A kalihinol analog disrupts apicoplast function and vesicular trafficking in P. falciparum malaria

Abstract

We report the discovery of MED6-189, an analog of the kalihinol family of isocyanoterpene natural products that is effective against drug-sensitive and drug-resistant Plasmodium falciparum strains, blocking both asexual replication and sexual differentiation. In vivo studies using a humanized mouse model of malaria confirm strong efficacy of the compound in animals with no apparent hemolytic activity or toxicity. Complementary chemical, molecular, and genomics analyses revealed that MED6-189 targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. Its high potency, excellent therapeutic profile, and distinctive mode of action make MED6-189 an excellent addition to the antimalarial drug pipeline.

Fig. 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 (right) (14, 15). (B) SYBR Green–based dose response assays were conducted on early-ring stage parasites (6 hours after 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), drug-resistant strains Dd2 (red), HB3 (black), W2 (gray), 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 after two consecutive erythrocytic cycles. The time points at which MED6-189 was introduced are depicted in green. HPS, hours post synchronization. (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 time points when drug was first introduced (n = 3 biological replicates, P < 0.05). (E) Percentage parasitemia after 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 biological replicates, two-way ANOVA, Tukey t test). (F) Inhibition of P. falciparum gametocyte development after MED6-189 treatment (300 nM) (blue) during early gametocytogenesis compared with the control (black) (P < 0.05, n = 3 biological replicates, two-way ANOVA).

Fig. 2.. MED6-189 localization and activity in combination with other anti-malarials.

(A) Cellular localization of MED6-131 (red) in D10-ACP-GFP P. falciparum transgenic parasites. Nuclei are stained with 4’,6-diamidino-2-phenylindole (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). Logarithmic growth of parasites (y axis) is plotted 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). (C) Normalized isobolograms demonstrating drug interaction (synergism, indifference, or antagonism) according to the Loewe additivity model between apicoplast inhibitors and MED6-189. Isobologram curves are expected to be parallel to the diagonal for additive drug pairs, concave for synergistic drug pairs, and convex for antagonistic drug pairs. (D) Rescue of 3D7 parasites exposed to DMSO (black), fosmidomycin (red), and MED6-189 (blue), along with several other known antimalarials supplemented with IPP 48 hours after synchronization (dotted). The analysis was performed using a two-way ANOVA, n = 3, with P < 0.05 significance.

Fig. 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 cutoff of 0.05. Transcripts associated with invasion and stress responses are highlighted in purple, and those related to apicoplast function are highlighted in green (the asterisk marks noncoding RNAs of unknown function). (B) Heatmap depicting the regulation of lipid metabolism in response to MED6-189. Metabolites significantly up-regulated in response to MED6-189 treatment are shown in green, and those down-regulated are shown in red. We used a log₂ transformation of 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 analog, MED6-118. The significance plot displays all proteins detected in at least two of the five independent MED6-118–based affinity purifications (APs). Scatterplots with gray dots depict QPROT-derived log₂(FC) and z-statistic values between MED6-118 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. (D) TPP melting-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. (E) Thermal profiles for two proteins not detected in MED6-118 pull-down but with significant changes in stability in the presence of MED6-189. For both (D) and (E), 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. Differences in melting temperatures (ΔT_m) along with arrow trends are reported when available (table S6).

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

(A) Graphical illustration of the methodology used to isolate MED6-189–resistant parasites. (B) Comparison of the growth rates of WT S. cerevisiae and transgenic clones with either overexpressed or down-regulated Sec13p after treatment with a vehicle (DMSO) or increasing concentrations of MED6-189. (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 polymerase chain reaction (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 WGS. (D) Predicted structure of the SEC13 protein highlighting the seven amino acid tandem repeat regions targeted for deletion (red). Single-letter abbreviations for the amino acid residues are as follows: G, Gly; M, Met; N, Asn; P, Pro; Q, Gln; and S, Ser. (E) Results of the sequencing analysis, confirming the successful deletion of the tandem repeat region of PfSec13 using CRISPR-Cas9 in an isolated clone. a.a., amino acid. (F) 3D7 WT and parental lines (green and blue, respectively) and 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 were analyzed using a Sigmoidal, 4PL (X represents concentration, n = 3, nonlinear regression, CI: 95%).

Fig. 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 with 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 ER by the Sec translocation complex (SEC61, SEC62, SEC63, SEC66), where it interacts with components of the ER transport machinery. The compound is 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.