Alam Khondoker, Farasyn Taleah, Crowe Alexandra, Ding Kai, Yue Wei
Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States of America.
Department of Biostatistics and Epidemiology, College of Public Health, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States of America.
PLoS One. 2017 Nov 6;12(11):e0186924. doi: 10.1371/journal.pone.0186924. eCollection 2017.
OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and-OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50-250 nM, 2-7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217βG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.
有机阴离子转运多肽1B1（OATP1B1）和有机阴离子转运多肽1B3（OATP1B3）介导多种药物（如他汀类药物）的肝脏摄取，并可介导转运体介导的药物相互作用（DDIs）。硼替佐米是美国食品药品监督管理局批准用于治疗多发性骨髓瘤的首个蛋白酶体抑制剂类药物。硼替佐米引起OATP介导的药物相互作用的可能性尚未得到评估。当前研究调查了泛素-蛋白酶体系统（UPS）在OATP1B1和OATP1B3降解中的作用，并确定了蛋白酶体抑制剂对OATP1B1和OATP1B3介导转运的影响。在用带有FLAG标签的OATP1B1/OATP1B3和带有血凝素（HA）标签的泛素共转染的人胚肾（HEK）293细胞中，观察到FLAG-OATP1B1/1B3与HA-泛素的共免疫沉淀，这表明OATP1B1和OATP1B3可被泛素修饰。尽管在HEK293-FLAG-OATP1B1和-OATP1B3细胞中，用硼替佐米处理（50 nM，7小时）阻断蛋白酶体活性会增加内源性泛素结合的FLAG-OATP1B1和FLAG-OATP1B3，但这种处理并不影响OATP1B1和OATP1B3的总蛋白水平，这表明在当前组成性条件下，UPS在OATP1B1和OATP1B3的降解中起次要作用。用硼替佐米（50 - 250 nM，2 - 7小时）预处理可显著降低[3H]CCK - 8（一种特异性OATP1B3底物）在HEK293 - OATP1B3和人三明治培养肝细胞（SCH）中的转运。然而，硼替佐米预处理对[3H]E217βG和[3H]匹伐他汀（OATP1B1和OATP1B3的双重底物）在HEK293 - OATP1B1/1B3细胞和/或人SCH中的转运影响可忽略不计。与溶剂对照处理相比，硼替佐米预处理显著降低了OATP1B3介导的CCK - 8转运的最大转运速度（Vmax）（92.25 ± 14.2对133.95 ± 15.5 pmol/mg蛋白/分钟），而不影响亲和常数（Km）值。用其他蛋白酶体抑制剂MG132、环氧霉素和卡非佐米处理也显著降低了OATP1B3介导的[3H]CCK - 8转运。总之，当前研究首次报道了OATP1B1和OATP1B3的泛素化以及硼替佐米对OATP1B3介导转运的明显底物依赖性抑制作用。数据表明硼替佐米引起OATP介导的药物相互作用的风险较低。
