Impact of Suramin on Key Pathological Features of Sporadic Alzheimer's Disease-Derived Forebrain Neurons.

Background: Alzheimer's disease (AD) is characterized by disrupted proteostasis and macroautophagy (hereafter "autophagy"). The pharmacological agent suramin has known autophagy modulation properties with potential efficacy in mitigating AD neuronal pathology. Objective: In the present work, we investigate the impact of forebrain neuron exposure to suramin on the Akt/mTOR signaling pathway, a major regulator of autophagy, in comparison with rapamycin and chloroquine. We further investigate the effect of suramin on several AD-related biomarkers in sporadic AD (sAD)-derived forebrain neurons. Methods: Neurons differentiated from ReNcell neural progenitors were used to assess the impact of suramin on the Akt/mTOR signaling pathway relative to the autophagy inducer rapamycin and autophagy inhibitor chloroquine. Mature forebrain neurons were differentiated from induced pluripotent stem cells (iPSCs) sourced from a late-onset sAD patient and treated with 100µM suramin for 72 h, followed by assessments for amyloid-ß, phosphorylated tau, oxidative/nitrosative stress, and synaptic puncta density. Results: Suramin treatment of sAD-derived neurons partially ameliorated the increased p-Tau(S199)/Tau ratio, and fully remediated the increased glutathione to oxidized nitric oxide ratio, observed in untreated sAD-derived neurons relative to healthy controls. These positive results may be due in part to the distinct increases in Akt/mTOR pathway mediator p-p70S6K noted with suramin treatment of both ReNcell-derived and iPSC-derived neurons. Longer term neuronal markers, such as synaptic puncta density, were unaffected by suramin treatment. Conclusions: These findings provide initial evidence supporting the potential of suramin to reduce the degree of dysregulation in sAD-derived forebrain neurons in part via the modulation of autophagy.

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.

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 is a safe autophagy inhibitor for sustaining the expression of antioxidant enzymes in trophoblasts.

Inhibition of autophagy contributes to the pathophysiology of preeclampsia. Although chloroquine (CHQ) is an autophagy inhibitor, it can reduce the occurrence of preeclampsia in women with systemic lupus erythematosus. To clarify this important clinical question, this study aimed to address the safety of CHQ in trophoblast cells from the viewpoint of homeostasis, in which the anti-oxidative stress (OS) response and autophagy are involved. We used Western blotting to evaluate the protein levels in the trophoblast cells. The expression levels of heme oxygenase-1 (HO-1), an anti-OS enzyme, mediate resistance to OS induced by hydrogen peroxide (H2O2) in trophoblast cell lines. Among the autophagy modulators, bafilomycin A1 (BAF), an autophagy inhibitor, but not autophagy activators, suppressed HO-1 expression in BeWo cells; CHQ did not suppress HO-1 expression in BeWo cells. To clarify the role of autophagy in HO-1 induction, we observed no difference in HO-1 induction by H2O2 between autophagy-normal and autophagy-deficient cells. As for the mechanism of HO-1 induction by OS, BAF suppressed HO-1 induction by downregulating the expression of neighbor of BRCA1 gene 1 (NBR1) in the selective p62-NBR1-nuclear factor erythroid 2-related factor 2 (Nrf2) autophagy pathway. CHQ did not inhibit HO-1 expression by sustaining NBR1 expression in human villous tissues compared to BAF treatment. In conclusion, CHQ is a safer medicine than BAF for sustaining NBR1, which resist against OS in trophoblasts by connecting selective autophagy and the anti-OS response.

Quercetin Promotes TFEB Nuclear Translocation and Activates Lysosomal Degradation of Ferritin to Induce Ferroptosis in Breast Cancer Cells.

Objective. To investigate the antiproliferative efficacy of quercetin on breast cell lines and its mechanism of ferroptosis regulation. Cells (MCF-7 and MDA-231) were treated with quercetin at 0.1, 1, and 10 µM, respectively. The cell counting kit-8 (CCK-8) assay was applied to assess cell viability, and the intracellular iron level, malondialdehyde (MDA), and carbonylated protein were measured. After treating the cells with quercetin, western blot was applied to determine the level of transcription factor EB (TFEB) and lysosomal-associated membrane protein 1 (LAMP-1) in cells. Meanwhile, western blot was performed to assess the nuclear translocation of TFEB protein in cells. TFEB siRNA and autophagy lysosomal inhibitor, chloroquine, were used to block ferroptosis induced by quercetin. Quercetin induced breast cancer cell death and upregulated the level of iron, MDA, and carbonyl protein in a concentration-dependent manner. Meanwhile, TFEB was highly expressed in the nucleus and lowly expressed in the cytoplasm. The high expression of TFEB promoted the expression of lysosome-related gene LAMP-1, which in turn promoted the degradation of ferritin and the release of ferric ions. The above pharmacodynamic effects of quercetin can be blocked by TFEB siRNA or chloroquine. Quercetin promotes TFEB expression and nuclear transcription, induces the onset of iron death, and thus exerts a pharmacological effect on killing breast cancer cells.

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.

Repurposing Itraconazole and Hydroxychloroquine to Target Lysosomal Homeostasis in Epithelial Ovarian Cancer.

Drug repurposing is an attractive option for oncology drug development. Itraconazole is an antifungal ergosterol synthesis inhibitor that has pleiotropic actions including cholesterol antagonism, inhibition of Hedgehog and mTOR pathways. We tested a panel of 28 epithelial ovarian cancer (EOC) cell lines with itraconazole to define its spectrum of activity. To identify synthetic lethality in combination with itraconazole, a whole-genome drop-out genome-scale clustered regularly interspaced short palindromic repeats sensitivity screen in two cell lines (TOV1946 and OVCAR5) was performed. On this basis, we conducted a phase I dose-escalation study assessing the combination of itraconazole and hydroxychloroquine in patients with platinum refractory EOC (NCT03081702). We identified a wide spectrum of sensitivity to itraconazole across the EOC cell lines. Pathway analysis showed significant involvement of lysosomal compartments, the trans-golgi network and late endosomes/lysosomes; similar pathways are phenocopied by the autophagy inhibitor, chloroquine. We then demonstrated that the combination of itraconazole and chloroquine displayed Bliss defined synergy in EOC cancer cell lines. Furthermore, there was an association of cytotoxic synergy with the ability to induce functional lysosome dysfunction, by chloroquine. Within the clinical trial, 11 patients received at least one cycle of itraconazole and hydroxychloroquine. Treatment was safe and feasible with the recommended phase II dose of 300 and 600 mg twice daily, respectively. No objective responses were detected. Pharmacodynamic measurements on serial biopsies demonstrated limited pharmacodynamic impact. In vitro, itraconazole and chloroquine have synergistic activity and exert a potent antitumor effect by affecting lysosomal function. The drug combination had no clinical antitumor activity in dose escalation. Significance: The combination of the antifungal drug itraconazole with antimalarial drug hydroxychloroquine leads to a cytotoxic lysosomal dysfunction, supporting the rational for further research on lysosomal targeting in ovarian cancer.

Effect of drug metabolism in the treatment of SARS-CoV-2 from an entirely computational perspective.

Understanding the effects of metabolism on the rational design of novel and more effective drugs is still a considerable challenge. To the best of our knowledge, there are no entirely computational strategies that make it possible to predict these effects. From this perspective, the development of such methodologies could contribute to significantly reduce the side effects of medicines, leading to the emergence of more effective and safer drugs. Thereby, in this study, our strategy is based on simulating the electron ionization mass spectrometry (EI-MS) fragmentation of the drug molecules and combined with molecular docking and ADMET models in two different situations. In the first model, the drug is docked without considering the possible metabolic effects. In the second model, each of the intermediates from the EI-MS results is docked, and metabolism occurs before the drug accesses the biological target. As a proof of concept, in this work, we investigate the main antiviral drugs used in clinical research to treat COVID-19. As a result, our strategy made it possible to assess the biological activity and toxicity of all potential by-products. We believed that our findings provide new chemical insights that can benefit the rational development of novel drugs in the future.

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.

Inhibition of lovastatin- and docosahexaenoic acid-initiated autophagy in triple negative breast cancer reverted resistance and enhanced cytotoxicity.

AIMS: Autophagy plays a complex role in breast cancer by suppressing or improving the efficiency of treatment. Triple-negative breast cancer (TNBC) cell line (MDA-MB-231) is associated with aggressive response and developing therapy resistance. MDA-MB-231 cells depend on autophagy for survival. Also, the potential benefits of autophagy inhibition in ameliorating developed chemotherapy resistance towards MDA-MB-231 remains to be elucidated. Despite showing anti-tumorigenic activities, the use of lovastatin and docosahexaenoic acid (DHA) for treating different types of cancers is still limited. We aimed to investigate the protective effect of autophagy inhibition by chloroquine (CQ) in MDA-MB-231 cells resistance treated with lovastatin or DHA. MAIN METHODS: MDA-MB-231 cells were treated with 30 µM lovastatin and/or 100 µM DHA for 48 h plus 20 µM CQ. Autophagic flux was assessed in association with the expression of multidrug resistance gene 1 (MDR1), transforming growth factor beta 1 gene (TGF-ß1), and autophagy-related 7 gene (ATG7). KEY FINDINGS: Both drugs exhibited dose-dependent cytotoxicity, enhanced the autophagic flux represented by increased LC3BII protein concentration and decreased p62 protein concentration, and up-regulated the expression of MDR1, TGF-ß1, and ATG7 genes. CQ addition enhanced the cytotoxicity of drugs and inhibited the autophagic flux which is detected by higher levels of LC3BII and p62 correlated with the reverted MDR1, TGF-ß1 and ATG7 genes expression. SIGNIFICANCE: Autophagy inhibition by CQ showed an ameliorative effect on lovastatin- and DHA-induced resistance and enhanced their cytotoxicity, providing a promising strategy in breast cancer therapy.

The antiviral mechanisms, effects, safety and adverse effects of chloroquine.

Chloroquine, a 4-aminoquinoline derivative, was initially used to treat malaria. It was later found to have immunomodulating, anti-infective, anti-thrombotic, anti-tumor, and metabolic effects. Recently, many studies have focused on the application of chloroquine in viral infections. Most in vitro studies suggested that chloroquine exerted some benefit in infections from viruses. However, animal experiment and clinical trials that attempted to use chloroquine in prevention or treatment of viral infections have reported disappointing results. It might be attributable to inadequate steady-state whole blood chloroquine concentration necessary for exerting its antiviral effects. A 16 µM/L steady-state whole blood concentration of chloroquine should suffice in antiviral treatment with minimal toxicity. Furthermore, chloroquine has both acute and cumulative toxicity. Hence, not only the appropriate treatment dose is crucial, the occurrence of adverse reactions should also be closely monitored and treated in time. Herein, we report the antiviral mechanisms, effects, safety and adverse effects of chloroquine.

Integrin αDß2 influences cerebral edema, leukocyte accumulation and neurologic outcomes in experimental severe malaria.

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDß2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDß2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.

Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS.

Tumor necrosis factor (TNF)­related apoptosis­inducing ligand (TRAIL) is a member of the TNF superfamily and is an antitumor drug that induces apoptosis in tumor cells with minimal or no effects on normal cells. Here, it is demonstrated that 6­shogaol (6­sho), a bioactive component of ginger, exerted anti­inflammatory and anticancer properties, attenuated tumor cell propagation and induced TRAIL­mediated cell death in liver cancer cells. The current study identified a potential pathway by revealing that TRAIL and 6­sho or chloroquine acted together to trigger reactive oxygen species (ROS) production, to upregulate tumor­suppressor protein 53 (p53) expression and to change the mitochondrial transmembrane potential (MTP). Treatment with N­acetyl­L­cysteine reversed these effects, restoring the MTP and attenuated ROS production and p53 expression. Interestingly, treatment with 6­sho increased p62 and microtubule­associated proteins 1A/1B light chain 3B­II levels, indicating an inhibited autophagy flux. In conclusion, attenuation of 6­sho­induced autophagy flux sensitized cells to TRAIL­induced apoptosis via p53 and ROS, suggesting that the administration of TRAIL in combination with 6­sho may be a suitable therapeutic method for the treatment of TRAIL­resistant Huh7 liver cells.

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.

Indomethacin Disrupts Autophagic Flux by Inducing Lysosomal Dysfunction in Gastric Cancer Cells and Increases Their Sensitivity to Cytotoxic Drugs.

NSAIDs inhibit tumorigenesis in gastrointestinal tissues and have been proposed as coadjuvant agents to chemotherapy. The ability of cancer epithelial cells to adapt to the tumour environment and to resist cytotoxic agents seems to depend on rescue mechanisms such as autophagy. In the present study we aimed to determine whether an NSAID with sensitizing properties such as indomethacin modulates autophagy in gastric cancer epithelial cells. We observed that indomethacin causes lysosomal dysfunction in AGS cells and promotes the accumulation of autophagy substrates without altering mTOR activity. Indomethacin enhanced the inhibitory effects of the lysosomotropic agent chloroquine on lysosome activity and autophagy, but lacked any effect when both functions were maximally reduced with another lysosome inhibitor (bafilomycin B1). Indomethacin, alone and in combination with chloroquine, also hindered the autophagic flux stimulated by the antineoplastic drug oxaliplatin and enhanced its toxic effect, increasing the rate of apoptosis/necrosis and undermining cell viability. In summary, our results indicate that indomethacin disrupts autophagic flux by disturbing the normal functioning of lysosomes and, by doing so, increases the sensitivity of gastric cancer cells to cytotoxic agents, an effect that could be used to overcome cancer cell resistance to antineoplastic regimes.

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.

The Lysosomal Protein Saposin†B Binds Chloroquine.

Chloroquine (CQ) has been widely used in the treatment of malaria since the 1950s, though toxicity and resistance is increasingly limiting its use in the clinic. More recently, CQ is also becoming recognized as an important therapeutic compound for the treatment of autoimmune disorders and has shown activity as an anticancer agent. However, the full extent of CQ pharmacology in humans is still unclear. Herein, we demonstrate that the lysosomal protein saposin B (sapB), critical for select lipid degradation, binds CQ with implications for both CQ function and toxicity. Using isothermal titration calorimetry (ITC) and fluorescence quenching experiments, CQ was shown to bind to the dimeric form of sapB at both pH 5.5 and pH 7.4 with an average binding affinity of 2.3×10(4) m(-1). X-ray crystallography confirmed this, and the first complete crystal structure of sapB with a bound small molecule (CQ) is reported. The results suggest that sapB might play a role in mitigating CQ-based toxicity and that sapB might itself be overwhelmed by CQ causing impaired lipid degradation.

Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.

A purine analog synergizes with chloroquine (CQ) by targeting Plasmodium falciparum Hsp90 (PfHsp90).

BACKGROUND: Drug resistance, absence of an effective vaccine, and inadequate public health measures are major impediments to controlling Plasmodium falciparum malaria worldwide. The development of antimalarials to which resistance is less likely is paramount. To this end, we have exploited the chaperone function of P. falciparum Hsp90 (PfHsp90) that serves to facilitate the expression of resistance determinants. METHODS: The affinity and activity of a purine analogue Hsp90 inhibitor (PU-H71) on PfHsp90 was determined using surface plasmon resonance (SPR) studies and an ATPase activity assay, respectively. In vitro, antimalarial activity was quantified using flow cytometry. Interactors of PfHsp90 were determined by LC-MS/MS. In vivo studies were conducted using the Plasmodium berghei infection mouse model. RESULTS: PU-H71 exhibited antimalarial activity in the nanomolar range, displayed synergistic activity with chloroquine in vitro. Affinity studies reveal that the PfHsp90 interacts either directly or indirectly with the P. falciparum chloroquine resistance transporter (PfCRT) responsible for chloroquine resistance. PU-H71 synergized with chloroquine in the P.berghei mouse model of malaria to reduce parasitemia and improve survival. CONCLUSIONS: We propose that the interaction of PfHsp90 with PfCRT may account for the observed antimalarial synergy and that PU-H71 is an effective adjunct for combination therapy.

Glutathione transport: a new role for PfCRT in chloroquine resistance.

AIMS: Chloroquine (CQ) kills Plasmodium falciparum by binding heme, preventing its detoxification to hemozoin in the digestive vacuole (DV) of the parasite. CQ resistance (CQR) is associated with mutations in the DV membrane protein P. falciparum chloroquine resistance transporter (PfCRT), mediating the leakage of CQ from the DV. However, additional factors are thought to contribute to the resistance phenotype. This study tested the hypothesis that there is a link between glutathione (GSH) and CQR. RESULTS: Using isogenic parasite lines carrying wild-type or mutant pfcrt, we reveal lower levels of GSH in the mutant lines and enhanced sensitivity to the GSH synthesis inhibitor l-buthionine sulfoximine, without any alteration in cytosolic de novo GSH synthesis. Incubation with N-acetylcysteine resulted in increased GSH levels in all parasites, but only reduced susceptibility to CQ in PfCRT mutant-expressing lines. In support of a heme destruction mechanism involving GSH in CQR parasites, we also found lower hemozoin levels and reduced CQ binding in the CQR PfCRT-mutant lines. We further demonstrate via expression in Xenopus laevis oocytes that the mutant alleles of Pfcrt in CQR parasites selectively transport GSH. INNOVATION: We propose a mechanism whereby mutant pfcrt allows enhanced transport of GSH into the parasite's DV. The elevated levels of GSH in the DV reduce the level of free heme available for CQ binding, which mediates the lower susceptibility to CQ in the PfCRT mutant parasites. CONCLUSION: PfCRT has a dual role in CQR, facilitating both efflux of harmful CQ from the DV and influx of beneficial GSH into the DV.