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The over-dosing use of chloroquine phosphate (CQ) poses severe threats to human beings and ecosystem due to the high persistence and biotoxicity. The discharge of CQ into wastewater would affect the biomass activity and process stability during the biological processes, e.g., anammox. However, the response mechanism of anammox consortia to CQ remain unknown. In this study, the accurate role of extracellular polymeric substances barrier in attenuating the negative effects of CQ, and the mechanism on cytotoxic behavior were dissected by molecular spectroscopy and computational chemistry. Low concentrations (≤6.0 mg/L) of CQ hardly affected the nitrogen removal performance due to the adaptive evolution of EPS barrier and anammox bacteria. Compact protein of EPS barrier can bind more CQ (0.24 mg) by hydrogen bond and van der Waals force, among which O-H and amide II region respond CQ binding preferentially. Importantly, EPS contributes to the microbiota reshape with selectively enriching Candidatus_Kuenenia for self-protection. Furthermore, the macroscopical cytotoxic behavior was dissected at a molecular level by CQ fate/distribution and computational chemistry, suggesting that the toxicity was ascribed to attack of CQ on functional proteins of anammox bacteria with atom N17 (f - =0.1209) and C2 (f + =0.1034) as the most active electrophilic and nucleophilic sites. This work would shed the light on the fate and risk of non-antibiotics in anammox process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

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-β, 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.

Background: Clinical observation suggests the atheroprotective effect of chloroquine and its derivatives, while its mechanism remains unclear. This study aimed to observe the protective effect of chloroquine against atherosclerosis and explore the underlying mechanism., Methods: Ataxia telangiectasia mutated (ATM) wild-type or haploinsufficient apolipoprotein-E-knockout (ATM +/+ ApoE -/- or ATM +/- ApoE -/- ) mice were treated with different dosages of chloroquine. Anti-CD25 antibody was used to deplete natural Tregs in ATM +/+ ApoE -/- mice. The atherosclerotic burden in different groups of mice was comprehensively evaluated by H&E staining and Masson staining. The effect of chloroquine on the regulatory T cells (Tregs) was assessed in vivo and in vitro by flow cytometry and immunohistochemical staining. The expression of related proteins was detected by real-time polymerase chain reaction and western blotting., Results: In ATM +/+ ApoE -/- mice, chloroquine alleviated atherosclerotic lesions, stabilized the plaque, and increased Treg counts in the atherosclerotic lesions and spleens. However, in ATM haploinsufficient mice (ATM +/- ApoE -/- ), chloroquine no longer prevented atherosclerosis or impacted Treg counts. Abolishing Treg cells using an anti-CD25 antibody in vivo abrogated the atheroprotective effect of chloroquine. In vitro, chloroquine promoted the differentiation of Tregs from naïve T cells, which was accompanied by enhanced ATM/AMP-activated protein kinase (AMPK) activity and reduced downstream mammalian target of rapamycin (mTOR) activity., Discussion: These findings suggest that chloroquine ameliorates atherosclerosis and stabilizes plaque by modulating Tregs differentiation through the regulation of the ATM/AMPK/mTOR pathway., Competing Interests: The authors declare no conflicts of interest in this work., (Thieme. All rights reserved.)

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 β-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 β-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-1β, 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Lysosomal trapping, a physicochemical process in which lipophilic cationic compounds are sequestered in lysosomes, can affect drug disposition and cytotoxicity. To better understand lysosomal trapping at the outer blood-retinal barrier (BRB), we investigated the distribution of LysoTracker Red (LTR), a probe compound for lysosomal trapping, in conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. LTR uptake by RPE-J cells was dependent on temperature and attenuated by ammonium chloride and protonophore, which decreased the pH gradient between the lysosome and cytoplasm, suggesting lysosomal trapping of LTR in RPE-J cells. The involvement of lysosomal trapping in response to cationic drugs, including neuroprotectants such as desipramine and memantine, was also suggested by an inhibition study of LTR uptake. Chloroquine, which is known to show ocular toxicity, induced cytoplasmic vacuolization in RPE-J cells with a half-maximal effective concentration of 1.35 μM. This value was 59 times lower than the median lethal concentration (= 79.1 μM) of chloroquine, suggesting that vacuolization was not a direct trigger of cell death. These results are helpful for understanding the lysosomal trapping of cationic drugs, which is associated with drug disposition and cytotoxicity in the outer BRB., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Japanese Society for the Study of Xenobiotics. All rights reserved.)

Malaria parasites break down host haemoglobin into peptides and amino acids in the digestive vacuole for export to the parasite cytoplasm for growth: interrupting this process is central to the mode of action of several antimalarial drugs. Mutations in the chloroquine (CQ) resistance transporter, pfcrt, located in the digestive vacuole membrane, confer CQ resistance in Plasmodium falciparum, and typically also affect parasite fitness. However, the role of other parasite loci in the evolution of CQ resistance is unclear. Here we use a combination of population genomics, genetic crosses and gene editing to demonstrate that a second vacuolar transporter plays a key role in both resistance and compensatory evolution. Longitudinal genomic analyses of the Gambian parasites revealed temporal signatures of selection on a putative amino acid transporter (pfaat1) variant S258L, which increased from 0% to 97% in frequency between 1984 and 2014 in parallel with the pfcrt1 K76T variant. Parasite genetic crosses then identified a chromosome 6 quantitative trait locus containing pfaat1 that is selected by CQ treatment. Gene editing demonstrated that pfaat1 S258L potentiates CQ resistance but at a cost of reduced fitness, while pfaat1 F313S, a common southeast Asian polymorphism, reduces CQ resistance while restoring fitness. Our analyses reveal hidden complexity in CQ resistance evolution, suggesting that pfaat1 may underlie regional differences in the dynamics of resistance evolution, and modulate parasite resistance or fitness by manipulating the balance between both amino acid and drug transport., (© 2023. The Author(s).)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lysosomes are known to influence cholesterol trafficking into endoplasmic reticulum (ER) membranes. Though intracellular cholesterol levels are known to influence the lipid biosynthetic responses in ER, the specific effects of lysosomal modulation on these outcomes is not known. To demonstrate this, C2C12 cells were treated with chloroquine, a lysosomotropic agent, and its effects on cellular biosynthetic capacity, structural and functional status of ER was determined. In addition to its known effects on autophagy reduction, chloroquine treatment induced accumulation of total cellular lipid and ER-specific cholesterol content. It was also observed that chloroquine caused an increase in smooth-ER content with defects in overall protein turnover. Further, since ER and mitochondria function in close association through ER membrane contact sites, it is likely that lysosomal modulation also brings about associated changes in mitochondria. In this regard, we found that chloroquine reduces mitochondrial membrane potential and mitochondrial dynamics. Collectively, the differential biosynthetic response of rise in lipid content, but not protein content, cannot be accounted by merely considering that chloroquine induced suppression of autophagy causes defects in organelle function. In this defective autophagy scenario, both biosynthetic responses such as lipid and protein synthesis are expected to be reduced rather than only the latter, as observed with chloroquine. These findings suggest that cholesterol trafficking/distribution within cellular organelles could act as an intracellular mediator of differential biosynthetic remodelling in interconnected organelles., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hydroxychloroquine (HCQ) is a lysosomotropic agent that is commonly used for treating Sjögren's disease (SjD). However, its efficacy is controversial because of the divergent response to the drug among patients. In a subgroup of SjD patients, lysosome-associated membrane protein 3 (LAMP3) is elevated in expression in the salivary glands and promotes lysosomal dysregulation and lysosome-dependent apoptotic cell death. In this study, chloroquine (CQ) and its derivative HCQ were tested for their ability to prevent LAMP3-induced apoptosis, in vitro and on a mouse model of SjD. In addition, efficacy of HCQ treatment was retrospectively compared between high LAMP3 mRNA expression in minor salivary glands and those with LAMP3 mRNA levels comparable with healthy controls. Study results show that CQ treatment stabilized the lysosomal membrane in LAMP3-overexpressing cells via deactivation of cathepsin B, resulting in decreased apoptotic cell death. In mice with established SjD-like phenotype, HCQ treatment also significantly decreased apoptotic cell death and ameliorated salivary gland hypofunction. Retrospective analysis of SjD patients found that HCQ tended to be more effective in improving disease activity index, symptom severity and hypergammaglobulinemia in patients with high LAMP3 expression compared those with normal LAMP3 expression. Taken together, these findings suggested that by determining salivary gland LAMP3 mRNA level, a patient's response to HCQ treatment could be predicted. This finding may provide a novel strategy for guiding the development of more personalized medicine for SjD., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

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 (H 2 O 2 ) 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 H 2 O 2 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2022. Published by Elsevier B.V.)

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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Okombo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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, β-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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (© 2022 Wiley Periodicals LLC.)

Burns can cause cell death and irreversible tissue damage. We examined the pathway of human dermis fibroblasts cell death caused by skin burns and the roles of chloroquine in human skin keratinocytes HaCaT wound healing. Western blot assays were performed to assess expression of proteins associated with autophagy, apoptosis, and endoplasmic reticulum stress in skin cells following burns. Changes in apoptosis-related proteins were assessed using flow cytometry, and wound cell migration was examined using wound healing assays. The burn animal model was used to test whether chloroquine would promote wound healing. In human burned fibroblasts, expression of LC3B-II and Cleave-caspase-7 was increased, whereas expression of Beclin-1, p62, and Grp78 was decreased. Severe burn induced ER stress and ERK phosphorylation, but PD98059 or necrostatin-1 treatment cells did not affect expression of autophagy LC3B-II protein and can induce apoptosis. Even though added with TGF-β and FGF did not repair autophagy caused by burns. Suggesting that autophagy and apoptosis were involved in heat-injured mechanism. Recombinant Wnt3a protein can help restore expression of β-catenin which reduced following burns in keratinocytes. Wnt3a protein can promote migration of keratinocytes after burns. Interesting, chloroquine increased expression of LC3B-II protein and restored cell migration activity after 24 h of burns. Consistently, surgical dressing containing chloroquine promoted wound healing in a burn animal mode. Autophagy and Wnt/β-catenin is two signalling pathways that participate in cell repair and wound healing in human fibroblasts, keratinocytes. Surgical dressing containing chloroquine can recover wound healing in burned rats., Competing Interests: The authors have declared that no competing interests exist.

The chloroquine resistance transporter, PfCRT, is an essential factor during intraerythrocytic development of the human malaria parasite Plasmodium falciparum. PfCRT resides at the digestive vacuole of the parasite, where hemoglobin taken up by the parasite from its host cell is degraded. PfCRT can acquire several mutations that render PfCRT a drug transporting system expelling compounds targeting hemoglobin degradation from the digestive vacuole. The non-drug related function of PfCRT is less clear, although a recent study has suggested a role in oligopeptide transport based on studies conducted in a heterologous expression system. The uncertainty about the natural function of PfCRT is partly due to a lack of a null mutant and a dearth of functional assays in the parasite. Here, we report on the generation of a conditional PfCRT knock-down mutant in P. falciparum. The mutant accumulated oligopeptides 2 to at least 8 residues in length under knock-down conditions, as shown by comparative global metabolomics. The accumulated oligopeptides were structurally diverse, had an isoelectric point between 4.0 and 5.4 and were electrically neutral or carried a single charge at the digestive vacuolar pH of 5.2. Fluorescently labeled dipeptides and live cell imaging identified the digestive vacuole as the compartment where oligopeptides accumulated. Our findings suggest a function of PfCRT in oligopeptide transport across the digestive vacuolar membrane in P. falciparum and associated with it a role in nutrient acquisition and the maintenance of the colloid osmotic balance. IMPORTANCE The chloroquine resistance transporter, PfCRT, is important for the survival of the human malaria parasite Plasmodium falciparum. It increases the tolerance to many antimalarial drugs, and it is essential for the development of the parasite within red blood cells. While we understand the role of PfCRT in drug resistance in ever increasing detail, the non-drug resistance functions are still debated. Identifying the natural substrate of PfCRT has been hampered by a paucity of functional assays to test putative substrates in the parasite system and the absence of a parasite mutant deficient for the PfCRT encoding gene. By generating a conditional PfCRT knock-down mutant, together with comparative metabolomics and uptake studies using fluorescently labeled oligopeptides, we could show that PfCRT is an oligopeptide transporter. The oligopeptides were structurally diverse and were electrically neutral or carried a single charge. Our data support a function of PfCRT in oligopeptide transport.

Purpose: We investigated the autophagic response of rat Müller rMC-1 cells during a short-term high glucose challenge., Methods: rMC-1 cells were maintained in 5 mM glucose (LG) or exposed to 25 mM glucose (HG). Western blot analysis was used to evaluate the expression levels of markers of autophagy (LC3-II, p62) and glial activation (AQP4), as well as the activation of TRAF2/JNK, ERK and AKT pathways. Autophagic flux assessment was performed using the autophagy inhibitor chloroquine. ROS levels were measured by flow cytometry using dichlorofluorescein diacetate. ERK involvement in autophagy induction was addressed using the ERK inhibitor FR180204. The effect of autophagy inhibition on cell viability was evaluated by SRB assay., Results: Activation of autophagy was observed in the first 2-6 h of HG exposure. This early autophagic response was transient, not accompanied by an increase in AQP4 or in the phospho-activation of JNK, a key mediator of cellular response to oxidative stress, and required ERK activity. Cells exposed to HG had a lower viability upon autophagy inhibition by chloroquine, as compared to those maintained in LG., Conclusion: A short-term HG challenge triggers in rMC-1 cells a process improving the ability to cope with stressful conditions, which involves ERK and an early and transient autophagy activation., (© 2022. The Author(s).)

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 μ M, 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., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Songbo An and Mingyue Hu.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: S. Marastoni reports a Canadian patent number 3,116,081 pending. A. Madariaga reports personal fees from Clovis and AstraZeneca outside the submitted work. Z.J. Li reports a Canadian patent number to CA3,116,081 pending. I. Colombo reports personal fees from MSD; other from GSK, AstraZeneca, MSD, Bayer, and Oasmia outside the submitted work. J.P. Bruce reports other from Bowhead Health outside the submitted work. M. Koritzinsky reports other from The Princess Margaret Cancer Foundation during the conduct of the study; in addition, M. Koritzinsky has a Canadian patent number number 3,116,081 pending. B.G. Wouters reports grants from Ontario Institute for Cancer Research, Canadian Institutes for Cancer Research, and Princess Margaret Cancer Foundation during the conduct of the study; other from Northern Biologics outside the submitted work; in addition, B.G. Wouters has a Canadian patent number 3,116,081 issued. A.M. Oza is PI and on clinical trial steer- ing committees for trials with Clovis, GSK, AstraZeneca—all uncompensated. A.M. Oza is uncompensated CEO of Ozmosis Research, a Not For Profit Clin- ical Trials Management company associated with UHN. A.M. Joshua reports non-financial support from Mayne Pharma during the conduct of the study; other from Pricilium outside the submitted work; in addition, A.M. Joshua has a Canadian patent number 3,116,081 pending. S. Lheureux reports grants from OICR - TRI Ovarian Cancer during the conduct of the study; grants and personal fees from AstraZeneca, GSK; personal fees from Eisai, Merck, Shattuck Labs; grants from Roche, and outside the submitted work. No other disclosures were reported., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: Despite the discovery of many important molecules in diabetic nephropathy, there has been very limited progress in the management of diabetic kidney diseases and the design of new drugs. To fill this gap, the present study explored the expression of SIRT2 in high-glucose murine kidney foot cells and its impact on cell biological functions., Methods: Expression levels of SIRT2 in the MPC-5 of murine kidney foot cells after high and normal glucose treatment or in cells targeted with siRNA were detected using qRT-PCR. Cellular proliferation and programmed cell death were analyzed via the CCK8 assay and flow cell technique, separately. Levels of autophagy markers were measured by western blotting, and chloroquine treatment was applied to the cells to observe the effect of SIRT2 on cell proliferation and apoptosis after treatment., Results: The expression level of SIRT2 was remarkably upregulated in the high-GLU group in contrast to the low-GLU group. The cell proliferation and autophagy levels were significantly reduced, and apoptosis was remarkably reinforced in the high-GLU group in contrast to the normal GLU group. However, knocking down the expression level of SIRT2 caused an increase in cell proliferation and cell autophagy levels and significantly weakened apoptosis. Chloroquine influenced cell proliferation and apoptosis in cells targeted with SIRT2 siRNA., Conclusion: SIRT2 expression was upregulated in hyperglycaemic murine kidney foot cells, and knocking down the expression level of SIRT2 affected the biological function of the cells. We found that SIRT2 may modulate cell proliferation and apoptosis by regulating cell autophagy., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2022 Shuang Liu et al.)

There is currently a dearth of specific therapies to treat respiratory infections caused by the three related species of coronaviruses viz . SARS-CoV-2, SARS-CoV and MERS-CoV. Prevention from disease is currently the safest and most convenient alternative available. The present study aimed to evaluate the preventive and therapeutic effect of fifteen phytoconstituents from medicinal plants of Ayurveda against coronaviruses by in silico screening. All the phytoconstituents exhibited rapid GI absorption and bioavailability and most of them had no toxicity versus reference drug chloroquine. BAS analyses revealed that most of the phytocomponents had favorable bioactivity scores towards biological target proteins. Principal component analysis revealed that most of the phytoconstituents fell close to chloroquine in 3D projection of chemical space. Affinity of phytoconstituents towards SARS-CoV-2 spike protein-human ACE2 complex decreased as isomeldenin > tinosporaside > EGCG whereas in case of unbound ACE2, the strength of binding followed the order isomeldenin > tinosporaside > ellagic acid. Towards SARS-CoV-2 main and papain-like proteases, the affinity decreased as isomeldenin > EGCG > tinosporaside and EGCG > tinosporaside > isomeldenin, respectively. Most phytoconstituents displayed significant binding kinetics to the selected protein targets than chloroquine. SAR analysis revealed that isomeldenin, tinosporaside, EGCG and ellagic acid bind to viral spike glycoproteins via H-bond, Pi-Pi, Pi-sigma and Pi-alkyl type interactions. Molecular dynamics simulation of isomeldenin and EGCG with SARS-CoV and SARS-CoV-2 spike glycoproteins exhibited low deviations throughout the 100 ns simulation indicating good stability and compactness of the protein-ligand complexes. Thus, the above four phytoconstituents have the potential to emerge as prophylactic and therapeutic agents against coronaviruses if investigated further in vitro and in vivo .

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., (© 2021. The Author(s).)

Quinacrine (QC) and chloroquine (CQ) have antimicrobial and antiviral activities as well as antimalarial activity, although the mechanisms remain unknown. QC increased the antimicrobial activity against yeast exponentially with a pH-dependent increase in the cationic amphiphilic drug (CAD) structure. CAD-QC localized in the yeast membranes and induced glucose starvation by noncompetitively inhibiting glucose uptake as antipsychotic chlorpromazine (CPZ) did. An exponential increase in antimicrobial activity with pH-dependent CAD formation was also observed for CQ, indicating that the CAD structure is crucial for its pharmacological activity. A decrease in CAD structure with a slight decrease in pH from 7.4 greatly reduced their effects; namely, these drugs would inefficiently act on falciparum malaria and COVID-19 pneumonia patients with acidosis, resulting in resistance. The decrease in CAD structure at physiological pH was not observed for quinine, primaquine, or mefloquine. Therefore, restoring the normal blood pH or using pH-insensitive quinoline drugs might be effective for these infectious diseases with acidosis.

Nurr1/NR4A2 is an orphan nuclear receptor transcription factor implicated as a drug target for neurological disorders including Alzheimer's and Parkinson's diseases. Previous studies identified small-molecule NR4A nuclear receptor modulators, but it remains unclear if these ligands affect transcription via direct binding to Nurr1. We assessed 12 ligands reported to affect NR4A activity for Nurr1-dependent and Nurr1-independent transcriptional effects and the ability to bind the Nurr1 ligand-binding domain (LBD). Protein NMR structural footprinting data show that amodiaquine, chloroquine, and cytosporone B bind the Nurr1 LBD; ligands that do not bind include C-DIM12, celastrol, camptothecin, IP7e, isoalantolactone, ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), and three high-throughput screening hit derivatives. Importantly, ligands that modulate Nurr1 transcription also show Nurr1-independent effects on transcription in a cell type-specific manner, indicating that care should be taken when interpreting the functional response of these ligands in transcriptional assays. These findings should help focus medicinal chemistry efforts that desire to optimize Nurr1-binding ligands.

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 (3CL pro ); 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 R 2 = 0.9039 and Q 2 = 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 3CL pro 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.

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-β1), 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-β1, 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-β1 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2020 The Korean Academy of Medical Sciences.)

In the current spread of novel coronavirus (SARS-CoV-2), antiviral drug discovery is of great importance. AutoDock Vina was used to screen potential drugs by molecular docking with the structural protein and non-structural protein sites of new coronavirus. Ribavirin, a common antiviral drug, remdesivir, chloroquine and luteolin were studied. Honeysuckle is generally believed to have antiviral effects in traditional Chinese medicine. In this study, luteolin (the main flavonoid in honeysuckle) was found to bind with a high affinity to the same sites of the main protease of SARS-CoV-2 as the control molecule. Chloroquine has been proved clinically effective and can bind to the main protease; this may be the antiviral mechanism of this drug. The study was restricted to molecular docking without validation by molecular dynamics simulations. Interactions with the main protease may play a key role in fighting against viruses. Luteolin is a potential antiviral molecule worthy of attention., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Quantification of hydroxychloroquine (HCQ) and its two metabolites desethylchloroquine and desethylhydroxychloroquine in human blood can provide insight into the pharmacokinetic/pharmacodynamic characteristics of HCQ for the treatment of systemic lupus erythematosus (SLE), which is crucial for the optimization of the therapy. A simple, sensitive and optimized high performance liquid chromatography with fluorescence detection method has been developed and validated for the simultaneous determination of HCQ and its two metabolites in human blood. After addition of internal standard chloroquine, the blood sample was deproteinized with 2-fold acetonitrile and separated on an YMC-Triart C18 column (250 × 4.6 mm, 5 μm) with a mobile phase of 20 mM sodium phosphate buffer solution containing 0.25% triethylamine (pH 8.0)-acetonitrile (60:40, v/v). The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 337 and 405 nm, respectively. The method was linear over the range of 3-3000 ng/mL for all three analytes and the chromatographic run time was 9 min. The values for intra- and inter-day precisions were ranged from 1.3 to 7.3. This method was successfully applied to quantify the concentrations of HCQ and its two metabolites in blood of 92 SLE patients., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

Background: In clinical practice, chloroquine and hydroxychloroquine are often co-administered with other drugs in the treatment of malaria, chronic inflammatory diseases, and COVID-19. Therefore, their metabolic properties and the effects on the activity of cytochrome P450 (P450, CYP) enzymes and drug transporters should be considered when developing the most efficient treatments for patients., Methods: Scientific literature on the interactions of chloroquine and hydroxychloroquine with human P450 enzymes and drug transporters, was searched using PUBMED.Gov (https://pubmed.ncbi.nlm.nih.gov/) and the ADME database (https://life-science.kyushu.fujitsu.com/admedb/)., Results: Chloroquine and hydroxychloroquine are metabolized by P450 1A2, 2C8, 2C19, 2D6, and 3A4/5 in vitro and by P450s 2C8 and 3A4/5 in vivo by N-deethylation. Chloroquine effectively inhibited P450 2D6 in vitro; however, in vivo inhibition was not apparent except in individuals with limited P450 2D6 activity. Chloroquine is both an inhibitor and inducer of the transporter MRP1 and is also a substrate of the Mate and MRP1 transport systems. Hydroxychloroquine also inhibited P450 2D6 and the transporter OATP1A2., Conclusions: Chloroquine caused a statistically significant decrease in P450 2D6 activity in vitro and in vivo, also inhibiting its own metabolism by the enzyme. The inhibition indicates a potential for clinical drug-drug interactions when taken with other drugs that are predominant substrates of the P450 2D6. When chloroquine and hydroxychloroquine are used clinically with other drugs, substrates of P450 2D6 enzyme, attention should be given to substrate-specific metabolism by P450 2D6 alleles present in individuals taking the drugs., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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 αDβ2 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 αDβ2 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., Competing Interests: The authors have declared that no competing interests exist.

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 worldwide 1 . Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy 2 . 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 parasite 3-9 . 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 resistance 5 , 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, respectively 6,9 , 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.

Aim: Extracellular vesicles (EVs) are desirable delivery vehicles for therapeutic cargoes. We aimed to load EVs with Cre recombinase protein and determine whether functional delivery to cells could be improved by using endosomal escape enhancing compounds. Materials & methods: Overexpressed CreFRB protein was actively loaded into EVs by rapalog-induced dimerization to CD81FKBP, or passively loaded by overexpression in the absence of rapalog. Functional delivery of CreFRB was analysed using a HEK293 Cre reporter cell line in the absence and presence of endosomal escape enhancing compounds. Results:  The EVs loaded with CreFRB by both active and passive mechanisms were able to deliver functional CreFRB to recipient cells only in the presence of endosomal escape enhancing compounds chloroquine and UNC10217938A. Conclusion:  The use of endosomal escape enhancing compounds in conjunction with EVs loaded with therapeutic cargoes may improve efficacy of future EV based therapeutics.

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 (CQ GREEN ) is commercially available and was assessed for its suitability., Methods: IC 50 values were determined for both CQ and CQ GREEN in two CQS and two CQR Plasmodium falciparum strains. Buffer solutions with varying pH were used to determine pH-dependent localization of CQ GREEN in infected red blood cells. Before CQS or CQR parasites were exposed to different pH buffers, they were pre-loaded with varying concentrations of CQ GREEN for up to 7 h. Intracellular accumulation was analysed using live cell confocal microscopy. CQ GREEN uptake rates were determined for the cytosol and DV in the presence and absence of verapamil., Results: In CQS strains, twofold higher IC 50 values were determined for the CQ GREEN analogue compared to CQ. No significant differences in IC 50 values were observed in CQR strains. Addition of verapamil reversed drug resistance of CQR strains to both CQ and CQ GREEN . Live cell imaging revealed that CQ GREEN 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 CQ GREEN . Nevertheless, CQ GREEN uptake rates in CQR strains were reduced by 50% compared to CQS strains., Conclusion: Although fluorescence of CQ GREEN was mainly seen in the cytosol of parasites, IC 50 assays showed comparable efficacy of CQ GREEN and CQ in parasite killing of CQS and CQR strains. Reduced uptake rates of CQ GREEN in CQR strains compared to CQS strains indicate parasite-specific responses to CQ GREEN exposure. The data contains valuable information when CQ GREEN is used as an analogue for CQ.

Biochemical methods of transfection, including calcium phosphate-mediated and diethylaminoethyl (DEAE)-dextran-mediated transfection, have been used for many years to deliver nucleic acids into cultured cells. Here, we briefly review the use of calcium phosphate-DNA coprecipitates for transfection., (© 2019 Cold Spring Harbor Laboratory Press.)

This protocol describes a calcium phosphate-mediated transfection method for use with plasmid DNAs and adherent cells. At the end of the protocol is an alternative method for high-efficiency generation of stable transfectants., (© 2019 Cold Spring Harbor Laboratory Press.)

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 P23 pro-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 P23 pro-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., (© 2019 Wiley Periodicals, Inc.)

Previous studies have demonstrated that both dorsal raphe nucleus (DR) and insular cortex (IC) are critical for somatic sensory information transmission and regulation, especially for pain, and neurons in the DR send projection fibers to the IC. However, whether these ascending connections are involved in the processing of itch sensation remains unknown. In order to provide evidence for that, fluoro-gold (FG) retrograde tracing combined with immunofluorescent histochemical staining was performed for revealing the chemical nature of the projection neurons and the FOS expression induced by acute itch stimulation via intradermal histamine or chloroquine injection in the mouse. Both FOS- and p-ERK-containing neurons were increased in the DR and IC in the acute itch mice compared to those in the sham group. After FG was injected into the IC, FG-labeled retrograde neuronal cell bodies were observed in the whole extent of the brainstem, especially in the DR. About 81% of the total number of FG-labeled neurons in the DR showed serotonin (5-HT)-immunopositive staining. About 32% FG-labeled 5-HT-ergic neurons within DR expressed FOS in chroloquine-induced acute itch, whereas only 6% FG-labeled 5-HT-ergic neurons within DR expressed FOS in histamine-induced acute itch. These results provide morphological evidence for that there are 5-HT-ergic projections from the DR to IC which might be involved in the sensory information processing of acute itch. These results are helpful for understanding functional roles of 5-HT-ergic ascending projection under the condition of acute itch., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Plasmodium falciparum malaria is the most severe form of malaria with several complications. The malaria pigment-hemozoin (Hz) is associated with severe anemia, cytokine dysfunction, and immunosuppression, thus making it an interesting target for developing new strategies for antimalarial therapy. Monocytes (MO) in circulation actively ingest Hz released by Plasmodium parasites and secrete pro- and anti-inflammatory cytokines. M1 and M2 types represent the two major forms of MO/macrophages (MQ) with distinct phenotypes and opposing functions. Imbalance in the polarization of these types is reported in many infectious diseases. Though the association of Hz with immunosuppression is well documented, its role in activation of MO in context of M1/M2 phenotypes remains to be addressed. We report here that natural Hz drives human MO toward M2-like phenotype as evidenced by the expression of M2 signature markers. Hz-fed MO showed elevated transcript and secreted level of IL-10, CCL17, CCL1, expression of mannose-binding lectin receptor (CD206), and arginase activity. Hz attenuated HLA-DR expression, nitric oxide, and reactive oxygen species production, which are the features of M1 phenotype. Our data also implicate the involvement of p38 MAPK, PI3K/AKT, and NF-κB signaling pathways in skewing of Hz-fed MO toward M2-like type and suppression of mitogen-stimulated lymphocyte proliferation. Importantly, antimalarial drugs-chloroquine and artemisinin-partially reversed activation of Hz-induced MO toward M2-like phenotype. Considering the limitations in the current therapeutic options for malaria, we propose that these drugs may be re-examined for their potential as immunomodulators and candidates for adjunctive treatment in malaria., (© 2018 National Centre for Cell Sciences. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Tumor necrosis factor (TNF)‑related apoptosis‑inducing 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 6‑shogaol (6‑sho), a bioactive component of ginger, exerted anti‑inflammatory and anticancer properties, attenuated tumor cell propagation and induced TRAIL‑mediated cell death in liver cancer cells. The current study identified a potential pathway by revealing that TRAIL and 6‑sho or chloroquine acted together to trigger reactive oxygen species (ROS) production, to upregulate tumor‑suppressor protein 53 (p53) expression and to change the mitochondrial transmembrane potential (MTP). Treatment with N‑acetyl‑L‑cysteine reversed these effects, restoring the MTP and attenuated ROS production and p53 expression. Interestingly, treatment with 6‑sho increased p62 and microtubule‑associated proteins 1A/1B light chain 3B‑II levels, indicating an inhibited autophagy flux. In conclusion, attenuation of 6‑sho‑induced autophagy flux sensitized cells to TRAIL‑induced apoptosis via p53 and ROS, suggesting that the administration of TRAIL in combination with 6‑sho may be a suitable therapeutic method for the treatment of TRAIL‑resistant Huh7 liver cells.

It is well established that chloroquine, a quinoline antimalarial, inhibits hemozoin formation in the malaria parasite. Counterintuitively, this archetypal antimalarial is also used in the treatment of diseases in which hemozoin biocrystallization does not play a role. Hence, we decided to investigate whether chloroquine possesses binding targets other than Fe(III) protoporphyrin IX in blood stage Plasmodium falciparum parasites and whether these are related to sites of accumulation within the parasite other than the digestive vacuole. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled fluorescent derivative of chloroquine, especially sensitive to regions outside the digestive vacuole and retaining the antiplasmodial pharmacophore, was synthesized to investigate subcellular localization in the parasite. Super-resolution microscopy revealed association with membranes including the parasite plasma membrane, the endoplasmic reticulum, and possibly also the mitochondrion. A drug-labeled affinity matrix was then prepared to capture protein binding targets of chloroquine. SDS-PAGE revealed a single prominent band between 200 and 250 kDa from the membrane-associated fraction. Subsequent proteomic analysis revealed that this band corresponded to P. falciparum multidrug resistance-associated protein (PfMRP1). Intrigued by this finding, we demonstrated pull-down of PfMRP1 by matrices labeled with Cinchona alkaloids quinine and quinidine. While PfMRP1 has been implicated in resistance to quinolines and other antimalarials, this is the first time that these drugs have been found to bind directly to this protein. Based on previous reports, PfMRP1, the only prominent protein found to bind to quinolines in this work, is likely to modulate the activity of these antimalarials in P. falciparum rather than act as a drug target.

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., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 (Aβ), 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, Aβ 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 Aβ 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

DNA supercoiling plays critical roles in several essential DNA metabolic pathways, such as replication, transcription and recombination. Typically plasmid DNA molecules are used to measure DNA supercoiling status inside bacterial cells. In this chapter, we describe how to isolate plasmid DNA molecules from E. coli cells and determine DNA supercoiling density by 1% agarose gel electrophoresis containing chloroquine using plasmid pACYC184 as an example.