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

Abstract

Human hepatic bile acid transporter Na+/taurocholate co-transporting 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 down-regulated 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 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.

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

“…TLC, after carriermediated uptake or passive diffusion, can bind to an intracellular TLC binding site of NTCP and thereby trans-inhibits myr-preS1 peptide and HDV binding from the outside of the cell. Interestingly, this trans-inhibitory effect of TLC also inhibited in vitro HDV infection of NTCP expressing HepG2 hepatoma cells (Lowjaga et al, 2021). In contrast, such a trans-inhibitory effect is not known for the substrates TC and DHEAS, what could explain their lower potential to inhibit myr-preS1 binding to NTCP.…”

“…However, TLC was much more potent in blocking the binding of the myr-preS1 peptide from NTCP compared with TC and DHEAS. This could also be explained by the particular trans-inhibitory potential of this compound at NTCP (Lowjaga et al, 2021). TLC, after carriermediated uptake or passive diffusion, can bind to an intracellular TLC binding site of NTCP and thereby trans-inhibits myr-preS1 peptide and HDV binding from the outside of the cell.…”

“…Furthermore, we identified taurolithocholic acid (TLC) as the first common substrate of all three carriers. (Lowjaga et al, 2021).…”

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

“…Recently, it was proposed that a large substrate/inhibitor entry zone exists in the outward oriented space between the “core” and the “panel” domains of NTCP that is characterized by multiple interaction domains for different kind of substrates such as TLC, TC, DHEAS, and different classes of inhibitors. Hence, TLC may have a potent inhibitory effect on the TC and DHEAS transport function of NTCP [ 39 , 40 ]. In addition, rapid accumulation of TLC mediates long-lasting trans -inhibition of the transporter and receptor functions of NTCP, most likely via an intracellular TLC-binding domain of NTCP [ 40 ].…”

“…Hence, TLC may have a potent inhibitory effect on the TC and DHEAS transport function of NTCP [ 39 , 40 ]. In addition, rapid accumulation of TLC mediates long-lasting trans -inhibition of the transporter and receptor functions of NTCP, most likely via an intracellular TLC-binding domain of NTCP [ 40 ]. Noteworthy, as a bile acid transporter, NTCP may also be responsible for the uptake of drugs and other xenobiotics, for instance rosuvastatin, fluvastatin, atorvastatin as well as pitavastatin, irbesartan, ezetimibe and losartan, thereby influencing bile acid circulation [ 41 , 42 , 43 , 44 , 45 ].…”