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Connexin-36-positive gap junctions in ventral tegmental area GABA neurons sustain opiate dependence.The European Journal of Neuroscience Jun 2024Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can...
Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 ) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.
Topics: Animals; Ventral Tegmental Area; Connexins; GABAergic Neurons; Gap Junctions; Male; Rats; Gap Junction delta-2 Protein; Opioid-Related Disorders; Mefloquine; Mice; Rats, Sprague-Dawley; Pedunculopontine Tegmental Nucleus
PubMed: 38679044
DOI: 10.1111/ejn.16366 -
Chembiochem : a European Journal of... Apr 2024The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action...
The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of μM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.
PubMed: 38668388
DOI: 10.1002/cbic.202400116 -
Antimicrobial Agents and Chemotherapy May 2024Capillary samples offer practical benefits compared with venous samples for the measurement of drug concentrations, but the relationship between the two measures varies... (Comparative Study)
Comparative Study
Comparison of lumefantrine, mefloquine, and piperaquine concentrations between capillary plasma and venous plasma samples in pregnant women with uncomplicated falciparum and vivax malaria.
Capillary samples offer practical benefits compared with venous samples for the measurement of drug concentrations, but the relationship between the two measures varies between different drugs. We measured the concentrations of lumefantrine, mefloquine, piperaquine in 270 pairs of venous plasma and concurrent capillary plasma samples collected from 270 pregnant women with uncomplicated falciparum or vivax malaria. The median and range of venous plasma concentrations included in this study were 447.5 ng/mL (8.81-3,370) for lumefantrine (day 7, = 76, median total dose received 96.0 mg/kg), 17.9 ng/mL (1.72-181) for desbutyl-lumefantrine, 1,885 ng/mL (762-4,830) for mefloquine (days 3-21, = 90, median total dose 24.9 mg/kg), 641 ng/mL (79.9-1,950) for carboxy-mefloquine, and 51.8 ng/mL (3.57-851) for piperaquine (days 3-21, = 89, median total dose 52.2 mg/kg). Although venous and capillary plasma concentrations showed a high correlation (Pearson's correlation coefficient: 0.90-0.99) for all antimalarials and their primary metabolites, they were not directly interchangeable. Using the concurrent capillary plasma concentrations and other variables, the proportions of venous plasma samples predicted within a ±10% precision range was 34% (26/76) for lumefantrine, 36% (32/89) for desbutyl-lumefantrine, 74% (67/90) for mefloquine, 82% (74/90) for carboxy-mefloquine, and 24% (21/89) for piperaquine. Venous plasma concentrations of mefloquine, but not lumefantrine and piperaquine, could be predicted by capillary plasma samples with an acceptable level of agreement. Capillary plasma samples can be utilized for pharmacokinetic and clinical studies, but caution surrounding cut-off values is required at the individual level.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT01054248.
Topics: Humans; Female; Mefloquine; Antimalarials; Pregnancy; Quinolines; Lumefantrine; Malaria, Falciparum; Adult; Malaria, Vivax; Young Adult; Ethanolamines; Fluorenes; Adolescent; Piperazines
PubMed: 38597636
DOI: 10.1128/aac.00093-24 -
Cell Chemical Biology Apr 2024Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular...
Identification of new druggable protein targets remains the key challenge in the current antimalarial development efforts. Here we used mass-spectrometry-based cellular thermal shift assay (MS-CETSA) to identify potential targets of several antimalarials and drug candidates. We found that falcilysin (FLN) is a common binding partner for several drug candidates such as MK-4815, MMV000848, and MMV665806 but also interacts with quinoline drugs such as chloroquine and mefloquine. Enzymatic assays showed that these compounds can inhibit FLN proteolytic activity. Their interaction with FLN was explored systematically by isothermal titration calorimetry and X-ray crystallography, revealing a shared hydrophobic pocket in the catalytic chamber of the enzyme. Characterization of transgenic cell lines with lowered FLN expression demonstrated statistically significant increases in susceptibility toward MK-4815, MMV000848, and several quinolines. Importantly, the hydrophobic pocket of FLN appears amenable to inhibition and the structures reported here can guide the development of novel drugs against malaria.
Topics: Humans; Antimalarials; Malaria; Phenols; Quinolines; Drug Development; Methylamines
PubMed: 38593807
DOI: 10.1016/j.chembiol.2024.03.002 -
Biomedicine & Pharmacotherapy =... May 2024Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication...
Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca, Mg, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca-regulated kinases, such as Ca/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.
Topics: Humans; Connexin 43; Gap Junctions; Phosphorylation; Allosteric Regulation; HeLa Cells; Molecular Docking Simulation
PubMed: 38593702
DOI: 10.1016/j.biopha.2024.116550 -
Biomedicines Feb 2024Mefloquine (MQ) is a quinoline-based anti-malarial drug used for chemoprophylaxis or as a treatment in combination with artesunate. Although MQ has clear anti-...
Mefloquine (MQ) is a quinoline-based anti-malarial drug used for chemoprophylaxis or as a treatment in combination with artesunate. Although MQ has clear anti- properties, it can induce neurotoxicity and undesired neuropsychiatric side effects in humans. Hence, this study aimed to characterize the neurotoxicity of MQ using human neuroblastoma SH-SY5Y cells. The effects of MQ on neuronal toxicity and cell viability were investigated over a concentration range of 1-100 µM using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The influence of MQ on cellular bioenergetics was examined by measuring cellular ATP levels and from the induction of reactive oxygen species (ROS). An in silico approach was used to assess the potential neurotoxicity of MQ mediated via binding to the active sites of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and then experimentally validated via in vitro enzymatic assays. MQ was cytotoxic to neuronal cells in a concentration and exposure duration dependent manner and induced a significant reduction in viability at concentrations of ≥25 µM after a 24 h exposure. MQ adversely impacted cellular bioenergetics and significantly depleted ATP production at concentrations of ≥1 µM after 24 h. MQ-induced cellular ROS production, which was correlated with the induction of apoptosis, as revealed by flow cytometry. In silico studies suggested that MQ was a dual cholinesterase inhibitor and one with remarkably potent binding to BuChE. Modelling data were supported by in vitro studies which showed that MQ inhibited both human AChE and BuChE enzymes. In summary, MQ is an antimalarial drug that may induce neurotoxicity by impacting cellular bioenergetics and perturbing the activity of cholinesterases at exposure concentrations relevant to human dosage.
PubMed: 38540118
DOI: 10.3390/biomedicines12030505 -
International Urology and Nephrology Mar 2024This study aimed to integrate bioinformatics technology to explore shared hub genes and related mechanisms between diabetes and tuberculosis and to provide a theoretical...
OBJECTIVE
This study aimed to integrate bioinformatics technology to explore shared hub genes and related mechanisms between diabetes and tuberculosis and to provide a theoretical basis for revealing the disease mechanisms in patients with both diabetes and tuberculosis.
METHODS
Differentially expressed genes and Venn analysis were used to identify shared genes between diabetes and tuberculosis. PPI network analysis was used to screen key hub genes. GO and KEGG analyses were used to analyze the potential biological functions of these key hub genes. Immune infiltration analysis was performed using the ssGSEA algorithm. EnrichR online analysis website was used to explore potential therapeutic drugs.
RESULTS
The dataset analysis showed that PSMB9, ISG15, RTP4, CXCL10, GBP2, and GBP3 were six hub genes shared by diabetes and tuberculosis, which not only could distinguish between the two disease samples but also had a high diagnostic rate. GO and KEGG analyses showed that these six genes mainly mediate immune-related biological processes such as interferon, interleukin, and chemokine receptor binding, as well as signaling pathways such as RIG-I-like receptor, NOD-like receptor, and proteasome. Immune infiltration analysis showed that high expression of TIL may mediate the development of both diabetes and tuberculosis. In addition, suloctidil HL60 UP, thioridazine HL60 UP, mefloquine HL60 UP, 1-NITROPYRENE CTD 00001569, and chlorophyllin CTD 00000324 were the candidate drugs predicted by this study that were most likely to target hub genes.
CONCLUSION
Six differentially expressed genes shared by both diseases (PSMB9, ISG15, RTP4, CXCL10, GBP2, and GBP3) may play a key role in the disease progression of patients with both diabetes and tuberculosis. Candidate drugs targeting these hub genes have therapeutic potential and are worthy of further research. In summary, this study reveals potential shared pathogenic mechanisms between tuberculosis and diabetes.
PubMed: 38512440
DOI: 10.1007/s11255-024-04024-6 -
Antimicrobial Agents and Chemotherapy May 2024, caused by the parasite (), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of is...
, caused by the parasite (), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP levels in a ratiometric manner, thereby facilitating measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein and stably integrated the sensors into the genome of the NF54 cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development.
Topics: Plasmodium falciparum; Adenosine Triphosphate; Antimalarials; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Quinine; Doxycycline; Artemisinins; Chloroquine; Hydrogen-Ion Concentration
PubMed: 38501806
DOI: 10.1128/aac.01690-23 -
Frontiers in Cellular and Infection... 2024The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this...
INTRODUCTION
The global evolution of resistance to Artemisinin-based Combination Therapies (ACTs) by malaria parasites, will severely undermine our ability to control this devastating disease.
METHODS
Here, we have used whole genome sequencing to characterize the genetic variation in the experimentally evolved Plasmodium chabaudi parasite clone AS-ATNMF1, which is resistant to artesunate + mefloquine.
RESULTS AND DISCUSSION
Five novel single nucleotide polymorphisms (SNPs) were identified, one of which was a previously undescribed E738K mutation in a 26S proteasome subunit that was selected for under artesunate pressure (in AS-ATN) and retained in AS-ATNMF1. The wild type and mutated three-dimensional (3D) structure models and molecular dynamics simulations of the P. falciparum 26S proteasome subunit Rpn2 suggested that the E738K mutation could change the toroidal proteasome/cyclosome domain organization and change the recognition of ubiquitinated proteins. The mutation in the 26S proteasome subunit may therefore contribute to altering oxidation-dependent ubiquitination of the MDR-1 and/or K13 proteins and/or other targets, resulting in changes in protein turnover. In light of the alarming increase in resistance to artemisin derivatives and ACT partner drugs in natural parasite populations, our results shed new light on the biology of resistance and provide information on novel molecular markers of resistance that may be tested (and potentially validated) in the field.
Topics: Animals; Artesunate; Mefloquine; Antimalarials; Parasites; Malaria, Falciparum; Mutation; Whole Genome Sequencing; Plasmodium falciparum
PubMed: 38495651
DOI: 10.3389/fcimb.2024.1353057 -
Journal For Immunotherapy of Cancer Mar 2024Ferroptosis plays an important role in enhancing the efficacy of anti-programmed cell death 1 (PD-1) immunotherapy; however, the molecular mechanisms by which tumor...
BACKGROUND
Ferroptosis plays an important role in enhancing the efficacy of anti-programmed cell death 1 (PD-1) immunotherapy; however, the molecular mechanisms by which tumor ferroptosis sensitizes melanoma and lung cancer to anti-PD-1 immunotherapy have not been elucidated.
METHODS
Cytotoxicity assays, colony formation assays, flow cytometry and animal experiments were used to evaluate the effects of mefloquine (Mef) on survival and ferroptosis in melanoma and lung cancer. RNA sequencing, Real-time quantitative PCR (qRT-PCR), western blotting, chromatin immunoprecipitation-qPCR and flow cytometry were used to determine the molecular mechanisms by which Mef regulates lysophosphatidylcholine acyltransferase 3 (LPCAT3). The relationship between LPCAT3 and the efficacy of anti-PD-1 immunotherapy was verified via a clinical database and single-cell RNA sequencing (ScRNA-Seq).
RESULTS
In this study, we discovered that Mef induces ferroptosis. Furthermore, treatment with Mef in combination with T-cell-derived interferon-γ (IFN-γ) enhanced tumor ferroptosis and sensitized melanoma and lung cancer cells to anti-PD-1 immunotherapy. Mechanistically, Mef upregulated the expression of LPCAT3, a key gene involved in lipid peroxidation, by activating IFN-γ-induced STAT1-IRF1 signaling, and knocking down LPCAT3 impaired the induction of ferroptosis by Mef+IFN-γ. Clinically, analysis of the transcriptome and single-cell sequencing results in patients with melanoma showed that LPCAT3 expression was significantly lower in patients with melanoma than in control individuals, and LPCAT3 expression was positively correlated with the efficacy of anti-PD-1 immunotherapy.
CONCLUSIONS
In conclusion, our study demonstrated a novel mechanism by which LPCAT3 is regulated, and demonstrated that Mef is a highly promising new target that can be utilized to enhance the efficacy of anti-PD-1 immunotherapy.
Topics: Animals; Humans; Melanoma; Mefloquine; Interferon-gamma; Ferroptosis; Cell Line, Tumor; Lung Neoplasms; Immunotherapy; STAT1 Transcription Factor; Interferon Regulatory Factor-1; 1-Acylglycerophosphocholine O-Acyltransferase
PubMed: 38471712
DOI: 10.1136/jitc-2023-008554