(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Abstract

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Keywords: PfATP4; drug discovery; malaria.

Conflict of interest statement

Conflict of interest statement: M.B.J.-D., M.G.G., M.S.M., A.R.-A., L.S., F-J.G., S.F., and I.A.-B. are employees of GlaxoSmithKline and are engaged in commercial development of antimalarial drugs, although not this compound.

Figures

Fig. 1.

SJ733 is an efficacious and safe orally active drug candidate. (A) SJ733 is efficacious against P. falciparum 3D70087/N9 in the nonobese diabetic Scid interleukin-2 receptor γ chain null (NSG) mouse model with the (+)-enantiomer exhibiting excellent potency (1.9 mg/kg); similar to pyrimethamine (ED90 0.9 mg/kg) and superior to chloroquine (ED90 4.3 mg/kg) in this model. (+)-SJ733 achieves this efficacy from exposure (AUCED90 1.5 µM⋅h) similar to that of chloroquine (AUCED90 3.1 µM⋅h) and superior to that of pyrimethamine (AUCED90 5.2 µM⋅h) in the same model. (B) (+)-SJ733 exhibits excellent exposure after oral administration in mouse, rat, and dog. In rodents, (+)-SJ733 reaches peak plasma concentrations of ∼5 µM within 1 h after 20–25 mg/kg doses and >20 µM in dogs following a 30 mg/kg oral dose. (C) (+)-SJ733 exhibits no significant toxicology at doses up to 200 mg/kg in rats with an exposure (AUC) ∼43-fold higher than that required to produce the maximum parasitological response (fastest rate of killing) and 220-fold that required to produce the ED90 in the mouse, indicating the potential for an excellent therapeutic ratio. (D) (+)-SJ733 potently and efficaciously blocks transmission of P. berghei from infected mice to mosquitos when the mice are treated 1 h before feeding the mosquitos as measured by counting oocysts from dissected mosquitos after the sexual stage is allowed to mature.

Fig. 2.

SJ733 targets the PfATP4 protein. (A) All SJ733-resistant strains of malaria generated worldwide contain mutations in the pfatp4 gene that cluster in a single region of the protein. The mutations are illustrated with a homology model of the protein structure for PfATP4 (gold/blue ribbon), built from the available SERCA crystal structures, with the location of mutations causing either high-fold resistance (red), medium-fold resistance (yellow), or low-fold resistance (blue) shown as solid fill. The blue helices represent those in the transmembrane domain and are indicated in the gene block diagram in C. (B) Theoretical docking studies with (+)-SJ733 reproducibly generate a single pose that places (+)-SJ733 in contact with the two residues inducing the highest fold resistance and the one residue selected by in vivo passage with cycling drug exposure. This pose is illustrated by the docked structure of (+)-SJ733 placed into the putative binding site on the PfATP4 structure, with residues conferring resistance color-coded as in A. A loop with sequence variability among Plasmodium spp. that may lead to differing sensitivities to (+)-SJ733 is highlighted in magenta. (C) Block diagram of pfatp4 gene structure and positioning of mutations detected in strains resistant to (+)-SJ733. The selected mutations are color-coded to match those in A, with the location in the coding sequence indicated. The blue cylinders represent the helical portions of the protein shown as blue on the ribbon diagram in A. (D) SJ733 disrupted resting [Na+]i within asexual blood-stage P. falciparum as illustrated by dose–response experiments with the active (+) and inactive (−) enantiomers. Resting [Na+]i was measured >60 min after addition of the compound to sodium-binding benzofuran isophthalate-loaded, saponin-isolated parasites. Each data point represents the mean final [Na+]i averaged from at least three independent experiments (shown ± SD). The relative potencies of the two SJ733 isomers in the [Na+]i assays shown here, on the two different strains (the SJ733-resistant mutant ATP4L350H and the wild-type parent) matched those seen in parasite growth assays.

Fig. 3.

SJ773 causes rapid clearance in vivo that is not dependent upon innate immunity. (A) (+)-SJ733 causes rapid clearance of parasites in vivo, with speed equivalent to artesunate, the fastest-acting antimalarial drug, as measured by clonal dilution assays. When total parasites present in blood are measured, all parasites are cleared systemically within 48 h of initiation of therapy. Similar pharmacodynamics are seen in P. berghei-infected animals (SI Appendix). (B) The in vivo clearance rate of (+)-SJ733 is independent of the presence of a spleen. When total parasites in blood are measured, all parasites are cleared within 48 h of initiation of treatment in both splenectomized and nonsplenectomized animals. Similar experiments in clodronate-treated animals infected with P. berghei indicate that macrophages are not required for rapid pharmacodynamics (SI Appendix). (C) (+)-SJ733 arrests growth of parasites rapidly in vitro, reaching a maximal effect within 24 h as measured by proliferation of a luciferase-labeled 3D7 strain. (D) (+)-SJ733 kills parasites in vitro, but does so with modest speed, equivalent to pyrimethamine as measured by clonal dilution assays. When viable parasites are measured, 96 h of continuous exposure above the EC99 is required for maximal effect.

Fig. 4.

SJ733 arrests parasite development and induces eryptosis selectively in infected erythrocytes. (A) (+)-SJ733 causes a significant proportion of infected erythrocytes to shrink and expose PS. Uninfected erythrocytes, whether treated with (+)-SJ733 or not (Bottom Left and Bottom Right), remain normally sized, do not have exposed PS, and do not possess substantial DNA, as evidenced by examining the cell population for forward scatter, Annexin V binding, and SYBR green binding in FACS. Cells are shown as a population contour plot that compared scatter with SYBR green binding and are color-coded to indicate Annexin binding. After infection, magnetically purified trophozoite/schizont-infected erythrocytes can be detected in three populations (large, with little exposed PS; small, with moderate levels of exposed PS; and intermediate-sized, with high levels of exposed PS), with the majority being in the small- and large-sized populations. Treatment with (+)-SJ733 causes the population distribution to shift strongly toward the intermediate-sized population with high amounts of exposed PS—consistent with strong induction of eryptosis. This shift is not seen with either control drugs or DMSO treatment. (B) When the eryptosis induction effect is examined for (+)-SJ733 by using a concentration-response experiment, the maximal effect is seen at a concentration of ∼40 nM and the EC50 at 30 nM—congruent with the doses causing similar levels of response for proliferation inhibition and parasite Na+ levels. Artesunate has no such effect. (C) (+)-SJ733 causes a significant increase in rigidity of infected, treated erythrocytes. As previously reported, infection of erythrocytes (iRBC) causes a significant increase in their rigidity (P < 0.001; Jonckheere–Terpstra test). After treatment with (+)-SJ733, these infected erythrocytes become significantly more rigid, with the degree of rigidity peaking at ∼7 h after treatment. *P < 0.001 (Jonckheere–Terpstra test). There are no detectable effects on unparasitized erythrocytes at any time point. (D) (+)-SJ733 induces a rapid arrest of parasite motility inside infected erythrocytes that is maintained for the period of treatment as shown by time-lapse microscopy of the treated and untreated cells. The first row of images shows typical motility and growth of a ring-stage parasite over 18 h, which is in stark contrast to the treated parasite in the second row of images that immediately arrests both motility and growth. In some cases the parasite can be observed to lyse within the erythrocyte as shown in the third row. In additional cases the infected, treated erythrocyte itself will lyse after lysis of the parasite as shown in the fourth row. No changes are noted to erythrocyte morphology after treatment of uninfected erythrocytes.

Fig. 5.

Induction of eryptosis by SJ733 triggers rapid clearance in vivo. When (+)-SJ733 is added to erythrocytes infected with Plasmodium spp., it prevents the action of the putative Na+ ATPase PfATP4 and causes a significant increase in cytosolic Na+ within the parasite, reaching maximal effect within 90 min after treatment. This process results in the immediate arrest of parasite motility and blockade of intracellular parasite replication that reaches maximal potency after 24 h of exposure. Simultaneously, infected, treated erythrocytes begin to enter eryptosis, as characterized by their shrinking and becoming more spherical, becoming significantly more rigid, and exposing PS on their plasma membrane. These effects maximize by 7 h after treatment. In vitro, the drug effects lead to complete arrest of replication within 24 h of treatment, followed by slow death, which is maximal by 96 h. In stark contrast to the in vitro setting, the induction of eryptosis leads to rapid clearance of the infected, treated erythrocytes in vivo.