Targeting Plasmodium PI(4)K to eliminate malaria

Abstract

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

Figure 1. Imidazopyrazines demonstrate potent activity across Plasmodium species and parasite stages

The central schematic illustrates the Plasmodium lifecycle in mosquitoes and the vertebrate host. Injected sporozoites infect the liver, proliferate and emerge into the bloodstream as merozoites; in some species hypnozoites may remain dormant in the liver. Merozoites undergo multiple cycles of asexual proliferation in RBCs, or at low frequency, differentiate into sexual-stage gametocytes. Mature gametocytes ingested by an Anopheles mosquito become gametes that mate and form oocysts, within which sporozoites develop that migrate to the salivary glands. a, Chemical structures of the imidazopyrazine analogs KAI407 (R=trifluoromethyl, R’=cyano), KDU691 (R=methylamide, R’=chloro), KAI715 and the quinoxaline BQR695. b, In vitro activity of imidazopyrazines against liver-stage schizonts of P. yoelii (means±s.d.; n=4). c, In vivo efficacy of KDU691 against luciferase-expressing P. berghei. Mice were either untreated (+) or given a single oral dose of 7.5 mg/kg KDU691 prior to sporozoite inoculation (t = 0 hr) or after a liver-stage infection was established (t=24, 36 or 48 hr post-infection), then monitored by bioluminescence (one representative mouse per group shown; n=8). d, In vitro inhibition of P. cynomolgi hypnozoites by imidazopyrazines (means±s.d.; n=4). e, Ex vivo analysis of imidazopyrazine activity against asexual blood-stages of P. falciparum (n=8) and P. vivax field isolates (n=6), shown as a boxplot (mean; interquartile range 25–75%) with whiskers (min-max). f, KDU691 inhibition of P. falciparum gametocyte conversion to female gametes after 24 hr incubation with compound, expressed as the percentage of Pfs25-positive female gametes (means; n=2). g, Transmission-blocking effect of KDU691, measured by the number of P. falciparum oocysts in Anopheles stephensi midguts (means±s.d.; n=20) infected with parasites exposed to either 0.1% DMSO, 1 μM DHA or 1 μM KDU691. Abbreviations: ATQ, atovaquone; PQ, primaquine; DHA, dihydroartemisinin.

Figure 2. PfPI4K is required for the completion of cytokinesis in asexual blood-stage schizonts

a, Microscopy of highly-synchronized parasites continuously treated with ~5×IC₅₀ drug (125 nM KAI407, 15 nM KAI715 or 150 nM KDU691) or DMSO vehicle. Representative images from a single experimental replicate are shown (n=3). b, Drug-free media was supplemented with 125 nM KAI407 in 2 hr intervals starting at the mid/late trophozoite stage (t=34 hr post-invasion). Parasitemia in the next lifecycle (t=72 hr) was normalized to untreated parasites (means±s.d.; n=4). Statistical significance was determined by Mann-Whitney U test: *P<0.05, **P< 0.01, ***P<0.001 (also for panel c). c, Percent reinvasion of RBCs after mechanical rupture of schizonts treated with 125 nM KAI407, 15 nM KAI715, 150 nM KDU691 or 1 μM E-64. (means±s.d.; n=4). d, PfATP4-GFP was used to visualize plasma membrane ingression around developing daughter merozoites, with nuclei stained by Hoechst 33342. Representative images from a single experimental replicate are shown (n=2). Scale bar, 5 μm. Parasites treated with 500 nM KAI407 for 4 hr had a disorganized membrane structure, without clearly defined daughter cells.

Figure 3. Resistance to imidazopyrazine and quinoxaline compounds is mediated by gene variations in PfPI4K and PfRab11A

a, Fold-change in IC₅₀ values between 11 drug-evolved clones and the drug-sensitive parent, for the imidazopyrazines, KAI407, KAI715, KDU691, and the quinoxaline BQR695 (means±s.d.; n=8). Statistically significant mean IC₅₀ values for each drug-resistant line were identified using the Mann-Whitney U test (also for panels c, d, e). CNVs and SNVs are noted. b, Schematic overview of the pfpi4k gene editing strategy to introduce putative resistance mutations into a wild type parasite. ZFNs target a 34-bp site on pfpi4k (thunderbolt); following cleavage, homology-dependent repair from a 1.7 kb donor sequence resulted in incorporation of the specific SNV (shown in red), as well as additional silent mutations at the ZFN cut site. c, Fold-change in IC₅₀ values of ZFN-edited lines H1484Y-, S1320L-and Y1356F-PfPI4K against KAI407, KDU691, KAI715 and BQR695 (means±s.d.; n=12). IC₅₀ values for other known antimalarials are provided in Extended Data Fig. 5a. d, Chromosomal integration of GFP fusions of wild type Rab11A, mutant Rab11A (D139Y) or wild type PI4K conferred resistance to imidazopyrazine and quinoxaline compounds. A control line expressing GFP alone was used to normalize the IC₅₀ shift (means±s.d.; n=6). e, Fold-change in IC₅₀ values of P. berghei in vitro liver-stage schizonts expressing PbPI4K-H1477Y (equivalent to PfPI4K-H1484Y). Data shown as means±s.d. (n=12).

Figure 4. Imidazopyrazines and quinoxalines bind the ATP-binding site of PI4K and alter PI4P intracellular distribution

a, In silico docking of a homology model of the PfPI4K catalytic domain showing KAI407 (green) and BQR695 (orange) binding to the ATP-binding pocket of PfPI4K (cartoon representation; grey). Resistance SNVs (yellow) are labeled. b, Zoomed view of the ATP-binding pocket rotated 90° along the y-axis, showing the predicted hydrogen bond (green dash) between the imidazole nitrogen in KAI407 and the amide bond from the V1357 backbone (grey sticks). c, d, Recombinant full-length P. vivax PI4K (PvPI4K) activity in the presence of (c) KDU691 or (d) BQR695 monitored across a range of ATP concentrations (1.25–40 μM). Data shown as means±s.d. (n=9). e, Recombinant PvPI4K mutants S1286L, Y1322F or H1450Y (equivalent to PfPI4K-S1320L, -Y1356F or -H1484Y respectively) were assayed against imidazopyrazine and quinoxaline compounds. Fold-change in IC₅₀ value is shown relative to wild type enzyme (means±s.d.; n=9). Statistical significance was determined by the Mann–Whitney U test: *P<0.05; **P<0.01; ***P<0.001 (also for panel h). f, Illustration of the PI4P-dependent localization of GFP-PH^Osh2. g, In vivo distribution of PI4P was detected in parasites expressing GFP-PH^Osh2. In untreated parasites, GFP-PH^Osh2 localized to intracellular foci (blue arrow) and the plasma membrane (white arrow). Treatment with 500 nM KAI407 or BQR695 for 4 hr depleted the intracellular pool and redistributed the probe to the plasma membrane. Representative images from a single experimental replicate are shown (n=3). Nuclei were stained with Hoechst 33342 (blue); DIC, differential interference contrast. Scale bar, 5 μm. h, Quantification of intracellular GFP-PH^Osh2-labeled foci after drug treatment (means±s.d.; n=3).

Figure 5. Proposed role of PfPI4K within asexual blood-stage parasites

Model depicting daughter merozoite biogenesis in a late-stage schizont, showing plasma membrane ingression and development of the underlying inner membrane complex (IMC). Delivery of post-Golgi secretory vesicles to the invaginating plasma membrane requires PfPI4K-driven production of PI4P, which likely recruits lipid-binding effector proteins in a Rab11A-dependent process. PVM, parasitophorous vacuolar membrane; N, nucleus.