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
基本信息
- 批准号:10638284
- 负责人:
- 金额:$ 48.33万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AffectBile AcidsBinding SitesBiochemicalBiological AssayBreast Cancer cell lineCarrier ProteinsCell LineCell membraneCellsCellular AssayChargeChemistryCommunitiesComplexContrast MediaCoupledCouplingCryoelectron MicroscopyDataData ReportingDiseaseDockingDoxorubicinDrug DesignDrug InteractionsDrug PrescriptionsElderlyEpitopesExcretory functionExhibitsFamilyFoodGoalsGrowthHealthHepaticHepatocyteHomologous GeneHormonesHumanImageIon TransportIonsIsomerismKineticsKnowledgeLifeLigand BindingLigandsLiteratureLiverMagnetic Resonance ImagingMalignant NeoplasmsMediatingMembraneMembrane Transport ProteinsMetabolic Clearance RateMetabolismMethodologyMethotrexateMicroscopicMissionModelingMolecular ConformationMotionNutrientOATP TransportersPaclitaxelPharmaceutical PreparationsPhenotypePhysiologicalPhysiologyPlasmaPlayPolymorphPost-Translational Protein ProcessingProcessPropertyProstaglandinsProtein IsoformsProteinsProtocols documentationPublic HealthRecordsReproducibilityResearchRiskRoleSignaling MoleculeSolidSpecificityStructureSulfateTestingTherapeuticTherapeutic EffectTyrosine Kinase InhibitorUnited States National Institutes of HealthXenobioticsabsorptionanti-cancerdocetaxelhydrophilicityinnovationinsightirinotecanmathematical modelmembermolecular dynamicsnoveloverexpressionprostate cancer metastasisprotein purificationprotein reconstitutionprotein structureproteoliposomesside effectstemstructural determinantsuptakevoltage
项目摘要
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OATP 1B 1在肝脏摄取和处置
内源性分子和药物。此外,OATP 1B 1参与许多药物相互作用(DDI),
它的多特异性。尽管经过数十年的研究,许多因素与配体结合的决定因素相关,
运输机制和能量学,以及这种重要的运输工具的结构尚未解决。的
长期目标是在微观和宏观水平上表征肝脏转运。由于
OATP 1B 1在肝脏清除中的功能重要性,这是研究的第一个靶点。核心假设是,
人OATP 1B 1的结构和对分离蛋白进行的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,
绝对需要蛋白质结构与明确的、可重复的活性测定相结合,
描述运输机制并解决文献中长期存在的相互矛盾的数据。
建立人OATP 1B 1的稳健表达和纯化方案有助于实现这两个目标。
具体目的:1)通过Cryo-EM解析OATP 1B 1的结构,并通过MD模拟分析其运动。(二)
功能性表征OATP 1B 1的转运机制。对于第一目标,
将筛选OATP 1B 1的Cryo-EM图像质量最高的结果,并将在
配体的存在和不存在。冷冻EM研究将通过MD模拟和对接来增强
这些研究将揭示仅仅通过结构分析无法获得的细节。不同的生化
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
OATP 1B 1的两个配体结合位点的配体(具有不同的化学性质)。B)求解多重结构
这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成
蛋白脂质体来表征OATP 1B 1的固有特性,例如用于运输的赋能离子,
这些结果将与细胞测定进行比较,
包括完整细胞机器和其它可能直接或间接影响
OATP1B1。本申请中提出的研究是创新的,申请人认为,
介绍了两种方法,从来没有探索任何OATP亚型。这些可能会发现一个
对OATP 1B 1的结构/功能关系的大量新见解。拟议的研究是
由于OATP 1B 1参与了几种常用处方药的DDI,有时会导致
危及生命的情况这项研究将揭示配体与OATP 1B 1结合的确切决定因素,
运输最终,这里产生的结构和功能知识有可能帮助设计
药物滥用倾向较低的药物。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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{{ truncateString('Aviv Paz', 18)}}的其他基金
Chemically engineered bilayers for cryoEM imaging of membrane proteins in continuous membranes
用于连续膜中膜蛋白冷冻电镜成像的化学工程双层
- 批准号:
10091731 - 财政年份:2019
- 资助金额:
$ 48.33万 - 项目类别:
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