Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT.

Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite's susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite's susceptibility to lumefantrine and mefloquine-a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite's digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine's access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.

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.

Piperaquine-resistant PfCRT mutations differentially impact drug transport, hemoglobin catabolism and parasite physiology in Plasmodium falciparum asexual blood stages.

The emergence of Plasmodium falciparum parasite resistance to dihydroartemisinin + piperaquine (PPQ) in Southeast Asia threatens plans to increase the global use of this first-line antimalarial combination. High-level PPQ resistance appears to be mediated primarily by novel mutations in the P. falciparum chloroquine resistance transporter (PfCRT), which enhance parasite survival at high PPQ concentrations in vitro and increase the risk of dihydroartemisinin + PPQ treatment failure in patients. Using isogenic Dd2 parasites expressing contemporary pfcrt alleles with differential in vitro PPQ susceptibilities, we herein characterize the molecular and physiological adaptations that define PPQ resistance in vitro. Using drug uptake and cellular heme fractionation assays we report that the F145I, M343L, and G353V PfCRT mutations differentially impact PPQ and chloroquine efflux. These mutations also modulate proteolytic degradation of host hemoglobin and the chemical inactivation of reactive heme species. Peptidomic analyses reveal significantly higher accumulation of putative hemoglobin-derived peptides in the PPQ-resistant mutant PfCRT isoforms compared to parental PPQ-sensitive Dd2. Joint transcriptomic and metabolomic profiling of late trophozoites from PPQ-resistant or -sensitive isogenic lines reveals differential expression of genes involved in protein translation and cellular metabolism. PPQ-resistant parasites also show increased susceptibility to an inhibitor of the P. falciparum M17 aminopeptidase that operates on short globin-derived peptides. These results reveal unique physiological changes caused by the gain of PPQ resistance and highlight the potential therapeutic value of targeting peptide metabolism in P. falciparum.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

Chloroquine inhibited Helicobacter pylori-related gastric carcinogenesis by YAP-ß-catenin-autophagy axis.

YAP participates in autophagy associated with many diseases. In this study, we demonstrate that YAP promotes autophagy by interacting with beclin 1, upregulating beclin 1 and LC3B-II protein expression, and promoting autophagosome formation after H. pylori infection in a vacuolating cytotoxin A-dependent manner. The protein levels of ß-catenin in the cytoplasm and nuclei of GES-1 cells and the mRNA levels of Axin2, Myc, Lgr5, and Ccnd1 were increased in H. pylori-infected cells or YAP-overexpressed cells, but were decreased in YAP-silenced cells. The ß-catenin inhibitor XAV939 significantly downregulated autophagy, whereas the activator LiCl showed opposite effects. An H. pylori-infected mouse model of gastric carcinoma was successfully established. The mouse model showed that H. pylori infection, when combined with NMU, promoted the tumorigenesis of gastric tissues; increased IL-1ß, IL-6, and TNF-α levels; promoted NO release; and increased the expression of beclin 1, LC3B-II more than NMU alone. Chloroquine inhibited these phenomena, but did not completely attenuate the effects of H. pylori. These results demonstrate that chloroquine can be used as a drug for the treatment of H. pylori-related gastric cancer, but the treatment should simultaneously remove H. pylori.

Rhein induces changes in the lysosomal compartment of HeLa cells.

Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy.

Lipopolysaccharide reduces melanin synthesis in vitiligo melanocytes by regulating autophagy.

Vitiligo is an autoimmune-related disease with a complex aetiology that involves innate immunity. Toll-like receptors (TLRs) are important parts of innate immunity and are related to a variety of autoimmune diseases, including vitiligo, through an unknown mechanism. In this study, we found that the TLR4 gene expression was increased in blood samples of patients with advanced stage vitiligo, and then, we evaluated the effect of TLR4 ligand lipopolysaccharide (LPS) on melanin synthesis in a vitiligo melanocyte cell line PIG3V and along with its mechanism. LPS suppressed melanin synthesis, downregulated the expression of melanin synthesis-related proteins and activated autophagy in vitiligo melanocytes. Inhibiting autophagy with 3-methyladenine or chloroquine blocked these effects. This suggests that LPS inhibits skin pigmentation by modulating autophagy, thus providing novel insights into the pathogenesis of vitiligo.

Intracellular cholesterol transport inhibition Impairs autophagy flux by decreasing autophagosome-lysosome fusion.

BACKGROUND: Autophagy is an intracellular degradation process crucial for homeostasis. During autophagy, a double-membrane autophagosome fuses with lysosome through SNARE machinery STX17 to form autolysosome for degradation of damaged organelle. Whereas defective autophagy enhances cholesterol accumulation in the lysosome and impaired autophagic flux that results Niemann-Pick type C1 (NPC1) disease. However, exact interconnection between NPC1 and autophagic flux remain obscure due to the existence of controversial reports. RESULTS: This study aimed at a comparison of the effects of three autophagic inhibitor drugs, including chloroquine, U18666A, and bafilomycin A1, on the intracellular cholesterol transport and autophagy flux. Chloroquine, an autophagic flux inhibitor; U1866A, a NPC1 inhibitor, and bafilomycin A, a lysosomotropic agent are well known to inhibit autophagy by different mechanism. Here we showed that treatment with U1866A and bafilomycin A induces lysosomal cholesterol accumulation that prevented autophagic flux by decreasing autophagosome-lysosome fusion. We also demonstrated that accumulation of cholesterol within the lysosome did not affect lysosomal pH. Although the clearance of accumulated cholesterol by cyclodextrin restored the defective autophagosome-lysosome fusion, the autophagy flux restoration was possible only when lysosomal acidification was not altered. In addition, a failure of STX17 trafficking to autophagosomes plays a key role in prevention of autophagy flux caused by intracellular cholesterol transport inhibitors. CONCLUSIONS: Our data provide a new insight that the impaired autophagy flux does not necessarily result in lysosomal cholesterol accumulation even though it prevents autophagosome-lysosome fusion. Video abstract.

Palmitic acid hinders extracellular traps of neutrophil from postpartum dairy cow in vitro.

Peripartum dairy cows experience negative energy balance, characterized by high concentrations of blood free fatty acids (FFA) and immune dysfunction. Palmitic acid (PA), the most abundant saturated fatty acid in cow blood, is not only an energy precursor, but causes cellular dysfunction when in excess. Neutrophil extracellular traps (NET) are one of the arsenals of weapons neutrophils use to fight invading pathogens. However, given the marked increase in circulating PA during the peripartum period, it remains to be determined what effect (if any) PA has on NET release. Thus, the objective of this study was to evaluate the effect of PA on NET release and the underlying mechanism in vitro. Phorbol-12-myristate-13-acetate (PMA; 100 ng/mL, 3 h) was used to induce the release of NET in vitro. We isolated neutrophils from the peripheral blood of 5 healthy postpartum dairy cows with similar parity (median = 3, range = 2-4), milk yield (median = 27.84 kg/d per cow, range = 25.79-31.43 kg/d per cow), days in milk (median = 7 d, range = 4-10 d), and serum FFA <0.25 mM, ß-hydroxybutyric acid <0.6 mM, and glucose >3.5 mM. Inhibition of double-stranded DNA (dsDNA) level, a marker of NET release, in response to PA was used to determine an optimal incubation time and concentration for in vitro experiments. Cells were maintained in RPMI-1640 basic medium without phenol red, treated with 600 µM PA for different times (4, 5, 6, and 7 h) in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased duration of PA treatment, with a peak response at 6 h. Thus, 6 h was selected as the challenge time. Then, cells were treated with different concentrations of PA (100, 200, 400, and 600 µM) for 6 h in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased dose of PA, with a peak response at 400 µM. Finally, 400 µM PA for 6 h was selected as the treatment for subsequent experiments. Protein abundance of citrullinated histone in the presence or absence of PMA was markedly lower in response to incubation with PA. Morphological observations by laser confocal microscopy and scanning electron microscopy showed that the ratio of NET-releasing cells decreased in response to incubation with PA. Autophagy is a potential key intermediate process in the regulation of NET by PA. To investigate the effect of PA on autophagy, we used chloroquine to block lysosomal degradation. Exogenous PA led to accumulation of sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and no further accumulation in the presence of chloroquine, all of which suggested an impairment of autophagic flux. To verify the role of autophagy in NET, we used rapamycin to promote autophagic flux; 100 nM rapamycin attenuated the suppressive effect of PA on NET release indicated by greater dsDNA levels, accumulation of citrullinated histone, and ratio of NET-releasing neutrophils. Overall, these data demonstrate PA inhibits NET release by suppressing autophagic flux, which provides information for understanding the immune dysfunction in postpartum cows.

Theoretical Insights into the Anti-SARS-CoV-2 Activity of Chloroquine and Its Analogs and In Silico Screening of Main Protease Inhibitors.

Corona virus disease (COVID-19) is a dangerous disease rapidly spreading all over the world today. Currently there are no treatment options for it. Drug repurposing studies explored the potency of antimalarial drugs, chloroquine and hydroxychloroquine, against SARS-CoV-2 virus. These drugs can inhibit the viral protease, called chymotrypsin-like cysteine protease, also known as Main protease (3CLpro); hence, we studied the binding efficiencies of 4-aminoquinoline and 8-aminoquinoline analogs of chloroquine. Six compounds furnished better binding energies than chloroquine and hydroxychloroquine. The interactions with the active site residues especially with Cys145 and His41, which are involved in catalytic diad for proteolysis, make these compounds potent main protease inhibitors. A regression model correlating binding energy and the molecular descriptors for chloroquine analogs was generated with R2 = 0.9039 and Q2 = 0.8848. This model was used to screen new analogs of primaquine and molecules from the Asinex compound library. The docking and regression analysis showed these analogs to be more potent inhibitors of 3CLpro than hydroxychloroquine and primaquine. The molecular dynamic simulations of the hits were carried out to determine the binding stabilities. Finally, we propose four compounds that show drug likeness toward SARS-CoV-2 that can be further validated through in vitro and in vivo studies.

Beneficial Effect of Chloroquine and Amodiaquine on Type 1 Diabetic Tubulopathy by Attenuating Mitochondrial Nox4 and Endoplasmic Reticulum Stress.

BACKGROUND: Oxidative stress induced by chronic hyperglycemia is recognized as a significant mechanistic contributor to the development of diabetic kidney disease (DKD). Nonphagocytic nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in many cell types and in the kidney tissue of diabetic animals. We designed this study to explore the therapeutic potential of chloroquine (CQ) and amodiaquine (AQ) for inhibiting mitochondrial Nox4 and diabetic tubular injury. METHODS: Human renal proximal tubular epithelial cells (hRPTCs) were cultured in high-glucose media (30 mM D-glucose), and diabetes was induced with streptozotocin (STZ, 50 mg/kg i.p. for 5 days) in male C57BL/6J mice. CQ and AQ were administered to the mice via intraperitoneal injection for 14 weeks. RESULTS: CQ and AQ inhibited mitochondrial Nox4 and increased mitochondrial mass in hRPTCs under high-glucose conditions. Reduced mitochondrial ROS production after treatment with the drugs resulted in decreased endoplasmic reticulum (ER) stress, suppressed inflammatory protein expression and reduced cell apoptosis in hRPTCs under high-glucose conditions. Notably, CQ and AQ treatment diminished Nox4 activation and ER stress in the kidneys of STZ-induced diabetic mice. In addition, we observed attenuated inflammatory protein expression and albuminuria in STZ-induced diabetic mice after CQ and AQ treatment. CONCLUSION: We substantiated the protective actions of CQ and AQ in diabetic tubulopathy associated with reduced mitochondrial Nox4 activation and ER stress alleviation. Further studies exploring the roles of mitochondrial Nox4 in the pathogenesis of DKD could suggest new therapeutic targets for patients with DKD.

Molecular docking studies of chloroquine and its derivatives against P23pro-zbd domain of chikungunya virus: Implication in designing of novel therapeutic strategies.

The arthropod-transmitted chikungunya virus has emerged as an epidemic menace that causes debilitating polyarthritis. With this life-threatening impact on humans, the possible treatment requires to cure the viral infectivity. But, devoid of any vaccine against the chikungunya virus (CHIKV), there is a need to develop a novel chemotherapeutic strategy to treat this noxious infection. CHIKV carries highly compact P23pro-zbd structure that possesses potential RNA-binding surface domains which extremely influences the use of RNA template during genome replication at the time of infection and pathogenesis. Therefore, computational approaches were used to explore the novel small molecule inhibitors targeting P23pro-zbd domain. The tertiary structure was modeled and optimized using in silico approaches. The results obtained from PROCHECK (93.1% residues in favored regions), ERRAT (87.480 overall model quality) and ProSA (Z-score: -11.72) revealed the reliability of the proposed model. Interestingly, a previously reported inhibitor, chloroquine possesses good binding affinities with the target domain. In-depth analysis revealed that chloroquine derivatives such as didesethyl chloroquine hydroxyacetamide, cletoquine, hydroxychloroquine exhibited a better binding affinity. Notably, MD simulation analysis exhibited that Thr1312, Ala1355, Ala1356, Asn1357, Asp1364, Val1366, Cys1367, Ala1401, Gly1403, Ser1443, Tyr1444, Gly1445, Asn1459, and Thr1463 residues are the key amino acid responsible for stable ligand-protein interaction. The results obtained from this study provide new insights and advances the understanding to develop a new approach to consider effective and novel drug against chikungunya. However, a detailed in vivo study is required to explore its drug likeliness against this life-threatening disease.

Chloroquine-treated dendritic cells require STAT1 signaling for their tolerogenic activity.

MS and EAE are T cell-driven autoimmune diseases of the CNS where IL-17-producing Th17 cells promote damage and are pathogenic. Conversely, tolerogenic DCs induce Treg cells and suppress Th17 cells. Chloroquine (CQ) suppresses EAE through the modulation of DCs by unknown mechanisms. Here, we show that STAT 1 is necessary for CQ-induced tolerogenic DCs (tolDCs) to efficiently suppress EAE. We observed that CQ induces phosphorylation of STAT1 in DCs in vivo and in vitro. Genetic blockage of STAT1 abrogated the suppressive activity of CQ-treated DCs. Opposed to its WT counterparts, CQ-treated STAT1-/- BMDCs were unable to suppress Th17 cells and increased EAE severity. Our findings show that STAT1 is a major signaling pathway in CQ-induced tolDCs and may shed light on new therapeutic avenues for the induction of tolDCs in autoimmune diseases such as MS.

Uptake of a fluorescently tagged chloroquine analogue is reduced in CQ-resistant compared to CQ-sensitive Plasmodium falciparum parasites.

BACKGROUND: Chloroquine (CQ) was the drug of choice for decades in the treatment of falciparum malaria until resistance emerged. CQ is suggested to accumulate in the parasite's digestive vacuole (DV), where it unfolds its anti-malarial properties. Discrepancies of CQ accumulation in CQ-sensitive (CQS) and CQ-resistant (CQR) strains are thought to play a significant role in drug susceptibility. Analysis of CQ transport and intracellular localization using a fluorescently tagged CQ analogue could provide much needed information to distinguish susceptible from resistant parasite strains. The fluorescently tagged CQ analogue LynxTag-CQ™GREEN (CQGREEN) is commercially available and was assessed for its suitability. METHODS: IC50 values were determined for both CQ and CQGREEN in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQGREEN in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQGREEN for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQGREEN uptake rates were determined for the cytosol and DV in the presence and absence of verapamil. RESULTS: In CQS strains, twofold higher IC50 values were determined for the CQGREEN analogue compared to CQ. No significant differences in IC50 values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQGREEN. Live cell imaging revealed that CQGREEN fluorescence was mainly seen in the cytosol of most parasites, independent of the concentration used. Incubation periods of up to 7 h did not influence intracellular localization of CQGREEN. Nevertheless, CQGREEN uptake rates in CQR strains were reduced by 50% compared to CQS strains. CONCLUSION: Although fluorescence of CQGREEN was mainly seen in the cytosol of parasites, IC50 assays showed comparable efficacy of CQGREEN and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQGREEN in CQR strains compared to CQS strains indicate parasite-specific responses to CQGREEN exposure. The data contains valuable information when CQGREEN is used as an analogue for CQ.

Synthesis of stable isotope-labeled chloroquine and amodiaquine and their metabolites.

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate 13 C and 15 N starting from U-13 C-labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7-chloro-1,2,3,4-tetrahydroquinolin-4-one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a similar fashion.

Iron is a substrate of the Plasmodium falciparum chloroquine resistance transporter PfCRT in Xenopus oocytes.

The chloroquine resistance transporter of the human malaria parasite Plasmodium falciparum, PfCRT, is an important determinant of resistance to several quinoline and quinoline-like antimalarial drugs. PfCRT also plays an essential role in the physiology of the parasite during development inside erythrocytes. However, the function of this transporter besides its role in drug resistance is still unclear. Using electrophysiological and flux experiments conducted on PfCRT-expressing Xenopus laevis oocytes, we show here that both wild-type PfCRT and a PfCRT variant associated with chloroquine resistance transport both ferrous and ferric iron, albeit with different kinetics. In particular, we found that the ability to transport ferrous iron is reduced by the specific polymorphisms acquired by the PfCRT variant as a result of chloroquine selection. We further show that iron and chloroquine transport via PfCRT is electrogenic. If these findings in the Xenopus model extend to P. falciparum in vivo, our data suggest that PfCRT might play a role in iron homeostasis, which is essential for the parasite's development in erythrocytes.

Molecular Mechanisms for Drug Hypersensitivity Induced by the Malaria Parasite's Chloroquine Resistance Transporter.

Mutations in the Plasmodium falciparum 'chloroquine resistance transporter' (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite's digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite's hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite's survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite's hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite's sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.

New phenylaniline derivatives as modulators of amyloid protein precursor metabolism.

The chloroquinoline scaffold is characteristic of anti-malarial drugs such as chloroquine (CQ) or amodiaquine (AQ). These drugs are also described for their potential effectiveness against prion disease, HCV, EBV, Ebola virus, cancer, Parkinson or Alzheimer diseases. Amyloid precursor protein (APP) metabolism is deregulated in Alzheimer's disease. Indeed, CQ modifies amyloid precursor protein (APP) metabolism by precluding the release of amyloid-beta peptides (Aß), which accumulate in the brain of Alzheimer patients to form the so-called amyloid plaques. We showed that AQ and analogs have similar effects although having a higher cytotoxicity. Herein, two new series of compounds were synthesized by replacing 7-chloroquinolin-4-amine moiety of AQ by 2-aminomethylaniline and 2-aminomethylphenyle moieties. Their structure activity relationship was based on their ability to modulate APP metabolism, Aß release, and their cytotoxicity similarly to CQ. Two compounds 15a, 16a showed interesting and potent effect on the redirection of APP metabolism toward a decrease of Aß peptide release (in the same range compared to AQ), and a 3-10-fold increased stability of APP carboxy terminal fragments (CTFα and AICD) without obvious cellular toxicity at 100 µM.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.