Search Results (65)

P-glycoprotein (Pgp) plays a significant role in the disposition of cardiac glycoside (CG) drugs across the cell membrane. The relatively narrow therapeutic indices of these drugs, coupled with the co-administration of drugs that inhibit Pgp’s transport mechanism, often cause an increased level of CG in the patient’s plasma, resulting in fatal arrhythmia. Therefore, understanding the underlying mechanism of the CG–Pgp interaction is necessary to circumvent Pgp-mediated transport and effectively design next-generation CGs. In this study, we conducted a comparative analysis to examine the interaction with Pgp and further understand the Pgp-mediated transport of digoxin, digitoxin, digoxigenin, and digitoxigenin. Through the drug-induced kinetic studies of Pgp, our findings suggest that each of the four drugs tested has a single binding site within Pgp. The CG–Pgp binding studies demonstrated that digoxin, digitoxin, and digoxigenin had relatively higher binding affinities. The CG-mediated conformational changes in Pgp indicated that each of the drugs shifts Pgp to an “outward-open” conformation in a nucleotide-dependent manner. STDD NMR indicated that the protons within the δ-lactone ring and the tri-D-digitoxose sugar moieties (glycones) predominantly interact with Pgp. Finally, a model was proposed for CG-induced Pgp-mediated ATP hydrolysis and transport by integrating our data with previously published Pgp-mediated CG transport results. Full article

Background/Objectives: The proton-coupled amino acid transporter (PAT1) is an intestinal absorptive solute carrier responsible for the oral bioavailability of some GABA-mimetic drug substances such as vigabatrin and gaboxadol. In the present work, we investigate if non-steroidal anti-inflammatory drug substances (NSAIDs) interact with substrate transport via human (h)PAT1. Methods: The transport of substrates via hPAT1 was investigated in Caco-2 cells using radiolabeled substrate uptake and in X. laevis oocytes injected with hPAT1 cRNA, measuring induced currents using the two-electrode voltage clamp technique. The molecular interaction between NSAIDs and hPAT1 was investigated using an AlphaFold2 model and molecular docking. Results: NSAIDs such as ibuprofen, diclofenac, and flurbiprofen inhibited proline uptake via hPAT1, with IC₅₀ values of 954 (logIC₅₀ 2.98 ± 0.1) µM, 272 (logIC₅₀ 2.43 ± 0.1) µM, and 280 (logIC₅₀ 2.45 ± 0.1) µM, respectively. Ibuprofen acted as a non-competitive inhibitor of hPAT1-mediated proline transport. In hPAT1-expressing oocytes, ibuprofen and diclofenac did not induce inward currents, and inhibited inward currents caused by proline. Molecular modeling pointed to a binding mode involving an allosteric site. Conclusions: NSAIDs interact with hPAT1 as non-translocated non-competitive inhibitors, and molecular modeling points to a binding mode involving an allosteric site distinct from the substrate binding site. The present findings could be used as a starting point for developing specific hPAT1 inhibitors. Full article

It has been previously established that breast cancer cells exhibit high expression of the monocarboxylate (lactate) transporters (MCT1 and/or MCT4) and carbonic anhydrase IX (CAIX) and form a functional metabolon for proton-coupled lactate export, thereby stabilizing intracellular pH. CD147 is the MCT accessory protein that facilitates the creation of the MCT/CAIX complex. This study describes how the small molecule Beta-Galactose 2C (BGal2C) blocks the physical and functional interaction between CAIX and either MCT1 or MCT4 in Xenopus oocytes, which reduces the rate of proton and lactate flux with an IC₅₀ of ~90 nM. This value is similar to the K_i for inhibition of CAIX activity. Furthermore, it is shown that BGal2C blocks hypoxia-induced lactate transport in MDA-MB-231 and MCF-7 breast cancer cells, both of which express CAIX. As in oocytes, BGal2C interferes with the physical interaction between CAIX and MCTs in both cell types. Finally, X-ray crystallographic studies highlight unique interactions between BGal2C and a CAIX-mimic that are not observed within the CAII active site and which may underlie the strong specificity of BGal2C for CAIX. These studies demonstrate the utility of a novel sulfonamide in interfering with elevated proton and lactate flux, a hallmark of many solid tumors. Full article

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc₁ complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway. Full article

α-glucosidase, a pharmacological target for type 2 diabetes mellitus (T2DM), is present in the intestinal brush border membrane and catalyzes the hydrolysis of sugar linkages during carbohydrate digestion. Since α-glucosidase inhibitors (AGIs) modulate intestinal metabolism, they may influence oxidative stress and glycolysis inhibition, potentially addressing intestinal dysfunction associated with T2DM. Herein, we report on a study of an ortho-carbonyl substituted hydroquinone series, whose members differ only in the number and position of methyl groups on a common scaffold, on radical-scavenging activities (ORAC assay) and correlate them with some parameters obtained by density functional theory (DFT) analysis. These compounds’ effect on enzymatic activity, their molecular modeling on α-glucosidase, and their impact on the mitochondrial respiration and glycolysis of the intestinal Caco-2 cell line were evaluated. Three groups of compounds, according their effects on the Caco-2 cells metabolism, were characterized: group A (compounds 2, 3, 5, 8, 9, and 10) reduces the glycolysis, group B (compounds 1 and 6) reduces the basal mitochondrial oxygen consumption rate (OCR) and increases the extracellular acidification rate (ECAR), suggesting that it induces a metabolic remodeling toward glycolysis, and group C (compounds 4 and 7) increases the glycolysis lacking effect on OCR. Compounds 5 and 10 were more potent as α-glucosidase inhibitors (AGIs) than acarbose, a well-known AGI with clinical use. Moreover, compound 5 was an OCR/ECAR inhibitor, and compound 10 was a dual agent, increasing the proton leak-driven OCR and inhibiting the maximal electron transport flux. Additionally, menadione-induced ROS production was prevented by compound 5 in Caco-2 cells. These results reveal that slight structural variations in a hydroquinone scaffold led to diverse antioxidant capability, α-glucosidase inhibition, and the regulation of mitochondrial bioenergetics in Caco-2 cells, which may be useful in the design of new drugs for T2DM and metabolic syndrome. Full article

We recently reported that cranberry proanthocyanidins (C-PACs) inhibit esophageal adenocarcinoma (EAC) by 83% through reversing reflux-induced bacterial, inflammatory and immune-implicated proteins and genes as well as reducing esophageal bile acids, which drive EAC progression. This study investigated whether C-PACs’ mitigation of bile reflux-induced transporter dysregulation mechanistically contributes to EAC prevention. RNA was isolated from water-, C-PAC- and reflux-exposed rat esophagi with and without C-PAC treatment. Differential gene expression was determined by means of RNA sequencing and RT-PCR, followed by protein assessments. The literature, coupled with the publicly available Gene Expression Omnibus dataset GSE26886, was used to assess transporter expression levels in normal and EAC patient biopsies for translational relevance. Significant changes in ATP-binding cassette (ABC) transporters implicated in therapeutic resistance in humans (i.e., Abcb1, Abcb4, Abcc1, Abcc3, Abcc4, Abcc6 and Abcc10) and the transport of drugs, xenobiotics, lipids, and bile were altered in the reflux model with C-PACs’ mitigating changes. Additionally, C-PACs restored reflux-induced changes in solute carrier (SLC), aquaporin, proton and cation transporters (i.e., Slc2a1, Slc7a11, Slc9a1, Slco2a1 and Atp6v0c). This research supports the suggestion that transporters merit investigation not only for their roles in metabolism and therapeutic resistance, but as targets for cancer prevention and targeting preventive agents in combination with chemotherapeutics. Full article

Proton transporters play a key role in maintaining the acidic tumor microenvironment; hence, their inhibition has been proposed as a new therapeutic treatment, although few methods can accurately assess their effect in vivo. In this study, we investigated whether MRI-CEST (Magnetic Resonance Imaging—Chemical Exchange Saturation Transfer) tumor pH imaging can be a useful tool to evaluate in vivo the therapeutic efficacy of several Proton Pump Inhibitors (PPIs) in breast cancer. Cell viability and extracellular pH assays were carried out in breast cancer cells cultured at physiological pH (7.4) or acid-adapted (pH of 6.5 and 6.8) following the exposure to inhibitors of V-ATPase (Lansoprazole, Esomeprazole) or NHE1 (Amiloride, Cariporide) at several concentrations. Next, triple-negative breast cancer 4T1 tumor-bearing mice were treated with Lansoprazole or Amiloride and MRI-CEST tumor pH imaging was utilized to assess the in vivo efficacy. Only Lansoprazole induced, in addition to breast cancer cell toxicity, a significant inhibition of proton extrusion. A significant reduction in tumor volume, prolonged survival, and increase in extracellular tumor pH after 1 and 2 weeks were observed after Lansoprazole treatment, whereas no significant changes were detected upon Amiloride treatment. Our results suggested that MRI-CEST tumor pH imaging can monitor the therapeutic efficacy of PPIs in breast cancer murine models. Full article

Lactose permease (LacY) from Escherichia coli belongs to the major facilitator superfamily. It facilitates the co-transport of β-galactosides, including lactose, into cells by using a proton gradient towards the cell. We now show that LacY is capable of scrambling glycerophospholipids across a membrane. We found that purified LacY reconstituted into liposomes at various protein to lipid ratios catalyzed the rapid translocation of fluorescently labeled and radiolabeled glycerophospholipids across the proteoliposome membrane bilayer. The use of LacY mutant proteins unable to transport lactose revealed that glycerophospholipid scrambling was independent of H⁺/lactose transport activity. Unexpectedly, in a LacY double mutant locked into an occluded conformation glycerophospholipid, scrambling activity was largely inhibited. The corresponding single mutants revealed the importance of amino acids G46 and G262 for glycerophospholipid scrambling of LacY. Full article

As mitochondria are negatively charged organelles, penetrating cations are used as parts of chimeric molecules to deliver specific compounds into mitochondria. In other words, they are used as electrophilic carriers for such chemical moieties as antioxidants, dyes, etc., to transfer them inside mitochondria. However, unmodified penetrating cations affect different aspects of cellular physiology as well. In this review, we have attempted to summarise the data about the side effects of commonly used natural (e.g., berberine) and artificial (e.g., tetraphenylphosphonium, rhodamine, methylene blue) penetrating cations on cellular physiology. For instance, it was shown that such types of molecules can (1) facilitate proton transport across membranes; (2) react with redox groups of the respiratory chain; (3) induce DNA damage; (4) interfere with pleiotropic drug resistance; (5) disturb membrane integrity; and (6) inhibit enzymes. Also, the products of the biodegradation of penetrating cations can be toxic. As penetrating cations accumulate in mitochondria, their toxicity is mostly due to mitochondrial damage. Mitochondria from certain types of cancer cells appear to be especially sensitive to penetrating cations. Here, we discuss the molecular mechanisms of the toxic effects and the anti-cancer activity of penetrating cations. Full article

When plants are exposed to environmental stress, their growth is inhibited. Under such conditions, controlled inhibition of growth is beneficial for plant survival. Jasmonic acid (JA) is a well-known phytohormone that limits plant growth, which has been confirmed in several species. However, its role in cucumber seedlings has not yet been comprehensively investigated. For this reason, we aimed to determine the involvement of JA in the regulation of proteins crucial for growth including plasma membrane proton pump (PM H⁺-ATPase), PM nitrate transporters, and nitrate reductase (NR). Treatment of cucumber seedlings with JA not only limited their growth but also increased the H₂O₂ content in their roots. The main sources of ROS generated for signalling purposes are PM NADPH oxidase (RBOH) and superoxide dismutase (SOD). Exposure of seedlings to JA induced the expression of some CsRBOH and SOD encoding genes, suggesting that ROS signalling can be activated by JA. As a consequence of JA exposure, the activity of all analysed proteins was inhibited and the expression of their genes was modified. The results indicate that reduction of PM H⁺-ATPase activity and the related decrease in nitrate uptake and assimilation are responsible for the root growth retardation of JA-treated plants. Full article

Brown adipocytes mainly utilize glucose and fatty acids to produce energy, which play key roles in thermogenesis. Furthermore, brown adipocytes also utilize other substrates, such as amino acids, for energy expenditure in various conditions. Here, we report the new physiological roles of proton-coupled amino acid transporters, SLC36A2 and SLC36A3, on global energy metabolism. The relative mRNA expression levels of both Slc36a2 and Slc36a3 were all highest in brown adipose tissue. We then generated global Slc36a2 and Slc36a3 knockout mice to investigate their functions in metabolism. Neither loss of Slc36a2 nor Slc36a3 affected the body weight and body composition of the mice. Slc36a2 knockout mice exhibited increased oxygen consumption during the daytime. After cold treatment, inhibition of Slc36a2 significantly decreased the mass of brown adipose tissue compared to wildtype mice, while it lowered the expression level of Cpt1a. Moreover, the serum lipid levels and liver mass were also decreased in Slc36a2 knockout mice after cold treatment. On the contrary, Slc36a3 knockout impaired glucose tolerance and up-regulated serum LDL-cholesterol concentration. Thus, SLC36A2 and SLC36A3 play central and different roles in the energy metabolism of the mice. Full article

Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative Acucp gene of the free-living protozoan amoeba, A. castellanii, encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete Acucp coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the S. cerevisiae strain InvSc1. Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the ΔSOD1 strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the A. castellanii UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins. Full article

Pleurotus ostreatus is widely cultivated in China. H₂O₂, as a signaling molecule, can regulate the formation of cap color, but its regulatory pathway is still unclear, severely inhibiting the breeding of dark-colored strains. In this study, 614 DEGs specifically regulated by H₂O₂ were identified by RNA-seq analysis. GO-enrichment analysis shows that DEGs can be significantly enriched in multiple pathways related to ATP synthesis, mainly including proton-transporting ATP synthesis complex, coupling factor F(o), ATP biosynthetic process, nucleoside triphosphate metabolic processes, ATP metabolic process, purine nucleoside triphosphate biosynthetic and metabolic processes, and purine ribonuclease triphosphate biosynthetic metabolic processes. Further KEGG analysis revealed that 23 DEGs were involved in cap color formation through the oxidative phosphorylation pathway. They were enriched in Complexes I, III, IV, and V in the respiratory chain. Further addition of exogenous uncoupling agents and ATP synthase inhibitors clarifies the important role of ATP synthesis in color formation. In summary, H₂O₂ may upregulate the expression of complex-encoding genes in the respiratory chain and promote ATP synthesis, thereby affecting the formation of cap color. The results of this study lay the foundation for the breeding of dark-colored strains of P. ostreatus and provide a basis for the color-formation mechanism of edible fungi. Full article

Acidification of cancerous tissue induced pharmacologically may slow tumor growth and can be detected using magnetic resonance imaging. Numerous studies have shown that pharmacologically inhibiting specific transporters, such as the Na⁺/H⁺ exchanger 1 (NHE1), can alter glycolitic metabolism and affect tumor acidosis. The sodium proton exchanger inhibitor Cariporide can acidify U87MG gliomas in mice. This study aimed to determine whether Cariporide could acidify C6 glioma tumors in rats with an intact immune system. C6 glioma cells were implanted in the right brain hemisphere of ten rats. Chemical exchange saturation transfer (CEST) MRI (9.4T) was acquired on days 7–8 and 14–15 after implantation to measure in vivo tissue intracellular pH (pH_i) within the tumors and on the contralateral side. pH_i was basic relative to contralateral tissue at both time points assessed using the amine and amide concentration-independent detection (AACID) value. On day 14–15, measurements were made before and up to 160 min after Cariporide injection (N = 6). Twenty minutes after drug injection, the average AACID value in the tumor significantly increased by ∼6.4% compared to pre-injection, corresponding to 0.31 ± 0.20 lower pH_i, while in contralateral tissue, AACID value increased significantly by ∼4.3% compared to pre-injection, corresponding to 0.22 ± 0.19 lower pH_i. Control rats without tumors showed no changes following injection of Cariporide dissolved in 10% or 1% DMSO and diluted in PBS. This study demonstrates the sensitivity of CEST-based pH-weighted imaging for monitoring the response of tumors to pharmacologically induced acidification. Full article

Telomerase is constitutively overexpressed in the majority of human cancers and telomerase inhibition provides a promising broad-spectrum anticancer therapeutic strategy. BIBR 1532 is a well-known synthetic telomerase inhibitor that blocks the enzymatic activity of hTERT, the catalytic subunit of telomerase. However, water insolubility of BIBR 1532 leads to low cellular uptake and inadequate delivery and thus, limits its anti-tumor effects. Zeolitic imidazolate framework-8 (ZIF-8) is considered as an attractive drug delivery vehicle for improved transport, release and anti-tumor effects of BIBR 1532. Herein, ZIF-8 and BIBR 1532@ZIF-8 were synthesized, respectively, and the physicochemical characterizations confirmed the successful encapsulation of BIBR 1532 in ZIF-8 coupled with an improved stability of BIBR 1532. ZIF-8 could alter the permeability of lysosomal membrane probably by the imidazole ring-dependent protonation. Moreover, ZIF-8 encapsulation facilitated the cellular uptake and release of BIBR 1532 with more accumulation in the nucleus. BIBR 1532 encapsulation with ZIF-8 triggered a more obvious growth inhibition of cancer cells as compared with free BIBR 1532. A more potent inhibition on hTERT mRNA expression, aggravated G0/G1 arrest accompanied with an increased cellular senescence were detected in BIBR 1532@ZIF-8-treated cancer cells. Our work has provided preliminary information on improving the transport, release and efficacy of water-insoluble small molecule drugs by using ZIF-8 as a delivery vehicle. Full article