Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Role of the Sodium-Dependent Organic Anion Transporter (SOAT/SLC10A6) in Physiology and Pathophysiology.

The sodium-dependent organic anion transporter (SOAT, gene symbol SLC10A6) specifically transports 3'- and 17'-monosulfated steroid hormones, such as estrone sulfate and dehydroepiandrosterone sulfate, into specific target cells. These biologically inactive sulfo-conjugated steroids occur in high concentrations in the blood circulation and serve as precursors for the intracrine formation of active estrogens and androgens that contribute to the overall regulation of steroids in many peripheral tissues. Although SOAT expression has been detected in several hormone-responsive peripheral tissues, its quantitative contribution to steroid sulfate uptake in different organs is still not completely clear. Given this fact, the present review provides a comprehensive overview of the current knowledge about the SOAT by summarizing all experimental findings obtained since its first cloning in 2004 and by processing SOAT/SLC10A6-related data from genome-wide protein and mRNA expression databases. In conclusion, despite a significantly increased understanding of the function and physiological significance of the SOAT over the past 20 years, further studies are needed to finally establish it as a potential drug target for endocrine-based therapy of steroid-responsive diseases such as hormone-dependent breast cancer.

Interactions of Na+/taurocholate cotransporting polypeptide with host cellular proteins upon hepatitis B and D virus infection: novel potential targets for antiviral therapy.

Na+/taurocholate cotransporting polypeptide (NTCP) is a member of the solute carrier (SLC) family 10 transporters (gene symbol SLC10A1) and is responsible for the sodium-dependent uptake of bile salts across the basolateral membrane of hepatocytes. In addition to its primary transporter function, NTCP is the high-affinity hepatic receptor for hepatitis B (HBV) and hepatitis D (HDV) viruses and, therefore, is a prerequisite for HBV/HDV virus entry into hepatocytes. The inhibition of HBV/HDV binding to NTCP and internalization of the virus/NTCP receptor complex has become a major concept in the development of new antiviral drugs called HBV/HDV entry inhibitors. Hence, NTCP has emerged as a promising target for therapeutic interventions against HBV/HDV infections in the last decade. In this review, recent findings on protein-protein interactions (PPIs) between NTCP and cofactors relevant for entry of the virus/NTCP receptor complex are summarized. In addition, strategies aiming to block PPIs with NTCP to dampen virus tropism and HBV/HDV infection rates are discussed. Finally, this article suggests novel directions for future investigations evaluating the functional contribution of NTCP-mediated PPIs in the development and progression of HBV/HDV infection and subsequent chronic liver disorders.

Tyrosine 146 of the Human Na+/Taurocholate Cotransporting Polypeptide (NTCP) Is Essential for Its Hepatitis B Virus (HBV) Receptor Function and HBV Entry into Hepatocytes.

Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1) is a hepatic bile acid uptake carrier participating in the enterohepatic circulation of bile acids. Apart from its transporter function, NTCP acts as the high-affinity liver-specific receptor for the hepatitis B virus (HBV), which attaches via its preS1-peptide domain of the large surface protein to NTCP, subsequently leading to endocytosis of the virus/NTCP-receptor complex. Although the process of NTCP-dependent HBV infection of hepatocytes has received much attention over the last decade, the precise molecular sites of the virus/NTCP interaction have not been fully identified. Inspection of the primary protein sequence of human NTCP revealed 139YIYSRGIY146 as a highly conserved tyrosine-rich motif. To study the role of Y139, Y141 and Y146 amino acids in NTCP biology, the aforementioned residues were substituted with alanine, phenylalanine or glutamate (mimicking phosphorylation) using site-directed mutagenesis. Similar to wt NTCP, the Y139A, Y141A, Y146A, Y141F, Y146F, and Y146E mutants were expressed at the plasma membrane of HEK293 cells and exhibited intact bile acid transport function. Y146A, Y146E, and Y146F demonstrated transport kinetics comparable to wild-type NTCP with Km values of 57.3-112.4 µM and Vmax values of 6683-7579 pmol/mg protein/min. Only Y141E was transport deficient, most likely due to an intracellular accumulation of the mutant protein. Most importantly, Y146A and Y146E mutation completely abrogated binding of the viral preS1-peptide to NTCP, while the Y146F mutant of NTCP showed some residual binding competence for preS1. Consequently, the NTCP mutants Y146A and Y146E, when expressed in HepG2 hepatoma cells, showed complete loss of susceptibility for in vitro HBV infection. In conclusion, tyrosine 146, and to some extent tyrosine 141, both belonging to the tyrosine-rich motif 139YIYSRGIY146 of human NTCP, are newly identified amino acid residues that play an essential role in the interaction of HBV with its receptor NTCP and, thus, in the process of virus entry into hepatocytes.

IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection.

The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1-3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein-protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.

Multitasking Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a Drug Target for HBV Infection: From Protein Engineering to Drug Discovery.

Hepatitis B virus (HBV) infections are among the major public health concerns worldwide with more than 250 million of chronically ill individuals. Many of them are additionally infected with the Hepatitis D virus, a satellite virus to HBV. Chronic infection frequently leads to serious liver diseases including cirrhosis and hepatocellular carcinoma, the most common type of liver cancer. Although current antiviral therapies can control HBV replication and slow down disease progress, there is an unmet medical need to identify therapies to cure this chronic infectious disease. Lately, a noteworthy progress in fighting against HBV has been made by identification of the high-affinity hepatic host receptor for HBV and HDV, namely Na+/taurocholate cotransporting polypeptide (NTCP, gene symbol SLC10A1). Next to its primary function as hepatic uptake transporter for bile acids, NTCP is essential for the cellular entry of HBV and HDV into hepatocytes. Due to this high-ranking discovery, NTCP has become a valuable target for drug development strategies for HBV/HDV-infected patients. In this review, we will focus on a newly predicted three-dimensional NTCP model that was generated using computational approaches and discuss its value in understanding the NTCP's membrane topology, substrate and virus binding taking place in plasma membranes. We will review existing data on structural, functional, and biological consequences of amino acid residue changes and mutations that lead to loss of NTCP's transport and virus receptor functions. Finally, we will discuss new directions for future investigations aiming at development of new NTCP-based HBV entry blockers that inhibit HBV tropism in human hepatocytes.

Organic Cation Transporter I and Na+ /taurocholate Co-Transporting Polypeptide are Involved in Retrorsine- and Senecionine-Induced Hepatotoxicity in HepaRG cells.

SCOPE: 1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity. METHODS AND RESULTS: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na+ /taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA-mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time-dependent manner and attenuated PA-mediated cytotoxic effects in HepaRG cells. CONCLUSION: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.

Rho/ROCK Inhibition Promotes TGF-ß3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation in vivo. The transforming growth factor (TGF)-ß, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-ß3-induced tenogenic differentiation. The MSC were incubated with and without TGF-ß3 (10 ng/ml), Y-27632 (10 µM), or both. On day 1 and day 3, the signalling pathway of TGF-ß and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-ß3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species (p < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-ß3 and ROCK inhibition were combined (p < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-ß3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-ß3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.

Functional Analysis of Rare Genetic Variants in the Negative Regulator of Intracellular Calcium Signaling RCAS/SLC10A7.

The solute carrier family 10 member SLC10A7 is a negative regulator of intracellular calcium signaling (RCAS). In cell culture, SLC10A7 expression is negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. SLC10A7-deficient cells have significantly increased calcium influx after treatment with thapsigargin for depletion of ER calcium stores, whereas SLC10A7/RCAS overexpression limits calcium influx. Genetic variants in the human SLC10A7 gene are associated with skeletal dysplasia and amelogenesis imperfecta and reveal loss of function on cellular calcium influx. More recently, an additional disease-related genetic variant (P303L) as well as some novel genetic variants (V235F, T221M, I136M, L210F, P285L, and G146S) have been identified. In the present study, these variants were expressed in HEK293 cells to study their subcellular localization and their effect on cellular calcium influx. All variants were properly sorted to the ER compartment and closely co-localized with the STIM protein, a functional component of SOCE. The variants P303L and L210F showed significantly reduced effects on cellular calcium influx compared to the wild type but still maintained some degree of residual activity. This might explain the milder phenotype of patients bearing the P303L variant and might indicate disease potential for the newly identified L210F variant. In contrast, all other variants behaved like the wild type. In conclusion, the occurrence of variants in the SLC10A7 gene should be considered in patients with skeletal dysplasia and amelogenesis imperfecta. In addition to the already established variants, the present study identifies another potential disease-related SLC10A7/RCAS variant, namely, L210F, which seems to be most frequent in South Asian populations.

Identification of Novel HBV/HDV Entry Inhibitors by Pharmacophore- and QSAR-Guided Virtual Screening.

The hepatic bile acid transporter Na+/taurocholate co-transporting polypeptide (NTCP) was identified in 2012 as the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Since then, this carrier has emerged as promising drug target for HBV/HDV virus entry inhibitors, but the synthetic peptide Hepcludex® of high molecular weight is the only approved HDV entry inhibitor so far. The present study aimed to identify small molecules as novel NTCP inhibitors with anti-viral activity. A ligand-based bioinformatic approach was used to generate and validate appropriate pharmacophore and QSAR (quantitative structure-activity relationship) models. Half-maximal inhibitory concentrations (IC50) for binding inhibition of the HBV/HDV-derived preS1 peptide (as surrogate parameter for virus binding to NTCP) were determined in NTCP-expressing HEK293 cells for 150 compounds of different chemical classes. IC50 values ranged from 2 µM up to >1000 µM. The generated pharmacophore and QSAR models were used for virtual screening of drug-like chemicals from the ZINC15 database (~11 million compounds). The 20 best-performing compounds were then experimentally tested for preS1-peptide binding inhibition in NTCP-HEK293 cells. Among them, four compounds were active and revealed experimental IC50 values for preS1-peptide binding inhibition of 9, 19, 20, and 35 µM, which were comparable to the QSAR-based predictions. All these compounds also significantly inhibited in vitro HDV infection of NTCP-HepG2 cells, without showing any cytotoxicity. The best-performing compound in all assays was ZINC000253533654. In conclusion, the present study demonstrates that virtual compound screening based on NTCP-specific pharmacophore and QSAR models can predict novel active hit compounds for the development of HBV/HDV entry inhibitors.

Mutational Analysis of the GXXXG/A Motifs in the Human Na+/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function.

Homodimerization is essential for plasma membrane sorting of the liver bile acid transporter NTCP and its function as Hepatitis B/D Virus (HBV/HDV) receptor. However, the protein domains involved in NTCP dimerization are unknown. NTCP bears two potential GXXXG/A dimerization motifs in its transmembrane domains (TMDs) 2 and 7. The present study aimed to analyze the role of these GXXXG/A motifs for the sorting, function, and dimerization of NTCP. The NTCP mutants G60LXXXA64L (TMD2), G233LXXXG237L (TMD7) and a double mutant were generated and analyzed for their interaction with wild-type NTCP using a membrane-based yeast-two hybrid system (MYTH) and co-immunoprecipitation (co-IP). In the MYTH system, the TMD2 and TMD7 mutants showed significantly lower interaction with the wild-type NTCP. In transfected HEK293 cells, membrane expression and bile acid transport activity were slightly reduced for the TMD2 mutant but were completely abolished for the TMD7 and the TMD2/7 mutants, while co-IP experiments still showed intact protein-protein interactions. Susceptibility for in vitro HBV infection in transfected HepG2 cells was reduced to 50% for the TMD2 mutant, while the TMD7 mutant was not susceptible for HBV infection at all. We conclude that the GXXXG/A motifs in TMD2 and even more pronounced in TMD7 are important for proper folding and sorting of NTCP, and so indirectly affect glycosylation, homodimerization, and bile acid transport of NTCP, as well as its HBV/HDV receptor function.

Functional and Pharmacological Comparison of Human and Mouse Na+/Taurocholate Cotransporting Polypeptide (NTCP).

The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM).In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.

Substrate Specificities and Inhibition Pattern of the Solute Carrier Family 10 Members NTCP, ASBT and SOAT.

Three carriers of the solute carrier family SLC10 have been functionally characterized so far. Na+/taurocholate cotransporting polypeptide NTCP is a hepatic bile acid transporter and the cellular entry receptor for the hepatitis B and D viruses. Its intestinal counterpart, apical sodium-dependent bile acid transporter ASBT, is responsible for the reabsorption of bile acids from the intestinal lumen. In addition, sodium-dependent organic anion transporter SOAT specifically transports sulfated steroid hormones, but not bile acids. All three carriers show high sequence homology, but significant differences in substrate recognition that makes a systematic structure-activity comparison attractive in order to define the protein domains involved in substrate binding and transport. By using stably transfected NTCP-, ASBT-, and SOAT-HEK293 cells, systematic comparative transport and inhibition experiments were performed with more than 20 bile acid and steroid substrates as well as different inhibitors. Taurolithocholic acid (TLC) was identified as the first common substrate of NTCP, ASBT and SOAT with K m values of 18.4, 5.9, and 19.3 µM, respectively. In contrast, lithocholic acid was the only bile acid that was not transported by any of these carriers. Troglitazone, BSP and erythrosine B were identified as pan-SLC10 inhibitors, whereas cyclosporine A, irbesartan, ginkgolic acid 17:1, and betulinic acid only inhibited NTCP and SOAT, but not ASBT. The HBV/HDV-derived myr-preS1 peptide showed equipotent inhibition of the NTCP-mediated substrate transport of taurocholic acid (TC), dehydroepiandrosterone sulfate (DHEAS), and TLC with IC50 values of 182 nM, 167 nM, and 316 nM, respectively. In contrast, TLC was more potent to inhibit myr-preS1 peptide binding to NTCP with IC50 of 4.3 µM compared to TC (IC50 = 70.4 µM) and DHEAS (IC50 = 52.0 µM). Based on the data of the present study, we propose several overlapping, but differently active binding sites for substrates and inhibitors in the carriers NTCP, ASBT, SOAT.

Hepatitis D Virus Entry Inhibitors Based on Repurposing Intestinal Bile Acid Reabsorption Inhibitors.

Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as high-affinity hepatic entry receptor for the Hepatitis B and D viruses (HBV/HDV) opened the field for target-based development of cell-entry inhibitors. However, most of the HBV/HDV entry inhibitors identified so far also interfere with the physiological bile acid transporter function of NTCP. The present study aimed to identify more virus-selective inhibitors of NTCP by screening of 87 propanolamine derivatives from the former development of intestinal bile acid reabsorption inhibitors (BARIs), which interact with the NTCP-homologous intestinal apical sodium-dependent bile acid transporter (ASBT). In NTCP-HEK293 cells, the ability of these compounds to block the HBV/HDV-derived preS1-peptide binding to NTCP (virus receptor function) as well as the taurocholic acid transport via NTCP (bile acid transporter function) were analyzed in parallel. Hits were subsequently validated by performing in vitro HDV infection experiments in NTCP-HepG2 cells. The most potent compounds S985852, A000295231, and S973509 showed in vitro anti-HDV activities with IC50 values of 15, 40, and 70 µM, respectively, while the taurocholic acid uptake inhibition occurred at much higher IC50 values of 24, 780, and 490 µM, respectively. In conclusion, repurposing of compounds from the BARI class as novel HBV/HDV entry inhibitors seems possible and even enables certain virus selectivity based on structure-activity relationships.

Long-term trans-inhibition of the hepatitis B and D virus receptor NTCP by taurolithocholic acid.

Human hepatic bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) represents the liver-specific entry receptor for the hepatitis B and D viruses (HBV/HDV). Chronic hepatitis B and D affect several million people worldwide, but treatment options are limited. Recently, HBV/HDV entry inhibitors targeting NTCP have emerged as promising novel drug candidates. Nevertheless, the exact molecular mechanism that NTCP uses to mediate virus binding and entry into hepatocytes is still not completely understood. It is already known that human NTCP mRNA expression is downregulated under cholestasis. Furthermore, incubation of rat hepatocytes with the secondary bile acid taurolithocholic acid (TLC) triggers internalization of the rat Ntcp protein from the plasma membrane. In the present study, the long-term inhibitory effect of TLC on transport function, HBV/HDV receptor function, and membrane expression of human NTCP were analyzed in HepG2 and human embryonic kidney (HEK293) cells stably overexpressing NTCP. Even after short-pulse preincubation, TLC had a significant long-lasting inhibitory effect on the transport function of NTCP, but the NTCP protein was still present at the plasma membrane. Furthermore, binding of the HBV/HDV myr-preS1 peptide and susceptibility for in vitro HDV infection were significantly reduced by TLC preincubation. We hypothesize that TLC rapidly accumulates in hepatocytes and mediates long-lasting trans-inhibition of the transport and receptor function of NTCP via a particular TLC-binding site at an intracellularly accessible domain of NTCP. Physiologically, this trans-inhibition might protect hepatocytes from toxic overload of bile acids. Pharmacologically, it provides an interesting novel NTCP target site for potential long-acting HBV/HDV entry inhibitors.NEW & NOTEWORTHY The hepatic bile acid transporter NTCP is a high-affinity receptor for hepatitis B and D viruses. This study shows that TLC rapidly accumulates in NTCP-expressing hepatoma cells and mediates long-lasting trans-inhibition of NTCP's transporter and receptor function via an intracellularly accessible domain, without substantially affecting its membrane expression. This domain is a promising novel NTCP target site for pharmacological long-acting HBV/HDV entry inhibitors.

Selective hepatitis B and D virus entry inhibitors from the group of pentacyclic lupane-type betulin-derived triterpenoids.

Current treatment options against hepatitis B and D virus (HBV/HDV) infections have only limited curative effects. Identification of Na+/taurocholate co-transporting polypeptide (NTCP) as the high-affinity hepatic receptor for both viruses in 2012 enables target-based development of HBV/HDV cell-entry inhibitors. Many studies already identified appropriate NTCP inhibitors. However, most of them interfere with NTCP's physiological function as a hepatic bile acid transporter. To overcome this drawback, the present study aimed to find compounds that specifically block HBV/HDV binding to NTCP without affecting its transporter function. A novel assay was conceptualized to screen for both in parallel; virus binding to NTCP (measured via binding of a preS1-derived peptide of the large HBV/HDV envelope protein) and bile acid transport via NTCP. Hits were subsequently validated by in vitro HDV infection studies using NTCP-HepG2 cells. Derivatives of the birch-derived pentacyclic lupane-type triterpenoid betulin revealed clear NTCP inhibitory potency and selectivity for the virus receptor function of NTCP. Best performing compounds in both aspects were 2, 6, 19, and 25. In conclusion, betulin derivatives show clear structure-activity relationships for potent and selective inhibition of the HBV/HDV virus receptor function of NTCP without tackling its physiological bile acid transport function and therefore are promising drug candidates.

Very High Dehydroepiandrosterone Sulfate (DHEAS) in Serum of an Overweight Female Adolescent Without a Tumor.

Introduction: An increase of serum dehydroepiandrosterone (DHEA) sulfate (DHEAS) is observed in premature adrenarche and congenital adrenal hyperplasia. Very high DHEAS levels are typical for adrenal tumors. Approximately 74% of DHEAS is hydrolyzed to DHEA by the steroid sulfatase (STS). The reverse reaction is DHEA sulfation. Besides these two enzyme reactions, the DHEAS transported through the cell membrane is important for its distribution and excretion. Case Presentation: We present a female adolescent with overweight and a very high DHEAS. The presence of a DHEAS-producing tumor was rejected using ultrasonography, Magnetic Resonance Tomography (MRT), and dexamethasone suppression. STS deficiency was suspected. Sequence analysis revealed a heterozygous nonsense mutation which predicts a truncation of the carboxyl region of the STS that is implicated in substrate binding. No partial gene deletion outside exon 5 was detected by multiplex ligation-dependent probe amplification. The bioassay revealed normal enzyme activity in the patient's leukocytes. A defect of transporter proteins was suggested. Both efflux [multidrug-resistance protein (MRP)2 and breast cancer-resistance protein (BCRP)] and uptake [organic anion-transporting polypeptide (OATP) and organic anion transporter (OAT) carriers] transporters were studied. Sequence analysis of exons revealed a heterozygous Q141K variant for BCRP. Conclusions: A novel heterozygous nonsense mutation in the STS gene and a known heterozygous missense variant in the BCRP gene were found. The heterozygous nonsense mutation in the STS gene is not supposed to be responsible for STS deficiency. The BCRP variant is associated with reduced efflux transport activity only in its homozygous state. The combination of the two heterozygous mutations could possibly explain the observed high levels of DHEAS and other sulfated steroids.

The orphan solute carrier SLC10A7 is a novel negative regulator of intracellular calcium signaling.

SLC10A7 represents an orphan member of the Solute Carrier Family SLC10. Recently, mutations in the human SLC10A7 gene were associated with skeletal dysplasia, amelogenesis imperfecta, and decreased bone mineral density. However, the exact molecular function of SLC10A7 and the mechanisms underlying these pathologies are still unknown. For this reason, the role of SLC10A7 on intracellular calcium signaling was investigated. SLC10A7 protein expression was negatively correlated with store-operated calcium entry (SOCE) via the plasma membrane. Whereas SLC10A7 knockout HAP1 cells showed significantly increased calcium influx after thapsigargin, ionomycin and ATP/carbachol treatment, SLC10A7 overexpression reduced this calcium influx. Intracellular Ca2+ levels were higher in the SLC10A7 knockout cells and lower in the SLC10A7-overexpressing cells. The SLC10A7 protein co-localized with STIM1, Orai1, and SERCA2. Most of the previously described human SLC10A7 mutations had no effect on the calcium influx and thus were confirmed to be functionally inactive. In the present study, SLC10A7 was established as a novel negative regulator of intracellular calcium signaling that most likely acts via STIM1, Orai1 and/or SERCA2 inhibition. Based on this, SLC10A7 is suggested to be named as negative regulator of intracellular calcium signaling (in short: RCAS).

In-vitro safety and off-target profile of the anti-parasitic arylmethylaminosteroid 1o.

Parasite-mediated diseases like malaria and schistosomiasis are growing health problems worldwide and novel drug candidates are urgently needed. In this study, the in-vitro safety profile of steroid compound 1o (sc1o), effective against the parasites Plasmodium falciparum and Schistosoma mansoni with an IC50 value of 5 nM, was characterized. We assessed viability/proliferation, apoptosis and cell cycle tests to determine the cytotoxic profile of sc1o in cancer cells. The mutagenic potential was determined with the AMES test. To identify off-target effects we investigated whether sc1o interacts with safety-relevant molecules such as cytochrome P450 (CYP) enzymes, phosphodiesterases (PDE), histone deacteylases (HDAC) and human ether-a-go-go related gene (hERG). Furthermore, to predict the potential bioavailability of sc1o, its effect on Caco-2 cell barrier integrity, by measurement of the transepithelial electrical resistance (TEER), was determined. Sc1o at 25 µM reduced cell viability, probably through cell-cycle arrest, but did not induce apoptosis in cancer cells. No adverse off-target effects nor mutagenic potential of sc1o were observed. Furthermore, sc1o did not disturb the integrity of the cell barrier, but exhibited low membrane permeability, apparently due to cell adherence. In conclusion, sc1o up to 10 µM showed a good in-vitro safety profile.

Highly diversified shrew hepatitis B viruses corroborate ancient origins and divergent infection patterns of mammalian hepadnaviruses.

Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human- and tupaia-derived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBV-specific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.