机械学见解Myrcludex B抑制NTCP。

StephenW

JHEP Rep. 2019 Aug 1;1(4):278-285. doi: 10.1016/j.jhepr.2019.07.006. eCollection 2019 Oct.
Mechanistic insights into the inhibition of NTCP by myrcludex B.
Donkers JM1, Appelman MD1, van de Graaf SFJ1,2.
Author information

1
    Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology and Metabolism, Amsterdam, the Netherlands.
2
    Amsterdam UMC, University of Amsterdam, Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology and Metabolism, Amsterdam, the Netherlands.

Abstract
Background & aims:

The sodium taurocholate co-transporting polypeptide (NTCP) is the entry receptor for the hepatitis B and delta virus (HBV/HDV) and the main hepatic uptake transporter of conjugated bile acids. Myrcludex B, a synthetic peptide mimicking the NTCP-binding domain of HBV, blocks HBV/HDV infection and inhibits NTCP-mediated bile acid uptake. In humans this increases systemic bile acid levels, which remain elevated for hours even after Myrcludex B is cleared from the circulation. Here, we investigated the dynamics of Myrcludex B-induced NTCP-mediated bile acid transport inhibition in mice and if/how the duration of this effect relates to NTCP protein turnover.
Methods:

Plasma bile acids were determined in Myrcludex B-treated OATP1a/1b-deficient mice. In vitro, plasma membrane-resident NTCP was labeled with biotin or fluorescein isothiocyanate (FITC)-labeled Myrcludex B and traced in time using hNTCP-overexpressing U2OS cells. Förster resonance energy transfer by fluorescent lifetime imaging microscopy was used to investigate whether Myrcludex B can transfer to newly synthesized NTCP.
Results:

Conjugated bile salt levels in plasma peaked 4 h after subcutaneous Myrcludex B administration. After 24 h, plasma bile salt levels were completely normalized, in line with restored NTCP-mediated bile acid transport in vitro. Biotin-labeled NTCP disappeared faster than Myrcludex B-FITC, with almost 40% of FITC signal remaining after 24 h. FITC fluorescence lifetime was strongly decreased upon expression of DY547-labeled acyl carrier protein-tagged NTCP, demonstrating transfer of pre-bound Myrcludex B-FITC to newly formed NTCP.
Conclusions:

The dynamics of NTCP protein turnover and Myrcludex B-induced plasma bile salt elevations are similar, suggesting that the Myrcludex B:NTCP interaction is very long-lived. Nevertheless, Myrcludex B is not completely degraded together with NTCP and can transfer to newly synthesized NTCP.
Lay summary:

The experimental drug Myrcludex B binds the sodium taurocholate co-transporting polypeptide (NTCP), the viral entry receptor for the hepatitis B and D virus (HBV/HDV), and thereby prevents infection, but also inhibits hepatic bile salt uptake leading to transiently elevated bile salt levels. This study describes that while the normalization of plasma bile salt levels likely depends on the protein turnover rate of NTCP, Myrcludex B partly escapes co-degradation with NTCP by transferring from one NTCP molecule to another. This is of importance to the HBV/HDV research field as it provides a potential explanation for the distinct kinetics and dose-dependence of Myrcludex B's effects on viral infection versus bile salt transport.

© 2019 The Authors.
KEYWORDS:

Bile acid; Myrcludex B; NTCP; OATP; hepatitis B virus; hepatitis Delta; transporter

PMID:
    32039379
PMCID:
    PMC7001544
DOI:
    10.1016/j.jhepr.2019.07.006

StephenW

JHEP代表.2019 Aug 1; 1（4）：278-285。 doi：10.1016 / j.jhepr.2019.07.006。 eCollection 2019十月
机械学见解Myrcludex B抑制NTCP。
Donkers JM1，Appelman MD1，van de Graaf SFJ1,2。
作者信息

1
    阿姆斯特丹UMC，阿姆斯特丹大学，Tytgat肝脏和肠道研究所，阿姆斯特丹胃肠病和新陈代谢，荷兰阿姆斯特丹。
2
    阿姆斯特丹UMC，阿姆斯特丹大学，胃肠病学和肝病学系，阿姆斯特丹胃肠病和新陈代谢，荷兰阿姆斯特丹。

抽象
背景与目的：

牛磺胆酸钠共转运多肽（NTCP）是乙型肝炎和三角洲病毒（HBV / HDV）的进入受体，也是结合胆汁酸的主要肝吸收转运蛋白。 Myrcludex B是一种模拟HBV NTCP结合域的合成肽，可阻断HBV / HDV感染并抑制NTCP介导的胆汁酸摄取。在人体中，这会增加全身胆汁酸水平，即使从循环系统中清除了Myrcludex B后，其体内胆汁酸水平仍会持续升高数小时。在这里，我们调查了Myrcludex B诱导的小鼠NTCP介导的胆汁酸转运抑制的动力学，以及这种作用的持续时间是否/如何与NTCP蛋白更新相关。
方法：

在Myrcludex B治疗的OATP1a / 1b缺陷型小鼠中测定血浆胆汁酸。在体外，用生物素或异硫氰酸荧光素（FITC）标记的Myrcludex B标记质膜上驻留的NTCP，并使用过表达hNTCP的U2OS细胞及时追踪。通过荧光寿命成像显微镜进行的Förster共振能量转移用于研究Myrcludex B是否可以转移至新合成的NTCP。
结果：

皮下注射Myrcludex B后4小时血浆中共轭胆汁盐水平达到峰值。 24小时后，血浆胆汁盐水平完全恢复正常，这与恢复的NTCP介导的胆汁酸体外转运一致。生物素标记的NTCP的消失速度比Myrcludex B-FITC快，在24小时后仍保留了近40％的FITC信号。当表达DY547标记的酰基载体蛋白标记的NTCP时，FITC荧光寿命大大降低，表明预结合的Myrcludex B-FITC转移至新形成的NTCP。
结论：

NTCP蛋白更新和Myrcludex B诱导的血浆胆汁盐升高的动力学相似，这表明Myrcludex B：NTCP相互作用是很长寿的。但是，Myrcludex B不能与NTCP一起完全降解，并且可以转移到新合成的NTCP中。
放置摘要：

实验药物Myrcludex B结合牛磺胆酸钠共转运多肽（NTCP），乙型和丁型肝炎病毒的病毒进入受体（HBV / HDV），从而预防感染，但也抑制肝胆盐摄入，导致瞬时升高胆汁盐水平。这项研究描述了血浆胆汁盐水平的正常化可能取决于NTCP的蛋白质更新速率，而Myrcludex B通过从一个NTCP分子转移到另一个分子，部分逃脱了与NTCP的共降解。这对于HBV / HDV研究领域非常重要，因为它为Myrcludex B对病毒感染与胆汁盐转运的影响的独特动力学和剂量依赖性提供了可能的解释。

©2019作者。
关键字：

胆汁酸Myrcludex B; NTCP; OATP；乙型肝炎病毒；肝炎三角洲运输者

PMID：
    32039379
PMCID：
    PMC7001544
DOI：
    10.1016 / j.jhepr.2019.07.006

StephenW

https://www.jhep-reports.eu/article/S2589-5559(19)30084-9/pdf