Structural insights into the HBV receptor and bile acid transporter NTCP

B型肝炎ウイルスの受容体“胆汁酸輸送体”の立体構造を解明. 京都大学プレスリリース. 2022-05-18.Roughly 250 million people are infected with hepatitis B virus (HBV) worldwide, and perhaps 15 million also carry the satellite virus HDV, which confers even greater risk of severe liver disease. Almost ten years ago the HBV receptor was identified as NTCP (sodium taurocholate co-transporting polypeptide), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large (L) protein. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria, and these models with ten transmembrane helices are believed to resemble strongly both NTCP and ASBT. Using cryo-electron microscopy we have solved the structure of NTCP bound to an antibody, clearly showing the transporter has no equivalent to the first transmembrane helix of other SLC10 models, leaving the N-terminus exposed on the extracellular face. Comparison of the different structures indicates a common mechanism of bile acid transport, but the NTCP structure also displays a pocket formed by residues known to interact with preS1, presenting new and enticing opportunities for structure-based drug design

Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

Current anti-hepatitis B virus (HBV) agents, which include nucleos(t)ide analogs and interferons, can significantly suppress HBV infection. However, there are limitations in the therapeutic efficacy of these agents, indicating the need to develop anti-HBV agents with different modes of action. In this study, through a functional cell-based chemical screening, we found that a thiazolidinedione, troglitazone, inhibits HBV infection independently of the compound's ligand activity for peroxisome proliferator-activated receptor γ (PPARγ). Analog analysis suggested chemical moiety required for the anti-HBV activity and identified ciglitazone as an analog having higher anti-HBV potency. Whereas, most of the reported HBV entry inhibitors target viral attachment to the cell surface, troglitazone blocked a process subsequent to viral attachment, i.e., internalization of HBV preS1 and its receptor, sodium taurocholate cotransporting polypeptide (NTCP). We also found that NTCP was markedly oligomerized in the presence of HBV preS1, but such NTCP oligomerization was abrogated by treatment with troglitazone, but not with pioglitazone, correlating with inhibition activity to viral internalization. Also, competitive peptides that blocked NTCP oligomerization impeded viral internalization and infection. This work represents the first report identifying small molecules and peptides that specifically inhibit the internalization of HBV. This study is also significant in proposing a possible role for NTCP oligomerization in viral entry, which will shed a light on a new aspect of the cellular mechanisms regulating HBV infection

Troglitazone Impedes the Oligomerization of Sodium Taurocholate Cotransporting Polypeptide and Entry of Hepatitis B Virus Into Hepatocytes

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A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction

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Inhibition of 20 S proteasome activity by homopiperazine derivatives.

<p>A. Purified erythrocyte-derived 20 S proteasome was incubated in the absence (control) or presence of the indicated HPDS at 5 µM. Chymotrypsin-like, caspase-like and trypsin-like activities were determined by measuring fluorescence generated from the cleavage of specific substrates. Results are represented as relative fluorescence units (RFU) with control set at 100%. The means ± S.D. (bars) of three independent experiments are shown. <i>P</i>-values were calculated by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test. Asterisks indicate p<0.05 against corresponding controls. B. RPMI8226 cells were treated with or without 5 µM HPDs, and analyzed for proteolytic activities as described above. C. RPMI8226 cells were cultured in the absence (control) or presence of 10 µM HPDs for 24 hours, and subjected to immunoblotting for ubiquitinated proteins and GAPDH (internal control).</p

Statistics of crystallographic analysis.

a<p>Completeness and <i>R-</i>merge, are given for overall data and for the highest resolution shell.</p><p>The highest resolution shells for the dataset were 2.54–2.50 Å.</p>b<p><i>R</i>merge = ∑ | <i>I_i</i> – <<i>I</i>> |/∑|<i>I_i</i>|; where <i>I_i</i> is intensity of an observation and <<i>I</i>> is the mean value of that reflection and the summations are over all equivalents.</p>c<p><i>R-work</i> = ∑<i>_h</i> || <i>Fo(h)</i> | – | <i>Fc(h)</i> ||/∑<i>_hFo(h)</i>; where <i>Fo</i> and <i>Fc</i> are the observed and calculated structure factor amplitudes, respectively. The <i>R-free</i> was calculated with 5% of the data excluded from the refinement.</p

Cytotoxic effects of K-7174 on bortezomib-resistant MM cells.

<p>We established wild-type (WT) and mutant (mutant) sublines from RPMI8226 by transducing with wild-type and mutated <i>PSMB5</i> cDNA, respectively, and analyzed the expression of VENUS by flow cytometry (A) and proteasome ß5 subunit by immunoblotting (B). The signal intensities of ß5 subunit (PSMB5) were quantified, normalized to those of the corresponding GAPDH, and shown as relative values in the panel B. C. Cell proliferation was measured by MTT assays after culturing each subline with either K-7174 or bortezomib at the indicated doses for 72 hours. Results are represented as relative absorbance with untreated control set at 100%. The means ± S.D. (bars) of three independent experiments are shown. <i>P</i>-values were calculated by one-way ANOVA with the Student-Newman-Keuls multiple comparisons test. Asterisks indicate p<0.01 against the WT subline. D. Each subline was cultured with either K-7174 or bortezomib (Bort) at the indicated doses for 48 hours. Whole cell lysates were subjected to immunoblotting for cellular protein ubiquitination, proteasome ß5 subunit (PSMB5) and GAPDH (internal control).</p

Comparison of K-7174 and bortezomib in cytotoxic activity and proteasome binding.

<p>A. Cell proliferation was measured by MTT assays after culturing with serially diluted K-7174, K-10487 and bortezomib for 72 hours. Absorbance at 450 nm was analyzed with a microplate reader, and expressed as a percentage of the value of corresponding untreated cells. The IC₅₀ value was defined as the concentration of each drug that produces 50% inhibition of cell growth. The means ± S.D. (bars) of three independent experiments are shown. Asterisks indicate “not determined”. B. Overall crystallographic structures showing the folds of ß1 to ß7 subunits in the proteasome bound with K-7174 (left panel) and bortezomib (right panel). Mutation sites observed in bortezomib-resistant cells are circled. C. Structure of the proteasome in complex with bortezomib (PDB cord 2F16) overlapped that with K-7174 described here. Only the protein atoms of the bortezomib-bound form are shown. Bortezomib-resistant mutant residues (Ala49, Thr21, Cys52 and Met45) are colored red and shown as a space-filling diagram.</p