Biophysical description of Bromosulfophthalein interaction with the 28-kDa glutathione transferase from Schistosoma japonicum.

Glutathione transferases (GSTs) are major detoxification enzymes vital for the survival and reproduction of schistosomes during infection in humans. Schistosoma encode two GST isoenzymes, the 26- and 28-kDa isoforms, that show different substrate specificities and cellular localisations. Bromosulfophthalein (BSP) has been identified and characterised as a potent 26-kDa Schistosoma japonicum GST (Sj26GST) inhibitor with an anthelmintic potential. This study describes the structure, function, and ligandin properties of the 28-kDa Schistosoma japonicum GST (Sj28GST) towards BSP. Enzyme kinetics show that BSP is a potent enzyme inhibitor, with a specific activity decreases from 60.4 µmol/min/mg to 0.0742 µmol/min/mg and an IC50 in the micromolar range of 0.74 µM. Far-UV circular dichroism confirmed that purified Sj28GST follows a typical GST fold, which is predominantly alpha-helical. Fluorescence spectroscopy suggests that BSP binding occurs at a site distinct from the glutathione-binding site (G-site); however, the binding does not alter the local G-site environment. Isothermal titration calorimetry studies show that the binding of BSP to Sj28GST is exergonic (∆G°= -33 kJ/mol) and enthalpically-driven, with a stoichiometry of one BSP per dimer. The stability of Sj28GST (∆G(H2O) = 4.7 kcal/mol) is notably lower than Sj26GST, owing to differences in the enzyme's dimeric interfaces. We conclude that Sj28GST shares similar biophysical characteristics with Sj26GST based on its kinetic properties and susceptibility to low concentrations of BSP. The study supports the potential benefits of re-purposing BSP as a potential drug or prodrug to mitigate the scourge of schistosomiasis.

Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies.

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure-function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 µmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

Bromosulfophthalein suppresses inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

OBJECTIVE: It has been reported that glutathione (GSH), the most abundant cellular antioxidant, can inhibit production of pro-inflammatory cytokines by activated macrophages. Bromosulfophthalein (BSP) has been recognized as an inhibitor of the efflux of reduced GSH from cells, leading to an increase in the intracellular GSH level. In this study, we evaluated, for the first time, whether BSP possessed anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages. MATERIALS AND METHODS: RAW 264.7 cells were treated with BSP and the levels of proinflammatory cytokines, GSH, and nitrite were assessed. Gene expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) was analyzed via quantitative RT-PCR. We also examined various inflammatory signaling pathways including Akt/forkhead box protein O1 (FoxO1)/toll-like receptor 4 (TLR4), mitogen-activated protein kinases (MAPKs), and Fas protein by Western blot and flow cytometry analysis. RESULTS: Our study demonstrated that BSP induced an increase in intracellular GSH level in LPS-stimulated macrophages. BSP inhibited production of nitric oxide and proinflammatory cytokines. BSP increased phosphorylation of Akt and nuclear exclusion of FoxO1 and suppressed TLR4 expression. Additionally, BSP decreased MAPKs activation and Fas expression. DISCUSSION AND CONCLUSION: Taken together, these data suggest that BSP can attenuate inflammation through multiple signaling pathways. These findings highlight the potential of BSP as a new anti-inflammatory agent.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Monitorable Mitochondria-Targeting DNAtrain for Image-Guided Synergistic Cancer Therapy.

It is highly desirable to realize real-time monitoring of the drug delivery/release process in cancer treatment. Herein, a monitorable mitochondria-specific DNAtrain (MitoDNAtrs) was developed for image-guided drug delivery and synergistic cancer therapy. In this system, mitochondria-targeting Cy5.5 dye served as the "locomotive" to guide the DNA "vehicle" selectively accumulating in the cancer cells in a detectable manner. More importantly, Cy5.5 showed reactive oxygen species (ROS) generation ability, which made it a promising adjuvant chemotherapy amplifier for cancer theranostics.

Interaction of Sulfonylureas with Liver Uptake Transporters OATP1B1 and OATP1B3.

Sulfonylureas (SUs) such as glibenclamide, gliclazide, glimepiride, glipizide and gliquidone are one of the first oral medicines available for the treatment of type 2 diabetes and are widely used for the treatment of hyperglycaemia. The hepatic transporters, organic anion transporting polypeptide 1B1 (OATP1B1) and organic anion transporting polypeptide 1B3 (OATP1B3), play an important role in the disposition of a variety of drugs by mediating their uptake from blood into hepatocytes. Drug-drug interactions mediated by OATP1B1/1B3 may result in the hepatic transporting change for drug substrates. The inhibitory effects of glibenclamide and glimepiride on sulfobromophthalein (BSP) uptake have been previously studied, and glibenclamide has been reported as the substrate of OATP1B3, but it remains unclear whether other SUs such as gliclazide, glipizide and gliquidone are substrates of OATP1B1 and OATP1B3. Here, we investigated the relationship between the five most commonly applied SUs (glibenclamide, gliclazide, glimepiride, glipizide, gliquidone) and OATP1B1 and OATP1B3. We performed uptake and inhibition assays in HEK293T cells stably expressing OATP1B1 or OATP1B3, respectively, and established a liquid chromatography-mass spectrometry (LC-MS) method for the simultaneous measurement of five SUs. We demonstrated that gliclazide and glimepiride are substrates of OATP1B1 and glibenclamide and glipizide are substrates of OATP1B3. We also confirmed the interaction between these SUs and rosuvastatin. No transporting was observed for gliquidone, suggesting that it is not a substrate of either transporter.

Uptake of perfluorooctanoic acid by Caco-2 cells: Involvement of organic anion transporting polypeptides.

The mechanism underlying the intestinal absorption of perfluorooctanoic acid (PFOA) was investigated using Caco-2 cells. The uptake of PFOA from the apical membrane of Caco-2 cells was fast, and pH, temperature, and concentration dependent, but Na+ independent. Coincubation with sulfobromophthalein (BSP), glibenclamide, estron-3-sulfate, cyclosporine A or rifamycin SV, which are typical substrates or inhibitors of organic anion transporting polypeptides (OATPs), significantly decreased the uptake of PFOA. However, coincubation with probenecid or p-aminohippuric acid, typical substrates of organic anion transporters, did not decrease the uptake of PFOA. Furthermore, coincubation with l-lactic acid or benzoic acid, substrates of monocarboxylic acid transporters, did not decrease PFOA uptake. The relationship between the initial uptake of PFOA and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The calculated Km and uptake clearance (Vmax/Km) values for PFOA were 8.3µM and 55.0µL/mg protein/min, respectively. This clearance value was about 3-fold greater than that of the non-saturable uptake clearance (Kd: 18.1µL/mg protein/min). Lineweaver-Burk plots revealed that BSP competitively inhibits the uptake of PFOA, with a Ki value of 23.1µM. These results suggest that the uptake of PFOA from the apical membranes of Caco-2 cells could be, at least in part, mediated by OATPs along with BSP.

Human organic anion transporter 2 is an entecavir, but not tenofovir, transporter.

Entecavir (ETV) and tenofovir (TFV) are essential nucleoside analogues in current hepatitis B virus (HBV) treatments. Since these drugs target the HBV polymerase that is localized within human hepatocytes, determining of their cellular uptake process is an important step in fully understanding their pharmacological actions. However, the human hepatic transporters responsible for their uptake have remained unidentified. Therefore, this study aimed at identifying the primary ETV and TFV uptake transporter(s) in human hepatocytes. In transport assays, temperature-sensitive ETV and TFV uptake by human hepatocytes were observed, and their uptake were strongly inhibited by bromosulfophthalein, which is an inhibitor of organic anion transporters/organic anion transporting polypeptides (OATs/OATPs). Given these results, ETV and TFV uptake activities in several human OAT/OATP expression systems were examined. The results showed that, among the transporters tested, only OAT2 possessed ETV transport activity. On the other hand, none of the transporters showed any TFV uptake activity. To summarize, our results identify that human OAT2 is an ETV transporter, thereby suggesting that it plays an important part in the mechanisms underlying ETV antiviral activity. Furthermore, although the hepatic TFV transporters remain unknown, our results have, at least, clarified that these two anti-HBV drugs have different hepatocyte entry routes.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression.

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine.

Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus.

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 µM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.