimalarials supplemented with IPP 48 post-synchronization (dotted lines). The analysis was performed using a 2-way ANOVA, n=3, with p<0.05 significance (See table S2C).

Figure 3.. Omics-based profiling of MED6-189 treated parasites.

A. Volcano plot representing transcriptomic changes induced by MED6-189 treatment. A total of 5712 transcripts were identified with an adjusted p-value cut-off of 0.05. Transcripts associated with invasion and stress responses are highlighted in purple and those related to apicoplast function in green (* represent ncRNAs of unknown function). B. Heatmap depicting the regulation of lipid metabolism in response to MED6-189. Metabolites significantly upregulated in response to MED6-189 treatment are shown in green and those downregulated in red. We used a Log₂ transformation to the data for the calculation of q-values (Benjamini-Hochberg adjusted p-values) and p-values using Welch's t-test or ANOVA. C. Protein pulldown assays using biotinylated kalihinol analogue, MED6-118. The significance plot displays all proteins detected in at least two of the five independent MED6-118-based affinity purifications (APs). Scatter plots with gray dots depict QPROT-derived log₂(FC) and Z-statistic values between MED6-APs and negative controls (table S4). Significantly enriched proteins with a Log₂(FC)≥1.5 and a Z score ≥1.645 or those not detected in controls are highlighted in green. Proteins for which thermal profiles are available are shown in red (table S5 and Supporting Information S2). Proteins localized to the apicoplast are indicated with a blue cross, while proteins of unknown function are marked with a gray “X”. A Venn diagram shows the protein overlap between the MED6-118 APs and controls. E. CETSA melt-curve analysis of P. falciparum lysates treated with MED6-189. The thermal profiles for four P. falciparum proteins significantly enriched in the MED6-118-based pull-downs are shown. Stabilization is assessed on the relative amount of soluble protein remaining (Y-axis) after thermal challenge (X-axis). Sample replicates are color-coded in shades of red for MED6-189-treated samples and blue for DMSO controls.

Figure 4.. Evidence for a role of Sec13 in susceptibility to MED6-189.

A. Graphical illustration of the methodology employed to isolate MED6-189 resistant parasites. B. Predicted structure of the Sec13 protein with the seven amino acid tandem repeat region targeted for deletion (red). C. Schematic representation of the CRISPR-Cas9-based replacement strategy used to introduce a deletion of the targeted repeat regions in the PfSec13 gene. The insertion was achieved through overlap extension PCR of fragments directly upstream and downstream of the target segment and subsequently formed by primer overlap extension PCR to replicate the desired deletion. The insertion was validated through whole genome sequencing. D. Results of the sequencing analysis, confirming the successful deletion of the tandem repeat region of PfSec13 using CRISPR-Cas9 in an isolated clone. E. 3D7 WT and parental lines (blue), resistant lines (red) maintained in the presence of DMSO or MED6-189 and transgenic PfSec13-mut clones (dashed) were subjected to a parasite survival assay. The curves depict parasite survival (y-axis) in response to serial drug dilution of MED6-189 (x-axis). Data was analyzed using a Sigmoidal, 4PL (X represents concentration, n=3, nonlinear regression, CI:95%). F. Comparison of the growth rates of wild type S. cerevisiae and transgenic clones with either overexpressed or down-regulated Sec13p following treatment with a vehicle (DMSO) or increasing concentrations of MED6-189.

Figure 5.. In vivo and broad-spectrum antimalarial efficacy of MED6-189.

A. The in vivo efficacy of MED6-189 was evaluated in a humanized mouse model infected with P. falciparum (blue) compared to untreated controls (black). B. Dose-dependent response of MED6-189 on P. knowlesi YH1 human erythrocyte infecting (black) and rhesus erythrocyte infecting (blue) parasites. The graphs illustrate the logarithmic growth of parasites (Y-axis) in response to varying drug concentrations (X-axis). Error bars represent standard deviations from two independent experiments conducted in triplicate. The regression line is derived from a nonlinear regression analysis (Variable slope with four parameters, least squares fit). C. Proposed mode of action of MED6-189 in P. falciparum-infected erythrocytes. The compound is imported into the endoplasmic reticulum (ER) via the Sec translocation complex (SEC61,62,63,66), where it interacts with components of the ER transport machinery. The compounds translocated into the apicoplast where it directly interacts with proteins involved in crucial apicoplast function, ultimately disrupting this vital organelle. The interactions of MED6-189 with components of the apicoplast function and trafficking systems lead to dysregulation of lipids, resulting in the disruption of key biological processes within the Plasmodium parasite.