Optimization and Characterization of N-Acetamide Indoles as Antimalarials That Target PfATP4

Abstract

To discover new antimalarials, a screen of the Janssen Jumpstarter library against Plasmodium falciparum uncovered the N-acetamide indole hit class. The structure-activity relationship of this chemotype was defined and culminated in the optimized frontrunner analog WJM664, which exhibited potent asexual stage activity and high metabolic stability. Resistant selection and whole-genome sequencing revealed mutations in PfATP4, which was validated as the target by showing that analogs exhibited reduced potency against parasites with resistance-conferring mutations in PfATP4, a metabolomic signature similar to that of the PfATP4 inhibitor KAE609, and inhibition of Na⁺-dependent ATPase activity consistent with on-target inhibition of PfATP4. WJM664 inhibited gamete development and blocked parasite transmission to mosquitoes but exhibited low efficacy in aPlasmodium berghei mouse model, which was attributed to ATP4 species differentiation and its moderate systemic exposure. Optimization of these attributes is required for N-acetamide indoles to be pursued for development as a curative and transmission-blocking therapy.

Figure 1

Structures of PfATP4 inhibitors and the biological activity of the N-acetamide indole hit compounds identified from a screen of the Jumpstarter Library against P. falciparum 3D7 parasites.

Scheme 1. General Synthetic Pathway to Access N-phenylacetamide Derivatives

Reagents and conditions: (a) TCFH, 1-methylimidazole, MeCN, 20 °C, 20 min, then hydrazine hydrate, 20 °C, 20 min, 61%; (b) triethyl orthoacetate, NH₄Cl, EtOH, reflux, 2 h, 82%; (c) NaH, 0 °C, 20 min, then ethyl bromoacetate, 0 °C, 20 °C, 16 h, 96%; (d) LiOH·H₂O, 1:1 THF/water, 96%; (e) substituted aniline, EDCI, Et₃N, MeCN, 20 °C, 16 h, or substituted aniline, T3P, pyridine, DMF, 20 °C, 16 h, 17–52%.

Scheme 2. General Synthetic Pathway to Access 6-Substituted Indole Derivatives

Reagents and conditions: (a) chloro acetyl chloride, DIPEA, DCM, 20 °C, 16 h, 90–97%; (b) NaH, DMF, 0 °C, 1 h, then 6-bromoindole, DMF, 20 °C, 16 h, 80–84%; (c) R²-boronic acid or R²-BPin ester, Pd(dppf)Cl₂·CH₂Cl₂, K₂CO₃, 1,4-dioxane, H₂O, 110 °C, 3 h, 5–78%.

Figure 2

Summary of the SAR.

Figure 3

PfATP4 homology model showing mutations observed in W454- and analog 80-resistant populations (red), PfATP4 drug-resistant (SJ733 and KAE609) strains (orange) and compound “49” resistant (purple) strains used in this study. The PfATP4 homology model was constructed from the rabbit sarcoendoplasmic reticulum calcium ATPase (SERCA) crystal structure (PDB 2C88). WJM664 (82) (green) was docked to the intramembrane groove of PfATP4 showing the possible binding site of the compound relative to the resistant mutations. The predicted transmembrane region is shown by the dashed line.

Figure 4

Differential analysis of Antimalarial Resistome Barcode Sequencing (AReBar) profiling of compound W452 (6) reveals enrichment of ATP4 mutant lines. (A) Plot showing barcode counts (x-axis) and log fold change (LFC) of individual barcoded lines (y-axis) after treatment with W452, relative to the untreated control on day 14. Two mutant lines, ATP4^G358S and ATP4^A353E/CARL^I1139K, were found with a significant response to compound (LFC > 2.5 and p < 0.001). Symbols indicate strain background, and colors indicate LFC relative to the no-drug control. Data shown in Table S10. (B) Barcode proportions of W452-treated (day 14) and untreated control samples (day 0 and 14), showing the expansion of the ATP4^G3582 line to 97.4% of the population.

Figure 5

Metabolomic fingerprint of analog 59 matches KAE609. A. Principal component analysis of the global metabolite profiles showing overlap of analog 59 with KAE609 (1). B. Heatmap analysis of the relative abundance of all metabolites revealed that both 59 and KAE609 induce widespread metabolic disruption. C. Pearson correlation of the average log₂ fold-change for all peptides significantly perturbed by analog 59 and KAE609 (1) (p < 0.05 and fold-change ≥1.5 or ≤0.67). Approximately 40% of perturbed peptides were mapped to the sequence of hemoglobin. D. Significant perturbations to pyrimidine biosynthesis and nucleotide metabolites (p < 0.05 and fold-change ≥1.5 or ≤0.67). E. Significant perturbations to central carbon metabolites (p < 0.05 and fold-change ≥1.5 or ≤0.67). For D-E data represents the log₂ fold-change of treated samples expressed relative to the average of the untreated control (n = 3–4).

Figure 6

Parasite reduction ratio and stage of asexual arrest analysis on selected N-acetamide indole analogs. A. Activity in a parasite reduction ratio assay in comparison to antimalarial drugs. Data represent the means and SDs for three replicate experiments using P. falciparum 3D7 parasites in an LDH assay. B. Microscopy images are representative of P. falciparum 3D7 parasite morphology determined by Giemsa-stained blood smears at 10 × EC₅₀ of each compound after treatment of ring-stage parasites. More representative microscopy images are shown in Figure S13. Compounds cause vacuolated parasites consistent with the morphology observed with the PfATP4 inhibitor SJ733. C. Quantification of asexual parasite growth over 48 h. Data represent the means and SDs for three experiments.

Figure 7

N-Acetamide indole analogs induce the lysis of red blood cells infected with P. falciparum 3D7 expressing the Hyp-1 Nluc fusion protein. Parasitized red blood cells were treated with the selected compounds, W454 (5) (5.7 μM) and 80 (0.03 μM) and PfATP4 inhibitors, KAE609 (1) (6.7 nM) and SJ733 (2) (0.7 μM), and chloroquine (0.15 μM) at a concentration of 10 × EC₅₀ for 8 h. The luminescence measured was proportional to the amount of red blood cell lysis. A. Red blood cell lysis over time. n = 1 data represent means and SD of 3 technical replicates. n = 2 and 3 are shown in Figure S14. B. Microscopic images from Giemsa-stained thin blood smears at 8 h. Quantification of the percentage of extracellular and intracellular parasites with each treatment at 8 h versus untreated parasites at 0 h. Replicates for cell lysis assay can be found in Figure S14.

Figure 8

N-Acetamide indole analogs inhibit Na⁺-ATPase activity in parasite membrane preparations, consistent with them targeting PfATP4. A. The effects of 0.2% v/v DMSO (solvent-only control), dihydroartemisinin (DHA; 50 nM; negative control), analog 9 (2 μM) and analog 72 (2 μM) on membrane ATPase activity under high-[Na⁺] conditions (152 mM Na⁺) and low-[Na⁺] conditions (2 mM Na⁺; stemming from the addition of 1 mM Na₂ATP) in the presence and absence of KAE609 (1) (50 nM). The data are from four independent experiments, each performed with different membrane preparations. The symbols show the data from individual experiments and the bars show the mean (+SEM). The P_i produced is expressed as a percentage of that measured in the 152 mM Na⁺ Control. In individual experiments, the P_i produced in the 152 mM Na⁺ control varied from 67 to 103 nmol per mg (total) protein per min. The prenormalized data for different compounds and conditions were compared using a repeated measures ANOVA with a post hoc Tukey test. For comparisons between different conditions for the same test compound (or for the control), significant differences are shown with black asterisks. For comparisons between the control and a compound under the same test condition, significant differences are shown with colored asterisks (in this case, gray asterisks, as the only significant differences were between the control and the N-acetamide indole compounds in the 152 mM Na⁺ condition). ***p < 0.001. B. The effects of a range of different concentrations of 9 (red circles) and 72 (black circles) on PfATP4-associated ATPase activity. The data were obtained from four independent experiments performed with different membrane preparations, with the exception of the two highest concentrations of analog 9, for which data are from three independent experiments. In A and B, the membranes were prepared from isolated trophozoite-stage 3D7 parasites. (C, D) The effects of a range of different concentrations of 72 (black; C) and 9 (red; D) on PfATP4-associated ATPase activity in membranes prepared from Dd2-Polδ-PfATP4^G358S parasites (open triangles) and their Dd2-Polδ parents (closed triangles). In C, the data are from four (Dd2-Polδ) or five (Dd2-Polδ-PfATP4^G358S) independent experiments (except for the four lowest concentrations, for which data are n = 2–3). In D, the data are from five independent experiments (except for the lowest four concentrations, for which data are n = 2–3). In B-D, the P_i production measured in the low-[Na⁺] (2 mM) condition was subtracted from that measured in the high-[Na⁺] (152 mM) condition in the presence of each of the different concentrations of analogs 9 and 72 to calculate the PfATP4-associated ATPase activity. The PfATP4-associated ATPase activity is expressed as a percentage of that obtained for the high-[Na⁺] (152 mM) Control, and shown as mean ± SEM.

Figure 9

Activity of WJM664 (82) in a standard membrane feeding assay. Oocyst counts from midguts dissected from Anopheles stephensi mosquitoes 7 days post a blood meal infected with P. falciparum NF54 stage V gametocytes treated with compound at the indicated concentration. Numbers indicate the total number of mosquito midguts dissected per treatment group. Red bars indicate average oocyst intensity and error bars represent SEM. WJM664 (82) at 100 and 500 nM versus the vehicle t test p = 0.04 and 0.001 respectively. The repeat experiment is shown in Figure S20.