Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.

Evidence for the early emergence of piperaquine-resistant Plasmodium falciparum malaria and modeling strategies to mitigate resistance.

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter's central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.

Structures of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) Isoforms and Their Interactions with Chloroquine.

Using a recently elucidated atomic-resolution cryogenic electron microscopy (cryo-EM) structure for the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein 7G8 isoform as template [Kim, J.; Nature 2019, 576, 315-320], we use Monte Carlo molecular dynamics (MC/MD) simulations of PfCRT embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to solve energy-minimized structures for 7G8 PfCRT and two additional PfCRT isoforms that harbor 5 or 7 amino acid substitutions relative to 7G8 PfCRT. Guided by drug binding previously defined using chloroquine (CQ) photoaffinity probe labeling, we also use MC/MD energy minimization to elucidate likely CQ binding geometries for the three membrane-embedded isoforms. We inventory salt bridges and hydrogen bonds in these structures and summarize how the limited changes in primary sequence subtly perturb local PfCRT isoform structure. In addition, we use the "AlphaFold" artificial intelligence AlphaFold2 (AF2) algorithm to solve for domain structure that was not resolved in the previously reported 7G8 PfCRT cryo-EM structure, and perform MC/MD energy minimization for the membrane-embedded AF2 structures of all three PfCRT isoforms. We compare energy-minimized structures generated using cryo-EM vs AF2 templates. The results suggest how amino acid substitutions in drug resistance-associated isoforms of PfCRT influence PfCRT structure and CQ transport.

Artemisinin-Based Drugs Target the Plasmodium falciparum Heme Detoxification Pathway.

Artemisinin (ART)-based antimalarial drugs are believed to exert lethal effects on malarial parasites by alkylating a variety of intracellular molecular targets. Recent work with live parasites has shown that one of the alkylated targets is free heme within the parasite digestive vacuole, which is liberated upon hemoglobin catabolism by the intraerythrocytic parasite, and that reduced levels of heme alkylation occur in artemisinin-resistant parasites. One implication of heme alkylation is that these drugs may inhibit parasite detoxification of free heme via inhibition of heme-to-hemozoin crystallization; however, previous reports that have investigated this hypothesis present conflicting data. By controlling reducing conditions and, hence, the availability of ferrous versus ferric forms of free heme, we modify a previously reported hemozoin inhibition assay to quantify the ability of ART-based drugs to target the heme detoxification pathway under reduced versus oxidizing conditions. Contrary to some previous reports, we find that artemisinins are potent inhibitors of hemozoin crystallization, with effective half-maximal concentrations approximately an order of magnitude lower than those for most quinoline-based antimalarial drugs. We also examine hemozoin and in vitro parasite growth inhibition for drug pairs found in the most commonly used ART-based combination therapies (ACTs). All ACTs examined inhibit hemozoin crystallization in an additive fashion, and all but one inhibit parasite growth in an additive fashion.

An ortholog of Plasmodium falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion.

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like organelle, termed the "Vacuolar Compartment/Plant-Like Vacuole" (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that contribute to the maturation of invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that the TgCRT protein plays a key role in maintaining the integrity of the parasite's endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in volume by ~15-fold and overlaps with adjacent endosome-like compartments. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in ΔTgCRT parasites. Expression of subtilisin protease 1 is significantly reduced, which impedes trimming of microneme proteins, and significantly decreases parasite invasion. Chemical or genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring integrity of the endolysosomal system, microneme protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in substrate transport that impacts VAC volume and the integrity of the endolysosomal system in T. gondii.

Altered Drug Transport by Plasmodium falciparum Chloroquine Resistance Transporter Isoforms Harboring Mutations Associated with Piperaquine Resistance.

Patterns of multiple amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT, UniProtKB Q8IBZ9) have previously been shown to mediate chloroquine resistance in P. falciparum malarial parasites. Recent reports suggest that novel mutations in PfCRT may mediate resistance to piperaquine (PPQ), which is used extensively as a partner drug in one prominent artemisinin combination therapy. How these novel PfCRT isoforms might mediate PPQ resistance (PPQR) is not known. Using codon optimization and other previously perfected methods for PfCRT analysis in yeast, we have expressed all known PPQR-associated PfCRT isoforms in Saccharomyces cerevisiae yeast and tested whether these isoforms catalyze PPQ transport. Relationships between relative PPQ and CQ transport are analyzed for these isoforms versus other previously recognized drug resistance-associated PfCRT isoforms.

Heterologous Expression, Purification, and Functional Analysis of the Plasmodium falciparum Phosphatidylinositol 4-Kinase IIIß.

Recently, we heterologously expressed, purified, and analyzed the function of the sole Plasmodium falciparum phosphatidylinositol 3-kinase (PI3K), found that the enzyme is a "class III" or "Vps34" PI3K, and found that it is irreversibly inhibited by Fe2+-mediated covalent, nonspecific interactions with the leading antimalarial drug, dihydroartemisinin [Hassett, M. R., et al. (2017) Biochemistry 56, 4335-4345]. One of several P. falciparum phosphatidylinositol 4-kinases [putative IIIß isoform (PfPI4KIIIß)] has generated similar interest as a druggable target; however, no validation of the mechanism of action for putative PfPI4K inhibitors has yet been possible due to the lack of purified PfPI4KIIIß. We therefore codon optimized the pfpi4kIIIß gene, successfully expressed the protein in yeast, and purified an N-lobe catalytic domain PfPI4KIIIß protein. Using an enzyme-linked immunosorbent assay strategy previously perfected for analysis of PfPI3K (PfVps34), we measured the apparent initial rate, Km,app(ATP), and other enzyme characteristics and found full activity for the construct and that PfPI4KIIIß activity is most consistent with the class IIIß designation. Because several novel antimalarial drug candidates with different chemical scaffolds have been proposed to target PfPI4KIIIß, we titrated enzyme inhibition for these candidates versus purified PfPI4KIIIß and PfVps34. We also analyzed the activity versus purified PfPI4KIIIß mutants previously expressed in P. falciparum selected for resistance to these drugs. Interestingly, we found that a putative PfPI4KIIIß inhibitor currently in advanced trials (MMV390048; MMV '0048) is a potent inhibitor of both PfVps34 and PfPI4KIIIß. These data are helpful for further preclinical optimization of an exciting new class of P. falciparum PI kinase inhibitor ("PfPIKi") antimalarial drugs.

Artesunate Activation by Heme in an Aqueous Medium.

The reaction between the antimalarial drug artesunate (ATS) and ferriprotoporphyrin_(IX) (FPIX) in the presence of glutathione (GSH) has been monitored by nuclear magnetic resonance (NMR) spectroscopy. By following the disappearance of resonances of protons near the endoperoxide group in ATS, the rate at which the drug is activated can be directly measured. In an aqueous medium, the rate of ATS activation is limited by the rate of reduction of the FPIX Fe(III) center by GSH. The reaction is observed to slow dramatically in the presence of other heme binding antimalarial drugs. These findings explain the long observed antagonism between artemisinin derivatives and quinoline-based drugs. This discovery suggests that combination therapy that involves artemisinin or any of its derivatives and a quinoline-based drug may be compromised.

Quantification of Free Ferriprotoporphyrin IX Heme and Hemozoin for Artemisinin Sensitive versus Delayed Clearance Phenotype Plasmodium falciparum Malarial Parasites.

We use Plasmodium falciparum culture synchronization, optimized heme and hemozoin extraction protocols, and mass spectrometry to quantify the abundance of free ferriprotoporphyrin IX (FPIX) heme and crystallized FPIX (hemozoin; Hz) for various growth stages of intraerythrocytic P. falciparum malarial parasites. Because of altered cell cycle kinetics for delayed clearance phenotype (DCP) parasites relative to that of the control, we test whether FPIX and Hz abundances differ for DCP and control parasites.

Dihydroartemisinin-Ferriprotoporphyrin IX Adduct Abundance in Plasmodium falciparum Malarial Parasites and the Relationship to Emerging Artemisinin Resistance.

Previously (Heller, L. E., and Roepe, P. D. Quantification of Free Ferriprotoporphyrin IX Heme and Hemozoin for Artemisinin Sensitive versus Delayed Clearance Phenotype Plasmodium falciparum Malarial Parasites. Biochemistry, DOI: 10.1021/acs.biochem.8b00959, preceding paper in this issue), we quantified free ferriprotoporphyrin IX (FPIX) heme abundance for control versus delayed clearance phenotype (DCP) intraerythrocytic Plasmodium falciparum malarial parasites. Because artemisinin drugs are activated by free FPIX, these data predict that the abundance of long-hypothesized toxic artemisinin drug-FPIX covalent adducts might differ for control versus DCP parasites. If so, this would have important repercussions for understanding the mechanism of the DCP, also known as emerging artemisinin resistance. To test these predictions, we studied in vitro formation of FPIX-dihydroartemisinin (DHA) adducts and then for the first time quantified the abundance of FPIX-DHA adducts formed within live P. falciparum versus the stage of intraerythrocytic development. Using matched isogenic parasite strains, we quantified the adduct for DCP versus control parasite strains and found that mutant PfK13 mediates lower adduct abundance for DCP parasites. The results suggest improved models for the molecular pharmacology of artemisinin-based antimalarial drugs and the molecular mechanism of the DCP.

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.

Inhibition of Human Class I vs Class III Phosphatidylinositol 3'-Kinases.

Most investigations of phosphatidylinositol 3'-kinase (PI3K) drug inhibition have been via assays based on ADP appearance or ATP consumption (e.g., Liu, Q., et al. ( 2011 ) J. Med. Chem. 54 , 1473 - 1480 ). However, at least some PI3K isoforms show basal ATPase activity in the absence of PI lipid substrate(s), which may complicate quantification of drug potency, isoform specificity of some drugs, and synergy for drug combinations. In this study, we probe the class I vs class III isoform specificity of a selected set of PI3K inhibitors using a simple, inexpensive, semi high-throughput assay that quantifies production of phosphatidylinositol 3'-phosphate (PI3P) from phosphatidylinositol. Results are compared to previous data largely generated using ATPase activity assays. Good agreement between EC50 values computed via ATPase assays vs the reported PI3P formation assay is found for most drugs, but with a few exceptions. Furthermore, for the first time, drug inhibition of class I vs class III enzymes is compared side-by-side with the same assay for the important class I-specific inhibitors GSK2126458 ("Omipalisib") and NVP-BGT226 ("BGT226") currently in clinical development for advanced solid tumors.

Analysis of Plasmodium vivax Chloroquine Resistance Transporter Mutant Isoforms.

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum malaria is widespread and has limited the use of CQ in many regions of the globe. Malaria caused by the related human parasite P. vivax is as widespread as is P. falciparum malaria and has been treated with CQ as extensively as has P. falciparum, suggesting that P. vivax parasites have been selected with CQ as profoundly as have P. falciparum parasites. Indeed, a growing number of clinical reports have presented data suggesting increased P. vivax CQR. Cytostatic (growth inhibitory) CQR for P. falciparum is caused by Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations, and it has been proposed that mutations in the PvCRT orthologue may simliarly cause P. vivax CQR via increasing CQ transport from the P. vivax digestive vacuole. Here we report the first quantitative analysis of drug transport mediated by all known mutant isoforms of Plasmodium vivax chloroquine resistance transporter (PvCRT) in order to test the protein's potential link to growing P. vivax CQR phenomena. Small, but statistically significant, differences in the transport of CQ and other quinoline antimalarial drugs were found for multiple PvCRT isoforms, relative to wild type PvCRT, suggesting that mutations in PvCRT can contribute to P. vivax CQR and other examples of quinoline antimalarial drug resistance.

Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3'-Kinase.

The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3'-kinase (PI3K), and sequence analysis suggests that the enzyme is a "class III"- or "Vps34"-type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3'-phosphate production from phosphatidylinositol ( Hassett et al., companion paper; DOI 10.1021/acs.biochem.7b00416 ) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme's catalytic and "N-lobe" domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs but only under conditions that cleave the drugs' endoperoxide bridge to generate reactive alkylating agents.

Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

BACKGROUND: Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). METHODS: The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. RESULTS: During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. CONCLUSION: These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT).

At least 53 distinct isoforms of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein are expressed in strains or isolates of P. falciparum malarial parasites from around the globe. These parasites exhibit a range of sensitivities to chloroquine (CQ) and other drugs. Mutant PfCRT is believed to confer cytostatic CQ resistance (CQR(CS)) by transporting CQ away from its DV target (free heme released upon hemoglobin digestion). One theory is that variable CQ transport catalyzed by these different PfCRT isoforms is responsible for the range of CQ sensitivities now found for P. falciparum. Alternatively, additional mutations in drug-selected parasites, or additional functions of PfCRT, might complement PfCRT-mediated CQ transport in conferring the range of observed resistance phenotypes. To distinguish between these possibilities, we recently optimized a convenient method for measuring PfCRT-mediated CQ transport, involving heterologous expression in Saccharomyces cerevisiae. Here, we use this method to quantify drug transport activity for 45 of 53 of the naturally occurring PfCRT isoforms. Data show that variable levels of CQR likely depend upon either additional PfCRT functions or additional genetic events, including perhaps changes that influence DV membrane potential. The data also suggest that the common K76T PfCRT mutation that is often used to distinguish a P. falciparum CQR phenotype is not, in and of itself, a fully reliable indicator of CQR status.

UV-triggered affinity capture identifies interactions between the Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) and antimalarial agents in live parasitized cells.

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.

Additional PfCRT mutations driven by selective pressure for improved fitness can result in the loss of piperaquine resistance and altered Plasmodium falciparum physiology.

IMPORTANCE: Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.

Function of resistance conferring Plasmodium falciparum chloroquine resistance transporter isoforms.

The function of Plasmodium falciparum chloroquine resistance transporter (PfCRT) can be quantified using a Saccharomyces cerevisiae model system [Baro, N. K., Pooput, C., and Roepe, P. D. (2011) Biochemistry 50, 6701-6710]. We further optimized this system to distinguish PfCRT isoforms found in P. falciparum strains and isolates from across the globe. We created and expressed 13 naturally occurring pfcrt alleles associated with a range of chloroquine resistant (CQR) phenotypes. Using galactose induction of PfCRT, we quantified PfCRT and chloroquine (CQ)-dependent yeast growth inhibition and [3H]CQ transport specifically due to a given PfCRT isoform. Surprisingly, we found poor correlation between these parameters and the CQ IC50 observed in strains of malaria harboring the same isoforms. This suggested that an increased level of CQ transport due to PfCRT mutation is necessary, but not sufficient, for the range of CQ IC50 values observed in globally distributed CQR P. falciparum isolates.

Relative to quinine and quinidine, their 9-epimers exhibit decreased cytostatic activity and altered heme binding but similar cytocidal activity versus Plasmodium falciparum.

The 9-epimers of quinine (QN) and quinidine (QD) are known to exhibit poor cytostatic potency against P. falciparum (Karle JM, Karle IL, Gerena L, Milhous WK, Antimicrob. Agents Chemother. 36:1538-1544, 1992). We synthesized 9-epi-QN (eQN) and 9-epi-QD (eQD) via Mitsunobu esterification-saponification and evaluated both cytostatic and cytocidal antimalarial activities. Relative to the cytostatic activity of QN and QD, we observed a large decrease in cytostatic activity (higher 50% inhibitory concentration [IC(50)s]) against QN-sensitive strain HB3, QN-resistant strain Dd2, and QN-hypersensitive strain K76I, consistent with previous work. However, we observed relatively small changes in cytocidal activity (the 50% lethal dose), similar to observations with chloroquine (CQ) analogues with a wide range of IC(50)s (see the accompanying paper [A. P. Gorka, J. N. Alumasa, K. S. Sherlach, L. M. Jacobs, K. B. Nickley, J. P. Brower, A. C. de Dios, and P. D. Roepe, Antimicrob. Agents Chemother. 57:356-364, 2013]). Compared to QN and QD, the 9-epimers had significantly reduced hemozoin inhibition efficiency and did not affect pH-dependent aggregation of ferriprotoporphyrin IX (FPIX) heme. Magnetic susceptibility measurements showed that the 9-epimers perturb FPIX monomer-dimer equilibrium in favor of monomer, and UV-visible (VIS) titrations showed that eQN and eQD bind monomer with similar affinity relative to QN and QD. However, unique ring proton shifts in the presence of zinc(II) protoporphyrin IX (ZnPIX) indicate that binding of the 9-epimers to monomeric heme is via a distinct geometry. We isolated eQN- and eQD-FPIX complexes formed under aqueous conditions and analyzed them by mass, fluorescence, and UV-VIS spectroscopies. The 9-epimers produced low-fluorescent adducts with a 2:1 stoichiometry (drug to FPIX) which did not survive electrospray ionization, in contrast to QN and QD complexes. The data offer important insight into the relevance of heme interactions as a drug target for cytostatic versus cytocidal dosages of quinoline antimalarial drugs and further elucidate a surprising structural diversity of quinoline antimalarial drug-heme complexes.