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; 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.

项目摘要 多态性转运蛋白OATP1B1在肝摄取和处置中起着重要作用 内源分子和药物。此外，OATP1B1参与了许多药物相互作用（DDI） 具有多种特异性。尽管进行了数十年的研究，但许多因素与确定配体结合有关的因素， 运输机制和能量学以及该重要转运蛋白的结构尚未解决。这 长期目标是在微观和宏观水平上表征肝脏运输。由于 OATP1B1在肝清除率中的功能重要性，这是第一个目标研究。中心假设是 在孤立蛋白上执行的人OATP1B1的结构和功能测定将明确定义 配体结合，转运和抑制的机制。这项研究的基本原理是 蛋白质结构以及明确的，可重复的活性测定绝对需要完全需要 表征运输机制并解决文献中报告的长期冲突数据。 建立人类OATP1B1的强大表达和纯化协议有助于追求两者 具体目的：1）通过冷冻EM解决OATP1B1的结构，并通过MD模拟分析其运动。 2） 在功能上表征OATP1B1的传输机制。对于第一个目标，最佳条件 将筛选OATP1B1的Cryo-Em的最高图像质量，并将数据获取 配体的存在和不存在。 Cryo-EM研究将通过MD模拟和对接进行增强 可以揭示仅通过结构分析无法访问的细节的研究。不同的生化 条件（APO和HOLO）增加了a）求解各种结合的结构决定者的可能性 配体（具有独特的化学）与OATP1B1的两个配体结合位点。 b）解决多重结构 构象是运输周期的一部分。对于第二个目标，我们将使用纯化的蛋白质重构为 蛋白脂质体以表征OATP1B1的固有特性，例如传输的能量离子， 膜电压的影响和pH。这些结果将与细胞测定法进行比较 包括完整的细胞机械以及可能直接或间接影响的其他质膜转运蛋白 OATP1B1。该应用程序认为，本申请中提出的研究具有创新性 引入了从未对任何OATP同工型探索过的两种方法。这些可能会发现 对OATP1B1的结构/功能关系的大量新见解。拟议的研究是 由于OATP1B1参与了几种常规药物的DDI，有时会导致 威胁生命的情况。这项研究将揭示配体与OATP1B1和 运输。最终，这里产生的结构和功能知识有可能帮助设计 对DDIS表现出较低前景的药物。