A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisininbased combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nucleasebased gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bisaminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates

Evolution of Fitness Cost-Neutral Mutant PfCRT Conferring P. falciparum 4-Aminoquinoline Drug Resistance Is Accompanied by Altered Parasite Metabolism and Digestive Vacuole Physiology

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite’s acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates

Synthesis, characterization, antiplasmodial evaluation and electrochemical studies of water-soluble heterobimetallic ferrocenyl complexes

Three new ferrocenyl-containing heterobimetallic complexes were synthesized using a sodium sulfonate-salicylaldimine mononuclear ferrocenyl complex and various metal precursors. Complexation with ruthenium(II), rhodium(III) and iridium(III) precursors yielded the heterobimetallic complexes, which display good water-solubility. The ferrocenyl ligand acts as a N,O-bidentate chelating ligand, coordinating to the metal center via the imine nitrogen and the deprotonated phenolic oxygen. The complexes were characterized using analytical and spectroscopic techniques. The compounds were evaluated for in vitro antiplasmodial activity against the NF54 chloroquine-sensitive strain of Plasmodium falciparum. The mono- and bimetallic complexes exhibit enhanced activity compared to the salicylaldimine hydrazone. The compounds were evaluated for their ability to inhibit β-haematin formation but were inactive, suggesting an alternative reason for their antiplasmodial activity. Electrochemical studies on the bimetallic complexes revealed a voltammetric wave corresponding to the oxidation of the ferrocenyl group and another at a more positive potential which inhibited the reversibility of the ferrocenyl oxidation

Identification and Mechanistic Evaluation of Hemozoin-Inhibiting Triarylimidazoles Active against <i>Plasmodium falciparum</i>

In a previous study, target based screening was carried out for inhibitors of β-hematin (synthetic hemozoin) formation, and a series of triarylimidazoles were identified as active against <i>Plasmodium falciparum</i>. Here, we report the subsequent synthesis and testing of derivatives with varying substituents on the three phenyl rings for this series. The results indicated that a 2-hydroxy-1,3-dimethoxy substitution pattern on ring A is required for submicromolar parasite activity. In addition, cell-fractionation studies revealed uncommonly large, dose-dependent increases of <i>P. falciparum</i> intracellular exchangeable (free) heme, correlating with decreased parasite survival for β-hematin inhibiting derivatives

Identification and SAR Evaluation of Hemozoin-Inhibiting Benzamides Active against <i>Plasmodium falciparum</i>

Quinoline antimalarials target hemozoin formation causing a cytotoxic accumulation of ferriprotoporphyrin IX (Fe­(III)­PPIX). Well-developed SAR models exist for β-hematin inhibition, parasite activity, and cellular mechanisms for this compound class, but no comparably detailed investigations exist for other hemozoin inhibiting chemotypes. Here, benzamide analogues based on previous HTS hits have been purchased or synthesized. Only derivatives containing an electron deficient aromatic ring and capable of adopting flat conformations, optimal for π–π interactions with Fe­(III)­PPIX, inhibited β-hematin formation. The two most potent analogues showed nanomolar parasite activity, with little CQ cross-resistance, low cytotoxicity, and high in vitro microsomal stability. Selected analogues inhibited hemozoin formation in <i>Plasmodium falciparum</i> causing high levels of free heme. In contrast to quinolines, introduction of amine side chains did not lead to benzamide accumulation in the parasite. These data reveal complex relationships between heme binding, free heme levels, cellular accumulation, and in vitro activity of potential novel antimalarials

Synthesis, characterization and pharmacological evaluation of ferrocenyl azines and their rhodium(I) complexes

Ferrocenyl azines containing salicylaldimine motifs were prepared by Schiff-base condensation of salicylaldehyde hydrazones and (dimethylamino)methyl ferrocenecarboxaldehyde. Their corresponding Rh(I) complexes were prepared by reaction of the various ferrocenyl azines with [RhCl(COD)]2 (where COD = 1,5-cyclooctadiene) to yield heterobimetallic complexes. The compounds were characterized using standard spectroscopic and analytical techniques. The characterization data suggests that the ferrocenyl azine acts as a bidentate donor. The rhodium(I) centre binds to the imine nitrogen and phenolic oxygen of the salicylaldimine, forming a neutral complex. The compounds were screened against the NF54 chloroquine-sensitive (CQS) and K1 chloroquine-resistant (CQR) strains of Plasmodium falciparum. The ferrocene-containing salicylaldimines exhibited weak to moderate activity across both parasite strains. The heterometallic complexes exhibited enhanced activity compared to the ferrocenyl azines in both strains. Most of the compounds exhibited enhanced activity in the resistant strain compared to the sensitive strain. Inhibition of haemozoin formation was considered as a possible mechanism of action of these compounds and indeed they exhibited β-haematin inhibition activity, albeit weaker than chloroquine. All compounds were also screened against the G3 strain of Trichomonas vaginalis. The compounds inhibited no more than 50% parasite growth at the tested concentration. One complex exhibited moderate cytotoxicity against WHCO1 oesophageal cancer cells

Fate of haem iron in the malaria parasite Plasmodium falciparum.

Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61+/-2% of the iron within parasitized erythrocytes, of which 92+/-6% is located within the food vacuole. Of this, 88+/-9% is in the form of haemozoin. (57)Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion