Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation.

Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7_0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7_1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability.

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention.

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity.

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P. falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger in vitro potency against liver-stage parasites. Unfortunately, in the in vivo transmission blocking assays, 5i,j were inactive against gametocytes.

Inferring a complete genotype-phenotype map from a small number of measured phenotypes.

Understanding evolution requires detailed knowledge of genotype-phenotype maps; however, it can be a herculean task to measure every phenotype in a combinatorial map. We have developed a computational strategy to predict the missing phenotypes from an incomplete, combinatorial genotype-phenotype map. As a test case, we used an incomplete genotype-phenotype dataset previously generated for the malaria parasite's 'chloroquine resistance transporter' (PfCRT). Wild-type PfCRT (PfCRT3D7) lacks significant chloroquine (CQ) transport activity, but the introduction of the eight mutations present in the 'Dd2' isoform of PfCRT (PfCRTDd2) enables the protein to transport CQ away from its site of antimalarial action. This gain of a transport function imparts CQ resistance to the parasite. A combinatorial map between PfCRT3D7 and PfCRTDd2 consists of 256 genotypes, of which only 52 have had their CQ transport activities measured through expression in the Xenopus laevis oocyte. We trained a statistical model with these 52 measurements to infer the CQ transport activity for the remaining 204 combinatorial genotypes between PfCRT3D7 and PfCRTDd2. Our best-performing model incorporated a binary classifier, a nonlinear scale, and additive effects for each mutation. The addition of specific pairwise- and high-order-epistatic coefficients decreased the predictive power of the model. We evaluated our predictions by experimentally measuring the CQ transport activities of 24 additional PfCRT genotypes. The R2 value between our predicted and newly-measured phenotypes was 0.90. We then used the model to probe the accessibility of evolutionary trajectories through the map. Approximately 1% of the possible trajectories between PfCRT3D7 and PfCRTDd2 are accessible; however, none of the trajectories entailed eight successive increases in CQ transport activity. These results demonstrate that phenotypes can be inferred with known uncertainty from a partial genotype-phenotype dataset. We also validated our approach against a collection of previously published genotype-phenotype maps. The model therefore appears general and should be applicable to a large number of genotype-phenotype maps.

The natural function of the malaria parasite's chloroquine resistance transporter.

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.

Biochemical characterization and chemical inhibition of PfATP4-associated Na+-ATPase activity in Plasmodium falciparum membranes.

The antimalarial activity of chemically diverse compounds, including the clinical candidate cipargamin, has been linked to the ATPase PfATP4 in the malaria-causing parasite Plasmodium falciparum The characterization of PfATP4 has been hampered by the inability thus far to achieve its functional expression in a heterologous system. Here, we optimized a membrane ATPase assay to probe the function of PfATP4 and its chemical sensitivity. We found that cipargamin inhibited the Na+-dependent ATPase activity present in P. falciparum membranes from WT parasites and that its potency was reduced in cipargamin-resistant PfATP4-mutant parasites. The cipargamin-sensitive fraction of membrane ATPase activity was inhibited by all 28 of the compounds in the "Malaria Box" shown previously to disrupt ion regulation in P. falciparum in a cipargamin-like manner. This is consistent with PfATP4 being the direct target of these compounds. Characterization of the cipargamin-sensitive ATPase activity yielded data consistent with PfATP4 being a Na+ transporter that is sensitive to physiologically relevant perturbations of pH, but not of [K+] or [Ca2+]. With an apparent Km for ATP of 0.2 mm and an apparent Km for Na+ of 16-17 mm, the protein is predicted to operate at below its half-maximal rate under normal physiological conditions, allowing the rate of Na+ efflux to increase in response to an increase in cytosolic [Na+]. In membranes from a cipargamin-resistant PfATP4-mutant line, the apparent Km for Na+ is slightly elevated. Our study provides new insights into the biochemical properties and chemical sensitivity of an important new antimalarial drug target.

Verapamil-Sensitive Transport of Quinacrine and Methylene Blue via the Plasmodium falciparum Chloroquine Resistance Transporter Reduces the Parasite's Susceptibility to these Tricyclic Drugs.

BACKGROUND: It is becoming increasingly apparent that certain mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) alter the parasite's susceptibility to diverse compounds. Here we investigated the interaction of PfCRT with 3 tricyclic compounds that have been used to treat malaria (quinacrine [QC] and methylene blue [MB]) or to study P. falciparum (acridine orange [AO]). METHODS: We measured the antiplasmodial activities of QC, MB, and AO against chloroquine-resistant and chloroquine-sensitive P. falciparum and determined whether QC and AO affect the accumulation and activity of chloroquine in these parasites. We also assessed the ability of mutant (PfCRT(Dd2)) and wild-type (PfCRT(D10)) variants of the protein to transport QC, MB, and AO when expressed at the surface of Xenopus laevis oocytes. RESULTS: Chloroquine resistance-conferring isoforms of PfCRT reduced the susceptibility of the parasite to QC, MB, and AO. In chloroquine-resistant (but not chloroquine-sensitive) parasites, AO and QC increased the parasite's accumulation of, and susceptibility to, chloroquine. All 3 compounds were shown to bind to PfCRT(Dd2), and the transport of QC and MB via this protein was saturable and inhibited by the chloroquine resistance-reverser verapamil. CONCLUSIONS: Our findings reveal that the PfCRT(Dd2)-mediated transport of tricyclic antimalarials reduces the parasite's susceptibility to these drugs.

Multiple drugs compete for transport via the Plasmodium falciparum chloroquine resistance transporter at distinct but interdependent sites.

Mutations in the "chloroquine resistance transporter" (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.

1H-NMR metabolite profiles of different strains of Plasmodium falciparum.

Although efforts to understand the basis for inter-strain phenotypic variation in the most virulent malaria species, Plasmodium falciparum, have benefited from advances in genomic technologies, there have to date been few metabolomic studies of this parasite. Using 1H-NMR spectroscopy, we have compared the metabolite profiles of red blood cells infected with different P. falciparum strains. These included both chloroquine-sensitive and chloroquine-resistant strains, as well as transfectant lines engineered to express different isoforms of the chloroquine-resistance-conferring pfcrt (P. falciparum chloroquine resistance transporter). Our analyses revealed strain-specific differences in a range of metabolites. There was marked variation in the levels of the membrane precursors choline and phosphocholine, with some strains having >30-fold higher choline levels and >5-fold higher phosphocholine levels than others. Chloroquine-resistant strains showed elevated levels of a number of amino acids relative to chloroquine-sensitive strains, including an approximately 2-fold increase in aspartate levels. The elevation in amino acid levels was attributable to mutations in pfcrt. Pfcrt-linked differences in amino acid abundance were confirmed using alternate extraction and detection (HPLC) methods. Mutations acquired to withstand chloroquine exposure therefore give rise to significant biochemical alterations in the parasite.

Chlorpheniramine Analogues Reverse Chloroquine Resistance in Plasmodium falciparum by Inhibiting PfCRT.

The emergence and spread of malaria parasites that are resistant to chloroquine (CQ) has been a disaster for world health. The antihistamine chlorpheniramine (CP) partially resensitizes CQ-resistant (CQR) parasites to CQ but possesses little intrinsic antiplasmodial activity. Mutations in the parasite's CQ resistance transporter (PfCRT) confer resistance to CQ by enabling the protein to transport the drug away from its site of action, and it is thought that resistance-reversers such as CP exert their effect by blocking this CQ transport activity. Here, a series of new structural analogues and homologues of CP have been synthesized. We show that these compounds (along with other in vitro CQ resistance-reversers) inhibit the transport of CQ via a resistance-conferring form of PfCRT expressed in Xenopus laevis oocytes. Furthermore, the level of PfCRT-inhibition was found to correlate well with both the restoration of CQ accumulation and the level of CQ resensitization in CQR parasites.

Diverse mutational pathways converge on saturable chloroquine transport via the malaria parasite's chloroquine resistance transporter.

Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.

Quinine dimers are potent inhibitors of the Plasmodium falciparum chloroquine resistance transporter and are active against quinoline-resistant P. falciparum.

Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRT(CQR)) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRT(CQR) can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT(CQR) and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H(+) ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRT(CQR) reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRT(CQR) from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRT(CQR). This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.

Mimicking the intramolecular hydrogen bond: synthesis, biological evaluation, and molecular modeling of benzoxazines and quinazolines as potential antimalarial agents.

The intramolecular hydrogen bond formed between a protonated amine and a neighboring H-bond acceptor group in the side chain of amodiaquine and isoquine is thought to play an important role in their antimalarial activities. Here we describe isoquine-based compounds in which the intramolecular H-bond is mimicked by a methylene linker. The antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based 1,3-quinazolin-4-ones were examined in vitro (against Plasmodium falciparum ) and in vivo (against Plasmodium berghei ). Compounds 6b,c caused modest inhibition of chloroquine transport via the parasite's "chloroquine resistance transporter" (PfCRT) in a Xenopus laevis oocyte expression system. In silico predictions and experimental evaluation of selected drug-like properties were also performed on compounds 6b,c. Compound 6c emerged from this work as the most promising analogue of the series; it possessed low toxicity and good antimalarial activity when administered orally to P. berghei -infected mice.

Optimization of 4-aminoquinoline/clotrimazole-based hybrid antimalarials: further structure-activity relationships, in vivo studies, and preliminary toxicity profiling.

Despite recent progress in the fight against malaria, the emergence and spread of drug-resistant parasites remains a serious obstacle to the treatment of infections. We recently reported the development of a novel antimalarial drug that combines the 4-aminoquinoline pharmacophore of chloroquine with that of clotrimazole-based antimalarials. Here we describe the optimization of this class of hybrid drug through in-depth structure-activity relationship studies. Antiplasmodial properties and mode of action were characterized in vitro and in vivo, and interactions with the parasite's 'chloroquine resistance transporter' were investigated in a Xenopus laevis oocyte expression system. These tests indicated that piperazine derivatives 4b and 4d may be suitable for coadministration with chloroquine against chloroquine-resistant parasites. The potential for metabolism of the drugs by cytochrome P450 was determined in silico, and the lead compounds were tested for toxicity and mutagenicity. A preliminary pharmacokinetic analysis undertaken in mice indicated that compound 4b has an optimal half-life.

Know your enemy: understanding the role of PfCRT in drug resistance could lead to new antimalarial tactics.

The prevention and treatment of malaria is heavily dependent on antimalarial drugs. However, beginning with the emergence of chloroquine (CQ)-resistant Plasmodium falciparum parasites 50 years ago, efforts to control the disease have been thwarted by failed or failing drugs. Mutations in the parasite's 'chloroquine resistance transporter' (PfCRT) are the primary cause of CQ resistance. Furthermore, changes in PfCRT (and in several other transport proteins) are associated with decreases or increases in the parasite's susceptibility to a number of other antimalarial drugs. Here, we review recent advances in our understanding of CQ resistance and discuss these in the broader context of the parasite's susceptibilities to other quinolines and related drugs. We suggest that PfCRT can be viewed both as a 'multidrug-resistance carrier' and as a drug target, and that the quinoline-resistance mechanism is a potential 'Achilles' heel' of the parasite. We examine a number of the antimalarial strategies currently undergoing development that are designed to exploit the resistance mechanism, including relatively simple measures, such as alternative CQ dosages, as well as new drugs that either circumvent the resistance mechanism or target it directly.

Quinoline antimalarials containing a dibemethin group are active against chloroquinone-resistant Plasmodium falciparum and inhibit chloroquine transport via the P. falciparum chloroquine-resistance transporter (PfCRT).

A series of 4-amino-7-chloroquinolines with dibenzylmethylamine (dibemethin) side chains were shown to inhibit synthetic hemozoin formation. These compounds were equally active against cultures of chloroquine-sensitive (D10) and chloroquine-resistant (K1) Plasmodium falciparum. The most active compound had an IC(50) value comparable to that of chloroquine, and its potency was undiminished when tested in three additional chloroquine-resistant strains. The three most active compounds exhibited little or no cytotoxicity in a mammalian cell line. When tested in vivo against mouse malaria via oral administration, two of the dibemethin derivatives reduced parasitemia by over 99%, with mice treated at 100 mg/kg surviving the full length of the experiment. Three of the compounds were also shown to inhibit chloroquine transport via the parasite's chloroquine-resistance transporter (PfCRT) in a Xenopus oocyte expression system. This constitutes the first example of a dual-function antimalarial for which the ability to inhibit both hemozoin formation and PfCRT has been demonstrated directly.

Functional characteristics of the malaria parasite's "chloroquine resistance transporter": implications for chemotherapy.

Chloroquine (CQ) was the best and most heavily used drug in the fight against malaria. However, the effectiveness of CQ has declined with the emergence and spread of CQ-resistant (CQR) Plasmodium falciparum parasites. The primary determinant of CQ resistance in P. falciparum is mutations in the parasite's 'chloroquine resistance transporter' (PfCRT). These mutations result in a marked reduction in the accumulation of CQ by the parasite; however the mechanism by which this is achieved was not understood. We have recently shown that the mutations confer upon PfCRT the ability to transport CQ away from its site of accumulation and action. Sensitive and resistance-conferring forms of the protein (PfCRT (CQS) and PfCRT (CQR) , respectively) were expressed at the surface of Xenopus laevis oocytes, and it was found that PfCRT (CQR) (but not PfCRT (CQS)) transports CQ. Here we discuss and expand upon our findings to address the question of whether PfCRT (CQR) behaves as a carrier or a channel, and how this distinction has significant implications for the treatment of CQR P. falciparum with CQ or CQ-like drugs. In particular we relate this to the example of Guinea-Bissau, where high doses of CQ are routinely used to treat CQR P. falciparum malaria.