Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase

Abstract

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

Fig. 1. In vitro potency of the compound MMV390048

(A) Structure of 2-aminopyridine MMV390048. (B) In vitro PRR depicting the number of viable parasites (P. falciparum 3D7 strain) over time after treatment with 10× IC₅₀ of MMV390048 compared to other antimalarial drugs. (C) IC₅₀ speed assay using unsynchronized culture of the P. falciparum NF54 strain (mean ± SD of n ≥ 3 independent assays), indicating activity at different incubation times. (D) Parasitemia as a function of time following once daily dosing for 4 days in the P. falciparum humanized SCID mouse model (n = 1 per dose level). Dosing was started on day 3 after infection. (All data points are included in tables S2 to S4.)

Fig. 2. In vitro and in vivo transmission-blocking potential of MMV390048

(A) P. falciparum sexual stages and the various readouts used in drug screening assays, including assays against early- and late-stage gametocytes, the male gamete formation assay, and the SMFA. (B) Game-tocyte viability assay was performed by measuring LDH activity of nonrecombinant stage IV and V gametocytes of P. falciparum. MMV390048 is in black (IC₅₀, 285 nM) and dihydroartemisinin is in blue (IC₅₀, 12.0 nM) (n = 2). (C) Gametocyte viability assay was performed by measuring luminescence of recombinant parasites expressing luciferase. Stage I to III gametocytes are in black (IC₅₀, 214 nM) and stage IV and V gametocytes are in red (IC₅₀, 140 nM) (n = 3). (D) Clearance rates for stage I to III (black) and stage IV and V (red) gametocytes treated with MMV390048 at its IC₅₀ concentration over a 3-day period indicated by the slopes of −6.09 and −15.49, respectively (n = 3). (E) Inhibition of exflagellation and gamete formation in the male gamete formation assay (n = 4). (F) Dose-response curve for MMV390048 in the indirect SMFA. The assay measures the number of oocysts that developed in the midgut of mosquitoes after feeding on P. falciparum stage V gametocyte–infected blood that was exposed to different concentrations of MMV390048 for 24 hours before feeding. In the direct SMFA (inset), mosquitoes fed on infected blood directly after it was treated with different concentrations of MMV390048 (n = 2). (G) Number of oocysts in the midgut of mosquitoes after feeding on P. berghei–infected mice that were treated with either MMV390048, vehicle, or control drugs during the mouse-to-mouse transmission study. (All data points are included in tables S10 to S12, S15, and S16).

Fig. 3. MMV390048 efficacy in P. cynomolgi–infected rhesus macaques and the phenotype of treated P. berghei liver-stage parasites

(A) Daily parasitemias for individual P. cynomolgi–infected monkeys that were treated with MMV390048 on day −1 before infection (prophylactic study). The black arrow indicates when chloroquine treatment (10 mg base/kg orally for 7 days) was started to clear primary infection. Control group monkeys relapsed on days 26 and 29, whereas monkeys treated with MMV390048 remained clear of parasitemia for the 100 days of observation. (B) Mean plasma concentrations of MMV390048 over time for the treated monkeys in the prophylactic study after a single oral treatment (20 mg/kg) with MMV390048. The concentration of MMV390048 remained above its IC₉₉ for 6.5 days (orange arrow). The in vivo IC₉₉ was estimated to be 353 ± 32 nM (138 ± 13 ng/ml) based on the in vitro IC₉₉ determined in the P. cynomolgi liver-stage assay and correcting for serum albumin (AlbuMAX) binding (54%) and plasma protein binding (86%). (C) Daily parasitemias for individual P. cynomolgi–infected monkeys in the radical cure study. Once infection was established, animals were treated with either MMV390048 or vehicle (20 mg/kg once daily for 5 days; brown arrows) with simultaneous treatment with chloroquine (10 mg base/kg orally for 7 days) to clear primary parasitemia (black arrow indicating start of chloroquine treatment). Control group monkeys relapsed on days 24 and 29, whereas MMV390048-treated monkeys relapsed on days 33 to 35. (D) Mean plasma concentrations of MMV390048 over time for the treatment group monkeys in the radical cure study. Brown arrows indicate treatment with MMV390048 (20 mg/kg oral dose daily for 5 days). The plasma concentration of MMV390048 remained above the P. cynomolgi liver-stage IC₉₉ for 15.8 days (orange arrow) and above the blood-stage MIC (11.5 ng/ml free concentration) for 20 days (black line). The MIC was calculated from the in vivo P. falciparum SCID mouse model and corrected for protein binding (86.1%) and blood-to-plasma partitioning (0.86). The blue arrow represents the time at which monkeys 3, 4, and 5 relapsed. (E) Image of an early liver-stage P. berghei parasite treated with 460 nM MMV390048 from 2 to 8 hours after inoculation. (F) The parasite displayed mislocalization of the parasitophorous vacuole membrane protein UIS4 from the surface to the parasite interior (red; visualized with goat polyclonal antibody and Alexa Fluor–conjugated secondary antibody) when treated with MMV390048 compared to vehicle. For visualization, cytoplasmic P. berghei heat shock protein 70 is displayed in green (visualized with 2E6 monoclonal antibody and Alexa Fluor–conjugated secondary antibody). Images are single confocal sections. LLOQ, lower limit of quantification. (All data points are included in tables S17 to S20.)

Fig. 4. Chemoproteomic identification of Plasmodium PI4K as the target of MMV390048

(A) Binding of MMV390048 to P. falciparum PI4K in parasite extracts. An analog of MMV390048, MMV666845, was covalently immobilized on Sepharose beads and used for affinity capture of potential target proteins from a P. falciparum blood-stage extract. The addition of excess MMV390048 (10 µM) to the P. falciparum extract competitively inhibited the binding of P. falciparum PI4K to the beads. No other protein was reproducibly inhibited. The addition of MMV034137, a closely related structural analog devoid of antimalarial activity, had no effect on the capture of P. falciparum PI4K. (B) MMV390048 binds to the ATP binding site of Plasmodium PI4K but not to human PI4Kα or PI4Kβ. A set of seven promiscuous ATP-competitive kinase inhibitors was covalently immobilized to beads (Kinobeads) and used for affinity capture of potential kinase targets from a P. falciparum blood-stage extract and from a human erythroleukemia K562 cell line. The plot illustrates pIC₅₀ values obtained in two independent replicates of the experiment (exp. 1 and 2). The addition of MMV390048 to the extract competitively inhibited the binding of P. falciparum PI4K to the beads. PIP4K2C was the only human kinase for which binding was also competitively inhibited by MMV390048 with an IC₅₀ value in the same range as the P. falciparum PI4K. (C) The addition of MMV390048 to the P. falciparum extract over a range of concentrations yielded an apparent dissociation constant (K_d^app) of 0.3 µM using MMV666845 for affinity capture and a calculated K_d^app of 0.1 µM using Kinobeads for affinity capture. (D) Structure-activity relationship of MMV390048 analogs revealed excellent correlation of antimalarial activity and binding to P. falciparum PI4K in parasite extracts. Fourteen compounds from the aminopyridine (MMV390048) series with increasing activity against P. falciparum were subjected to profiling using immobilized MMV666845 beads [see also (A)]. With increased potency against P. falciparum, the compounds showed increased competition for binding to P. falciparum PI4K and thus reduced its binding to the bead matrix. r, Pearson’s correlation coefficient; P, P value (calculated probability). (All data points are included in tables S26 and S27.)