Effects of Commonly used Surfactants, Poloxamer 188 and Tween 80, on the Drug Transport Capacity of Intestinal Glucose Transporters.

Some glycoside drugs can be transported through intestinal glucose transporters (IGTs). The surfactants used in oral drug preparations can affect the function of transporter proteins. This study aimed to investigate the effect of commonly used surfactants, Poloxamer 188 and Tween 80, on the drug transport capacity of IGTs. Previous studies have shown that gastrodin is the optimal drug substrate for IGTs. Gastrodin was used as a probe drug to evaluate the effect of these two surfactants on intestinal absorption in SD rats through pharmacokinetic and in situ single-pass intestinal perfusion. Then, the effects of the two surfactants on the expression of glucose transporters and tight-junction proteins were examined using RT-PCR and western blotting. Additionally, the effect of surfactants on intestinal permeability was evaluated through hematoxylin-eosin staining. The results found that all experimental for Poloxamer 188 (0.5%, 2.0% and 8.0%) and Tween 80 (0.1% and 2.0%) were not significantly different from those of the blank group. However, the AUC(0-∞) of gastrodin increased by approximately 32% when 0.5% Tween 80 was used. The changes in IGT expression correlated with the intestinal absorption of gastrodin. A significant increase in the expression of IGTs was observed at 0.5% Tween 80. In conclusion, Poloxamer 188 had minimal effect on the drug transport capacity of IGTs within the recommended limits of use. However, the expression of IGTs increased in response to 0.5% Tween 80, which significantly enhanced the drug transport capacity of IGTs. However, 0.1% and 2.0% Tween 80 had no significant effect.

A Quantity-Dependent Nonlinear Model of Sodium Cromoglycate Suppression on Beta-Conglycinin Transport.

Understanding the transport mechanism is crucial for developing inhibitors that block allergen absorption and transport and prevent allergic reactions. However, the process of how beta-conglycinin, the primary allergen in soybeans, crosses the intestinal mucosal barrier remains unclear. The present study indicated that the transport of beta-conglycinin hydrolysates by IPEC-J2 monolayers occurred in a time- and quantity-dependent manner. The beta-conglycinin hydrolysates were absorbed into the cytoplasm of IPEC-J2 monolayers, while none were detected in the intercellular spaces. Furthermore, inhibitors such as methyl-beta-cyclodextrin (MßCD) and chlorpromazine (CPZ) significantly suppressed the absorption and transport of beta-conglycinin hydrolysates. Of particular interest, sodium cromoglycate (SCG) exhibited a quantity-dependent nonlinear suppression model on the absorption and transport of beta-conglycinin hydrolysates. In conclusion, beta-conglycinin crossed the IPEC-J2 monolayers through a transcellular pathway, involving both clathrin-mediated and caveolae-dependent endocytosis mechanisms. SCG suppressed the absorption and transport of beta-conglycinin hydrolysates by the IPEC-J2 monolayers by a quantity-dependent nonlinear model via clathrin-mediated and caveolae-dependent endocytosis. These findings provide promising targets for both the prevention and treatment of soybean allergies.

Effects of Three Kinds of Carbohydrate Pharmaceutical Excipients-Fructose, Lactose and Arabic Gum on Intestinal Absorption of Gastrodin through Glucose Transport Pathway in Rats.

BACKGROUND: Some glucoside drugs can be transported via intestinal glucose transporters (IGTs), and the presence of carbohydrate excipients in pharmaceutical formulations may influence the absorption of them. This study, using gastrodin as probe drug, aimed to explore the effects of fructose, lactose, and arabic gum on intestinal drug absorption mediated by the glucose transport pathway. METHODS: The influence of fructose, lactose, and arabic gum on gastrodin absorption was assessed via pharmacokinetic experiments and single-pass intestinal perfusion. The expression of sodium-dependent glucose transporter 1 (SGLT1) and sodium-independent glucose transporter 2 (GLUT2) was quantified via RT‒qPCR and western blotting. Alterations in rat intestinal permeability were evaluated through H&E staining, RT‒qPCR, and immunohistochemistry. RESULTS: Fructose reduced the area under the curve (AUC) and peak concentration (Cmax) of gastrodin by 42.7% and 63.71%, respectively (P < 0.05), and decreased the effective permeability coefficient (Peff) in the duodenum and jejunum by 58.1% and 49.2%, respectively (P < 0.05). SGLT1 and GLUT2 expression and intestinal permeability remained unchanged. Lactose enhanced the AUC and Cmax of gastrodin by 31.5% and 65.8%, respectively (P < 0.05), and increased the Peff in the duodenum and jejunum by 33.7% and 26.1%, respectively (P < 0.05). SGLT1 and GLUT2 levels did not significantly differ, intestinal permeability increased. Arabic gum had no notable effect on pharmacokinetic parameters, SGLT1 or GLUT2 expression, or intestinal permeability. CONCLUSION: Fructose, lactose, and arabic gum differentially affect intestinal drug absorption through the glucose transport pathway. Fructose competitively inhibited drug absorption, while lactose may enhance absorption by increasing intestinal permeability. Arabic gum had no significant influence.

Effects of allopurinol and febuxostat on uric acid transport and transporter expression in human umbilical vein endothelial cells.

Uric acid induces radical oxygen species formation, endothelial inflammation, and endothelial dysfunction which contributes to the progression of atherosclerosis. Febuxostat inhibits BCRP- and allopurinol stimulates MRP4-mediated uric acid efflux in human embryonic kidney cells. We hypothesized that endothelial cells express uric acid transporters that regulate intracellular uric acid concentration and that modulation of these transporters by febuxostat and allopurinol contributes to their different impact on cardiovascular mortality. The aim of this study was to explore a potential difference between the effect of febuxostat and allopurinol on uric acid uptake by human umbilical vein endothelial cells. Febuxostat increased intracellular uric acid concentrations compared with control. In contrast, allopurinol did not affect intracellular uric acid concentration. In line with this observation, febuxostat increased mRNA expression of GLUT9 and reduced MRP4 expression, while allopurinol did not affect mRNA expression of these uric acid transporters. These findings provide a possible pathophysiological pathway which could explain the higher cardiovascular mortality for febuxostat compared to allopurinol but should be explored further.

Pleurotus eryngii polysaccharides alleviate aflatoxin B1-induced liver inflammation in ducks involving in remodeling gut microbiota and regulating SCFAs transport via the gut-liver axis.

Aflatoxin B1 (AFB1) is one of the most widespread contaminants in agricultural commodities. Pleurotus eryngii (PE) is widely used as a feed additive for its anti-inflammatory properties, and its major active substance is believed to be polysaccharides. This study aims to explore the underlying mechanism of dietary PE polysaccharides alleviating AFB1-induced toxicity in ducks. The major monosaccharide components of PE polysaccharides were identified as glucose, mannose, galactose, glucuronic acid, and fucose. The results showed that dietary PE polysaccharides could alleviate liver inflammation, alleviate intestinal barrier dysfunction, and change the imbalanced gut microbiota induced by AFB1 in ducks. However, PE polysaccharides failed to exert protective roles on the liver and intestine injury induced by AFB1 in antibiotic-treated ducks. The PE + AFB1-originated microbiota showed a positive effect on intestinal barrier and inflammation, the SCFAs transport via the gut-liver axis, and liver inflammation compared with the AFB1-originated microbiota in ducks. These findings provided a possible mechanism that PE polysaccharides alleviated AFB1-induced liver inflammation in ducks by remodeling gut microbiota, regulating microbiota-derived SCFAs transport via the gut-liver axis, and inhibiting inflammatory gene expressions in the liver, which may provide new insight for therapeutic methods against AFB1 exposure in animals.

Oral sericin ameliorates type 2 diabetes through passive intestinal and bypass transport into the systemic circulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Boiled silkworm cocoons have been used to treat 'Xiaoke disease' (diabetes mellitus) recorded in Chinese medicine for over 800 years. In recent years, it has been found that the active substance silk sericin (SS) has therapeutic benefits in treating type 2 diabetes mellitus (T2DM). SS promotes pancreatic islet signalling, the proliferation of pancreatic islet cells, and insulin secretion. It is inferred that SS enters the bloodstream after oral administration and plays a role in the body's circulation. As a natural protein, SS needs to resist digestion by proteases in the gastrointestinal tract and cross the gastrointestinal barrier after oral administration. It is currently unclear how SS crosses the gastrointestinal barrier and whether it exerts therapeutic effects on T2DM by entering the circulation. AIM OF THE STUDY: To study how SS crosses the gastrointestinal barrier and whether it enters the body circulation to exert a therapeutic effect on T2DM. MATERIALS AND METHODS: SS was extracted from silkworm cocoons using an alkaline method with sodium carbonate. The antidigestive capacity of SS was detected using SDS-PAGE gel electrophoresis experiments. The mode of uptake and translocation of orally consumed SS in vivo was analysed using the AP-side to BL-side and BL-side-AP-side translocations, apparent Permeability coefficient (Papp), and Exocytosis rates (ER). The study compared the differences between the adSS group and the adSS + EDTA group by using Ethylenediaminetetraacetic acid (EDTA) to separate the tight junctions between Caco-2 cells. The aim was to analyze whether the transport mode of oral filaggrin proteins in vivo could be absorbed by bypass transport. By administering SS through oral and intraperitoneal injection to type 2 diabetic mice, we measured its concentration in the blood, as well as blood glucose and insulin levels, to determine its effectiveness in treating diabetes and its ability to enter the body's circulation for treatment. RESULTS: The molecular weight of SS decreased from 10k∼25 kDa to 10k∼15 kDa after in vitro simulated gastrointestinal fluid digestion, indicating its good antidigestive properties. The apparent Papp was greater than 1 × 10-6 cm·s-1, and the ER was between 0.5 and 1.5, indicating that adSS was well-absorbed and mainly passively transported. The Caco-2 cell model showed that the addition of EDTA promoted the transport of adSS, resulting in significantly larger Papp and ER values, indicating that adSS was absorbed by bypass transport. After oral administration of SS, the concentration of SS in the blood was lower than after intraperitoneal injection, which is 60% of intraperitoneal administration. Mice with a T2DM model who were administered SS for 5 weeks showed significant improvement in insulin resistance and glucose tolerance. Additionally, the pancreatic tissue appeared more regular. In the treatment of T2DM, injections of SS have been shown to be more effective than oral administration. Both oral and intraperitoneal injections have been partially involved in the circulation. CONCLUSIONS: SS is enzymatically cleaved by proteolytic enzymes in the gastrointestinal tract. The smaller molecules are partially absorbed into the body's circulation through passive and paracrine transport, exerting a therapeutic effect on T2DM.

Interactions between corn starch and lingonberry polyphenols and their effects on starch digestion and glucose transport.

This study investigated the interaction mechanism between corn starch (CS) and lingonberry polyphenols (LBP) during starch gelatinization, focusing on their effects on starch structure and physicochemical properties. Moreover, it explored the effect of this interaction on starch digestion and glucose transport. The results indicated that LBP interacted non-covalently with CS during starch gelatinization, disrupted the short-range ordered structure of starch, decreased gelatinization enthalpy of starch, and formed a dense network structure. Furthermore, the incorporation of LBP remarkably reduced the digestibility of CS. In particular, the addition of 10 % LBP decreased the terminal digestibility (C∞) from 77.87 % to 60.43 % and increased the amount of resistant starch (RS) by 21.63 %. LBP was found to inhibit α-amylase and α-glucosidase in a mixed manner. Additionally, LBP inhibited glucose transport in Caco-2 cells following starch digestion. When 10 % LBP was added, there was a 34.17 % decrease in glucose transport compared with starch digestion without LBP. This study helps establish the foundation for the development of LBP-containing starch or starch-based healthy foods and provides new insights into the mechanism by which LBP lowers blood glucose.

MCF-7 cell - derived exosomes were involved in protecting source cells from the damage caused by tributyltin chloride via transport function.

Tributyltin chloride (TBTC) is a ubiquitous environmental pollutant with various adverse effects on human health. Exosomes are cell - derived signaling and substance transport vesicles. This investigation aimed to explore whether exosomes could impact the toxic effects caused by TBTC via their transport function. Cytotoxicity, DNA and chromosome damage caused by TBTC on MCF-7 cells were analyzed with CCK-8, flow cytometry, comet assay and micronucleus tests, respectively. Exosomal characterization and quantitative analysis were performed with ultracentrifugation, transmission electron microscope (TEM) and bicinchoninic acid (BCA) methods. TBTC content in exosomes was detected with Liquid Chromatography-Mass Spectrometry (LC-MS). The impacts of exosomal secretion on the toxic effects of TBTC were analyzed. Our data indicated that TBTC caused significant cytotoxicity, DNA and chromosome damage effects on MCF-7 cells, and a significantly increased exosomal secretion. Importantly, TBTC could be transported out of MCF-7 cells by exosomes. Further, when exosomal secretion was blocked with GW4869, the toxic effects of TBTC were significantly exacerbated. We concluded that TBTC promoted exosomal secretion, which in turn transported TBTC out of the source cells to alleviate its toxic effects. This investigation provided a novel insight into the role and mechanism of exosomal release under TBTC stress.

Kinetic Properties and Pharmacological Modulation of High- and Low-Affinity Dopamine Transport in Striatal Astrocytes of Adult Rats.

Astrocytes actively participate in neurotransmitter homeostasis by bidirectional communication with neuronal cells, a concept named the tripartite synapse, yet their role in dopamine (DA) homeostasis remains understudied. In the present study, we investigated the kinetic and molecular mechanisms of DA transport in cultured striatal astrocytes of adult rats. Kinetic uptake experiments were performed using radiolabeled [3H]-DA, whereas mRNA expression of the dopamine, norepinephrine, organic cation and plasma membrane monoamine transporters (DAT, NET, OCTs and PMAT) and DA receptors D1 and D2 was determined by qPCR. Additionally, astrocyte cultures were subjected to a 24 h treatment with the DA receptor agonist apomorphine, the DA receptor antagonist haloperidol and the DA precursor L-DOPA. [3H]-DA uptake exhibited temperature, concentration and sodium dependence, with potent inhibition by desipramine, nortriptyline and decynium-22, suggesting the involvement of multiple transporters. qPCR revealed prominent mRNA expression of the NET, the PMAT and OCT1, alongside lower levels of mRNA for OCT2, OCT3 and the DAT. Notably, apomorphine significantly altered NET, PMAT and D1 mRNA expression, while haloperidol and L-DOPA had a modest impact. Our findings demonstrate that striatal astrocytes aid in DA clearance by multiple transporters, which are influenced by dopaminergic drugs. Our study enhances the understanding of regional DA uptake, paving the way for targeted therapeutic interventions in dopaminergic disorders.

Overexpression of OsTIP1;2 confers arsenite tolerance in rice and reduces root-to-shoot translocation of arsenic.

Tonoplast Intrinsic Proteins (TIPs) are vital in transporting water and solutes across vacuolar membrane. The role of TIPs in the arsenic stress response is largely undefined. Rice shows sensitivity to the arsenite [As[III]] stress and its accumulation at high concentrations in grains poses severe health hazards. In this study, functional characterization of OsTIP1;2 from Oryza sativa indica cultivar Pusa Basmati-1 (PB-1) was done under the As[III] stress. Overexpression of OsTIP1;2 in PB-1 rice conferred tolerance to As[III] treatment measured in terms of enhanced shoot growth, biomass, and shoot/root ratio of overexpression (OE) lines compared to the wild-type (WT) plants. Moreover, seed priming with the IRW100 yeast cells (deficient in vacuolar membrane As[III] transporter YCF1) expressing OsTIP1;2 further increased As[III] stress tolerance of both WT and OE plants. The dithizone assay showed that WT plants accumulated high arsenic in shoots, while OE lines accumulated more arsenic in roots than shoots thereby limiting the translocation of arsenic to shoot. The activity of enzymatic and non-enzymatic antioxidants also increased in the OE lines on exposure to As[III]. The tissue-specific localization showed OsTIP1;2 promoter activity in root and root hairs, indicating its possible root-specific function. After As[III] treatment in hydroponic medium, the arsenic translocation factor (TF) for WT was around 0.8, while that of OE lines was around 0.2. Moreover, the arsenic content in the grains of OE lines reduced significantly compared to WT plants.

Digestion of surfactants does not affect their ability to inhibit P-gp-mediated transport in vitro.

While various non-ionic surfactants at low concentrations have been shown to increase the transport of P-gp substrates in vitro, in vivo studies in rats have shown that a higher surfactant concentration is needed to increase the oral absorption of e.g. the P-gp substrates digoxin and etoposide. The aim of the present study was to investigate if intestinal digestion of surfactants could be the reason for this deviation between in vitro and in vivo data. Therefore, Kolliphor EL, Brij-L23, Labrasol and polysorbate 20 were investigated for their ability to inhibit P-gp and increase digoxin absorption in vitro. Transport studies were performed in Caco-2 cells, while P-gp inhibition and cell viability assays were performed in MDCKII-MDR1 cells. Polysorbate 20, Kolliphor EL and Brij-L23 increased absorptive transport and decreased secretory digoxin transport in Caco-2 cells, whereas only polysorbate 20 and Brij-L23 showed P-gp inhibiting properties in the MDCKII-MDR1 cells. Polysorbate 20 and Brij-L23 were chosen for in vitro digestion prior to transport- or P-gp inhibiting assays. Brij-L23 was not digestible, whereas polysorbate 20 reached a degree of digestion around 40%. Neither of the two surfactants showed any significant difference in their ability to affect absorptive or secretory transport of digoxin after pre-digestion. Furthermore, the P-gp inhibiting effects of polysorbate 20 were not decreased significantly. In conclusion, the mechanism behind the non-ionic surfactant mediated in vitro P-gp inhibition seemed independent of the intestinal digestion and the results presented here did not suggest it to be the cause of the observed discrepancy between in vitro and in vivo.

Azelaic acid can efficiently compete for the auxin binding site TIR1, altering auxin polar transport, gravitropic response, and root growth and architecture in Arabidopsisthaliana roots.

The present study investigates the phytotoxic potential of azelaic acid (AZA) on Arabidopsis thaliana roots. Effects on root morphology, anatomy, auxin content and transport, gravitropic response and molecular docking were analysed. AZA inhibited root growth, stimulated lateral and adventitious roots, and altered the root apical meristem by reducing meristem cell number, length and width. The treatment also slowed down the roots' gravitropic response, likely due to a reduction in statoliths, starch-rich organelles involved in gravity perception. In addition, auxin content, transport and distribution, together with PIN proteins' expression and localisation were altered after AZA treatment, inducing a reduction in auxin transport and its distribution into the meristematic zone. Computational simulations showed that AZA has a high affinity for the auxin receptor TIR1, competing with auxin for the binding site. The AZA binding with TIR1 could interfere with the normal functioning of the TIR1/AFB complex, disrupting the ubiquitin E3 ligase complex and leading to alterations in the response of the plant, which could perceive AZA as an exogenous auxin. Our results suggest that AZA mode of action could involve the modulation of auxin-related processes in Arabidopsis roots. Understanding such mechanisms could lead to find environmentally friendly alternatives to synthetic herbicides.

Inhibition of human drug transporter activities by succinate dehydrogenase inhibitors.

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

The inflammatory injury of porcine small intestinal epithelial cells induced by deoxynivalenol is related to the decrease in glucose transport.

The present study aimed to investigate the effects of deoxynivalenol (DON) stimulation on inflammatory injury and the expression of the glucose transporters sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter protein 2 (GLU2) in porcine small intestinal epithelial cells (IPEC-J2). Additionally, the study aimed to provide initial insights into the connection between the expression of glucose transporters and the inflammatory injury of IPEC-J2 cells. DON concentration and DON treatment time were determined using the CCK­8 assay. Accordingly, 1.0 µg/mL DON and treatment for 24 h were chosen for subsequent experiments. Then IPEC-J2 cells were treated without DON (CON, N = 6) or with 1 µg/mL DON (DON, N = 6). Lactate dehydrogenase (LDH) content, apoptosis rate, and proinflammatory cytokines including interleukin (IL)-1ß, Il-6, and tumor necrosis factor α (TNF-α) were measured. Additionally, the expression of AMP-activated protein kinase α1 (AMPK-α1), the content of glucose, intestinal alkaline phosphatase (AKP), and sodium/potassium-transporting adenosine triphosphatase (Na+/K+-ATPase) activity, and the expression of SGLT1 and GLU2 of IPEC-J2 cells were also analyzed. The results showed that DON exposure significantly increased LDH release and apoptosis rate of IPEC-J2 cells. Stimulation with DON resulted in significant cellular inflammatory damage, as evidenced by a significant increase in proinflammatory cytokines (IL-1ß, IL-6, and TNF-α). Additionally, DON caused damage to the glucose absorption capacity of IPEC-J2 cells, indicated by decreased levels of glucose content, AKP activity, Na+/K+-ATPase activity, AMPK-α1 protein expression, and SGLT1 expression. Correlation analysis revealed that glucose absorption capacity was negatively correlated with cell inflammatory cytokines. Based on the findings of this study, it can be preliminarily concluded that the cell inflammatory damage caused by DON may be associated with decreased glucose absorption.

Glucose is one of the most basic nutrients necessary to sustain animal life and plays a crucial role in animal body composition and energy metabolism. Previous studies suggested a link between glucose absorption and inflammatory injury. In the present study, deoxynivalenol (DON) stimulation caused severe inflammatory injury and reduced the glucose absorption capacity of IPEC-J2 cells. Pearson's correlation analysis revealed a negative correlation between glucose absorption capacity and cell inflammatory cytokines. Ultimately, it can be speculated that the cellular inflammatory response triggered by DON may be related to the altered expression of glucose transporters.

The decreased interface tension increased the transmembrane transport of soy hull polysaccharide-derived SCFAs in the Caco-2 cells.

Polysaccharides impact intestinal fermentation and regulate interfacial properties which affect absorption and transportation. Short-chain fatty acids (SCFAs), the main metabolites of soy hull polysaccharide lysate, are readily absorbed by the body and perform various physiological functions. We analysed the interfacial properties and transport of soy hull polysaccharide-derived SCFAs in the Caco-2 cell model to clarify the transmembrane transport mechanism. The results showed that the interfacial properties of the co-culture system were influenced by both transit time and concentration of SCFAs, the uptake and transit rates of SCFAs at 1-3 h increased significantly with time (P < 0.05). With increasing transit time and concentration, the transit rates of SCFAs on the apical side (AP) â†’ basolateral side (BL) and BL â†’ AP sides increased and then stabilised, the transit rate of the AP â†’ BL side was higher than that of the BL â†’ AP side. Proteomic analysis showed that soy hull polysaccharide-derived SCFAs resulted in the differential expression of 285 upregulated and 501 downregulated after translocation across Caco-2 cells. The differentially expressed proteins were mainly enriched in ribosomes, oxidative phosphorylation, nuclear transport, and SNARE vesicular transport. This study lays the theoretical foundation for understanding the structure-activity relationship of soy hull polysaccharides in the intestine.

A new antibiotic traps lipopolysaccharide in its intermembrane transporter.

Gram-negative bacteria are extraordinarily difficult to kill because their cytoplasmic membrane is surrounded by an outer membrane that blocks the entry of most antibiotics. The impenetrable nature of the outer membrane is due to the presence of a large, amphipathic glycolipid called lipopolysaccharide (LPS) in its outer leaflet1. Assembly of the outer membrane requires transport of LPS across a protein bridge that spans from the cytoplasmic membrane to the cell surface. Maintaining outer membrane integrity is essential for bacterial cell viability, and its disruption can increase susceptibility to other antibiotics2-6. Thus, inhibitors of the seven lipopolysaccharide transport (Lpt) proteins that form this transenvelope transporter have long been sought. A new class of antibiotics that targets the LPS transport machine in Acinetobacter was recently identified. Here, using structural, biochemical and genetic approaches, we show that these antibiotics trap a substrate-bound conformation of the LPS transporter that stalls this machine. The inhibitors accomplish this by recognizing a composite binding site made up of both the Lpt transporter and its LPS substrate. Collectively, our findings identify an unusual mechanism of lipid transport inhibition, reveal a druggable conformation of the Lpt transporter and provide the foundation for extending this class of antibiotics to other Gram-negative pathogens.

A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Screening for endocrine disrupting chemicals inhibiting monocarboxylate 8 (MCT8) transporter facilitated thyroid hormone transport using a modified nonradioactive assay.

Early neurodevelopmental processes are strictly dependent on spatial and temporally modulated of thyroid hormone (TH) availability and action. Thyroid hormone transmembrane transporters (THTMT) are critical for regulating the local concentrations of TH, namely thyroxine (T4) and 3,5,3'-tri-iodothyronine (T3), in the brain. Monocarboxylate transporter 8 (MCT8) is one of the most prominent THTMT. Genetically induced deficiencies in expression, function or localization of MCT8 are associated with irreversible and severe neurodevelopmental adversities. Due to the importance of MCT8 in brain development, studies addressing chemical interferences of MCT8 facilitated T3 uptake are a crucial step to identify TH system disrupting chemicals with this specific mode of action. Recently a non-radioactive in vitro assay has been developed to rapidly screen for endocrine disrupting chemicals (EDCs) acting upon MCT8 mediated transport. This study explored the use of an UV-light digestion step as an alternative for the original ammonium persulfate (APS) digestion step. The non-radioactive TH uptake assay, with the incorporated UV-light digestion step of TH, was then used to screen a set of 31 reference chemicals and environmentally relevant substances to detect inhibition of MCT8-depending T3 uptake. This alternative assay identified three novel MCT8 inhibitors: methylmercury, bisphenol-AF and bisphenol-Z and confirmed previously known MCT8 inhibitors.

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.