Structure meets function for OATP1B1, a transporter involved in the uptake of endogenous and xenobiotic materials and drugs

OATP1B1 的结构与功能相结合，OATP1B1 是一种参与内源性和外源性物质和药物摄取的转运蛋白

• 批准号: 10638284
• 负责人: Aviv Paz
• 金额: $ 48.33万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638284
• 关键词: Affect; Bile Acids; Binding Sites; Biochemical; Biological Assay; Breast Cancer cell line; Carrier Proteins; Cell Line; Cell membrane; Cells; Cellular Assay; Charge; Chemistry; Communities; Complex; Contrast Media; Coupled; Coupling; Cryoelectron Microscopy; Data; Data Reporting; Disease; Docking; Doxorubicin; Drug Design; Drug Interactions; Drug Prescriptions; Elderly; Epitopes; Excretory function; Exhibits; Family; Food; Goals; Growth; Health; Hepatic; Hepatocyte; Homologous Gene; Hormones; Human; Image; Ion Transport; Ions; Isomerism; Kinetics; Knowledge; Life; Ligand Binding; Ligands; Literature; Liver; Magnetic Resonance Imaging; Malignant Neoplasms; Mediating; Membrane; Membrane Transport Proteins; Metabolic Clearance Rate; Metabolism; Methodology; Methotrexate; Microscopic; Mission; Modeling; Molecular Conformation; Motion; Nutrient; OATP Transporters; Paclitaxel; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Plasma; Play; Polymorph; Post-Translational Protein Processing; Process; Property; Prostaglandins; Protein Isoforms; Proteins; Protocols documentation; Public Health; Records; Reproducibility; Research; Risk; Role; Signaling Molecule; Solid; Specificity; Structure; Sulfate; Testing; Therapeutic; Therapeutic Effect; Tyrosine Kinase Inhibitor; United States National Institutes of Health; Xenobiotics; absorption; anti-cancer; docetaxel; hydrophilicity; innovation; insight; irinotecan; mathematical model; member; molecular dynamics; novel; overexpression; prostate cancer metastasis; protein purification; protein reconstitution; protein structure; proteoliposomes; side effect; stem; structural determinants; uptake; voltage

Project summary The polymorphic transporter OATP1B1 plays significant roles in the hepatic uptake and disposition of endogenous molecules and drugs. Further, OATP1B1 is involved in numerous drug-drug interactions (DDIs) due to its multispecificity. Despite decades of research, many factors related to the determinants of ligand binding, transport mechanism and energetics, as well as the structure of this important transporter are unresolved. The long-term goal is to characterize hepatic transport at the microscopic and macroscopic levels. Due to the functional importance of OATP1B1 in liver clearance, this is the first target studied. The central hypothesis is that structures of human OATP1B1 and functional assays performed on isolated proteins will unequivocally define the mechanism of ligand binding, transport, and inhibition. The rationale that underlies this research is that protein structures coupled with unambiguous, reproducible activity assays are absolutely required to fully characterize the mechanism of transport and resolve long-standing conflicting data reported in the literature. Establishing robust expression and purification protocols for human OATP1B1 facilitates pursuing the two Specific Aims: 1) Resolve structures of OATP1B1 by Cryo-EM and analyze their motions by MD simulations. 2) Functionally characterize the transport mechanism of OATP1B1. For the 1st Aim, the optimal conditions that result in the highest image quality for Cryo-EM of OATP1B1 would be screened, and data would be acquired in the presence and absence of ligands. The Cryo-EM studies will be augmented by MD simulations and docking studies that would reveal details that are not accessible by mere structural analysis. The different biochemical conditions (apo and holo) increase the likelihood of A) solving the structural determinants of the binding of various ligands (with distinct chemistries) to the two ligand-binding sites of OATP1B1. B) solving the structures of multiple conformations that are part of the transport cycle. For the 2nd Aim, we will use purified protein reconstituted into proteoliposomes to characterize the inherent properties of OATP1B1 such as the energizing ion for transport, the influence of the membrane voltage, and pH. Those results would be compared with cellular assays that include intact cellular machinery, and other plasma membrane transporters that might directly or indirectly affect OATP1B1. The research proposed in this application is innovative, in the applicant’s opinion because it introduces two methodologies that have never been explored for any OATP isoform. These could uncover a plethora of novel insights into the structure/function relationship of OATP1B1. The proposed research is significant since OATP1B1 is involved in DDIs of several commonly prescribed drugs, sometimes resulting in life-threatening situations. This study would unravel the exact determinants of ligand binding to OATP1B1 and transport. Ultimately, the structural and functional knowledge generated here has the potential to aid in the design of drugs that exhibit a lower propensity for DDIs.

项目概要 多态性转运蛋白 OATP1B1 在肝脏摄取和处置中发挥重要作用 此外，OATP1B1 还参与多种药物相互作用 (DDI)。 尽管经过数十年的研究，许多因素与配体结合的决定因素有关， 运输机制和能量学以及这一重要转运蛋白的结构尚未解决。 长期目标是在微观和宏观水平上表征肝脏运输。 OATP1B1 在肝脏清除中的功能重要性，这是研究的第一个目标。中心假设是： 人类 OATP1B1 的结构和对分离蛋白质进行的功能测定将明确定义 配体结合、运输和抑制的机制是这项研究的基本原理。 蛋白质结构与明确的、可重复的活性测定相结合是完全必要的 描述运输机制并解决文献中报告的长期存在的相互矛盾的数据。 为人类 OATP1B1 建立稳健的表达和纯化方案有助于追求这两个目标 具体目标： 1) 通过冷冻电镜解析 OATP1B1 的结构并通过 MD 模拟分析其运动 2)。 从功能上表征 OATP1B1 的转运机制 对于第一个目标，最佳条件是 结果将筛选 OATP1B1 冷冻电镜的最高图像质量，并在 MD 模拟和对接将增强冷冻电镜研究。 这些研究将揭示仅通过结构分析无法获得的细节。 条件（apo 和 Holo）增加了 A) 解决各种结合的结构决定因素的可能性 B) 解决多个配体的结构。 对于第二个目标，我们将使用重组的纯化蛋白质。 蛋白脂质体来表征 OATP1B1 的固有特性，例如用于运输的供能离子， 这些结果将与细胞测定进行比较。 包括完整的细胞机器和其他可能直接或间接影响的质膜转运蛋白 申请人认为，本申请中提出的研究具有创新性，因为它 引入了两种从未针对任何 OATP 同工型进行过探索的方法，这些方法可能会发现一个 OATP 同工酶。 拟议的研究对 OATP1B1 的结构/功能关系有大量新颖的见解。 意义重大，因为 OATP1B1 参与几种常用处方药的 DDI，有时会导致 这项研究将揭示配体与 OATP1B1 结合的确切决定因素和 最终，这里产生的结构和功能知识有可能有助于设计。 表现出较低 DDI 倾向的药物。