The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs

Abstract

The emergence of drug resistance is a major limitation of current antimalarials. The discovery of new druggable targets and pathways including those that are critical for multiple life cycle stages of the malaria parasite is a major goal for developing next-generation antimalarial drugs. Using an integrated chemogenomics approach that combined drug resistance selection, whole-genome sequencing, and an orthogonal yeast model, we demonstrate that the cytoplasmic prolyl-tRNA (transfer RNA) synthetase (PfcPRS) of the malaria parasite Plasmodium falciparum is a biochemical and functional target of febrifugine and its synthetic derivative halofuginone. Febrifugine is the active principle of a traditional Chinese herbal remedy for malaria. We show that treatment with febrifugine derivatives activated the amino acid starvation response in both P. falciparum and a transgenic yeast strain expressing PfcPRS. We further demonstrate in the Plasmodium berghei mouse model of malaria that halofuginol, a new halofuginone analog that we developed, is active against both liver and asexual blood stages of the malaria parasite. Halofuginol, unlike halofuginone and febrifugine, is well tolerated at efficacious doses and represents a promising lead for the development of dual-stage next-generation antimalarials.

Figure 1. Identification and confirmation of Pfc PRS of P. falciparum as a target of halofuginone

(A) Shown are chemical structures for febrifugine and its analogs halofuginone (relative stereochemistry) and MAZ1310 (relative stereochemistry). (B) Independent selection experiments under intermittent and dose-adjusted drug pressure starting with the Dd2 lab strain of P. falciparum yielded two highly resistant clones (HFGR-I and HFGR-II). (C) Whole genome sequencing identified nonsynonomous mutations in the highly resistant clones HFGR-I and HFGR-II that map to the same amino acid codon, L482, in PfcPRS (PF3D7_1213800).

Figure 2. Confirmation of Pf cPRS as the functional target of halofuginone using a heterologous yeast model

(A) +/−YHR020w (ScPRS) heterozygous S. cerevisiae was transformed with a YHR020w-containing plasmid, and haploid spores were selected for genomic deletion of YHR020w. The intermediate strain was transformed with a second plasmid with an orthogonal selection marker and YHR020w, wildtype PfcPRS (codon optimized) or mutant PfcPRS (codon optimized), and subsequently selected for loss of the first plasmid. (B) Only transgenic S. cerevisiae expressing wildtype PfcPRS (green) displayed dose-dependent sensitivity to halofuginone, whereas strains expressing ScPRS (blue) or the L482H PfcPRS mutant (red) were insensitive to halofuginone treatment up to 100 µM (all strains were pdr1,3 deleted). The control compound MAZ1310 did not affect growth of PfcPRS expressing yeast (orange). (C) Halofuginone and febrifugine treatment or amino acid starvation (-AA) induce phosphorylation of eIF2α (p-eIF2α) after 90 minutes. Western blot analysis of phosphorylated eIF2α and total eIF2α protein in drug-treated asynchronous Dd2 P. falciparum cultures is shown. Histone H3 is the loading control and the blot is representative of two independent replicates. (D) Halofuginone (HGF) treatment induced pronounced eIF2α phosphorylation in PfcPRS but not in ScPRS-expressing S. cerevisiae.

Figure 3. Models of the ternary complex of PRS with ATP and halofuginone

Shown are molecular dynamic simulations of the ternary complex of PRS with ATP and halofuginone (HFG) for: (A) the PfcPRS of P. falciparum, (B) the ScPRS of S. cerevisiae and (C) the PfcPRS L482H mutant of P. falciparum. The differential binding affinity of halofuginone to PfcPRS and ScPRS can be traced to a T512S mutation in ScPRS that results in a differential ATP binding geometry. This modification in turn changes the interaction of ATP with halofuginone and results in a reorientation of the loop consisting of residues 318–337. Specifically, F335, which stacks against the aromatic ring of halofuginone, is in a different position in the two structures. Additionally, the position of the triphosphate is different, which in turn changes the orientation of Arg401. (C) Effect of the L482H resistance mutation on the interactions of halofuginone in the active site of PfcPRS. Leu482 is adjacent to the proline binding pocket, and although it does not directly participate in the hydrogen bond network formed between halofuginone and PfcPRS, it does support the binding geometry of the amino acid residues that directly interact with halofuginone. The histidine in the L482H mutant provides an alternative hydrogen bond acceptor, thus destabilizing the network. All residues are numbered based on PfcPRS.

Figure 4. Halofuginol is active against the asexual erythrocytic and liver stages of the malaria parasite in vitro

(A) Chemical structures of halofuginol (relative stereochemistry), and epi-halofuginol (relative stereochemistry). (B) In vitro activity of halofuginone, febrifugine, MAZ1310, halofuginol, and epi-halofuginol against P. falciparum strain 3D7 erythrocytic stage parasites. Growth inhibition was quantified after 72 hours by SYBR® green staining. (C) In vitro dose-response for halofuginol after treatment of luciferase-expressing P. berghei ANKA liver stage parasites that have infected HepG2 cells.

Figure 5. Halofuginol is active against the asexual erythrocytic and liver stages of the malaria parasite in vivo

(A) Blood parasitemia at day 5 post-infection in mice treated with halofuginol (i.p. in saline, n = 5; p.o. in water, n = 7; or vehicle, n = 5) q.d. for 4 days and 10 days, respectively. Treatment with halofuginol began 24 h after inoculation with 10⁶ red blood cells infected with GFP-expressing P. berghei ANKA parasites. Blood parasite numbers were analyzed by FACS. (B) In vivo potency of halofuginol in the P. berghei mouse model of malaria. Shown is the relative parasitemia in mouse liver 44 h after infection with luciferase-expressing P. berghei sporozoites. Mice were treated 1 h post infection with halofuginol (25 mg/kg p.o.) or vehicle (10% hydroxypropyl-beta-cyclodextrin in 100 mM pH 5.0 citrate buffer). Parasite load was quantified relative to vehicle control by luminescence measurements. Data are displayed as mean relative to vehicle treated control, with the mean of the control group set to 100% (n = 10).(C) Relative parasitemia in mouse livers 44 h after infection with P. berghei sporozoites. Mice were treated 1hr post infection with halofuginol (i.p. in saline, p.o. in water, n = 4) Parasite load was quantified relative to vehicle control by qRT-PCR of P. berghei 18S rRNA. Data are displayed as mean relative to vehicle treated control, with the mean of the control group set to 100%.(D) Mice were maintained for 14 days post infection or until they developed blood stage malaria. Significance values (*** p < 0.001, **** p < 0.0001) were calculated (Graphpad PRISM) by ordinary one-way ANOVA (A-C) and Log-rank (Mantel-Cox) test (D).