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Computationally Guided Design of Thermostable mutants of Neurotensin receptor1

神经降压素受体 1 热稳定突变体的计算引导设计

基本信息

批准号：
8327192
负责人：
Nagarajan Vaidehi
金额：
$ 31.9万
依托单位：
BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2014-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8327192
关键词：
Adenosine A2A Receptor Adrenergic Receptor Algorithms Binding Biochemical Budgets Calibration Cells Cities Collaborations Computer Simulation Computing Methodologies Crystallization Data Detergents Disease Drug Delivery Systems Drug Design Drug abuse Environment Feedback Functional disorder Funding G-Protein-Coupled Receptors Goals Growth Growth Factor Homology Modeling Hydrogen Bonding In Vitro Interdisciplinary Study Laboratories Ligand Binding Ligands Malignant Neoplasms Marketing Membrane Methods Modeling Molecular Conformation Molecular Structure Mutagenesis Mutation National Institute of Neurological Disorders and Stroke Nature Neurotensin Neurotensin Receptors Peptides Pharmaceutical Preparations Pharmacology Phase Play Point Mutation Potential Energy Process Proteins Rattus Research Personnel Role Sampling Scanning Schizophrenia Side Signal Pathway Solutions Structure Techniques Testing Time United States National Institutes of Health Work base cancer cell cost design experience flexibility improved in vivo inhibitor/antagonist innovation insight interdisciplinary approach interdisciplinary collaboration meetings mimetics mutant protein purification receptor reconstitution research study stem valylleucine

项目摘要

DESCRIPTION (provided by applicant): Neurotensin receptors belong to the superfamily of peptide activated G protein-coupled receptors (GPCRs) and they are important drug targets to diseases such as schizophrenia and even cancer. The complexity in drug design for NTS1 and for many peptide activated GPCRs partly stems from the paucity of structural information, which could provide vital information for rational drug design. The barriers to pursuing structural and biophysical studies of membrane bound neurotensin receptors are due primarily to three challenges that are: 1.obtaining sufficient levels of expressed mammalian receptors, 2. functionally reconstituting them into membrane mimetics and 3. most importantly, stabilizing a particular inactive or active state conformation due to the conformational flexibility of GPCRs. While steps (1) and (2) are surmountable, step (3) the conformational flexibility in GPCRs, severely hampers the protein purification process for further crystallization or any other biophysical studies of GPCRs. Therefore meeting challenge (3) is critical in solving the macromolecular structure and ligand binding to GPCRs and requires a confluence of predictive computational models accompanied by experimental techniques. We propose to develop a computational method "LiticonDesign" that would enable design of thermally stable mutants of rat neurotensin receptor 1 (NTS1) (class A GPCRs in general). LiticonDesign will be based on the "Liticon", a predictive method that allows accurate modeling of various functional ligand stabilized conformational states of GPCRs. The predicted mutants of NTS1 will be expressed and tested for thermal stability and derive sufficient quantities of the pure functional receptor for structural studies. The results of this proposed work will provide a breakthrough method to obtain stable and functional pure protein of NTS1 which could be extended to other class A GPCRs. The proposed interdisciplinary collaboration is between Dr. Nagarajan Vaidehi (PI) at City of Hope, Dr. Reinhard Grisshammer NINDS/NIH and Dr. Christopher Tate (Univ. of Cambridge UK). Dr. Vaidehi has extensive experience in developing modeling techniques for GPCRs, while Dr. Tate is an expert in deriving thermostable mutants of GPCRs. Dr. Grisshammer is an expert on neurotensin receptors and their pharmacology. The budget is allocated only for Dr. Vaidehi, since Dr. Grisshammer is funded by the intramural funding in NINDS and Dr. Tate has sufficient funding for this work. The proposed work is ongoing in his laboratory.

描述（申请人提供）：神经紧张素受体属于肽激活G蛋白偶联受体（gpcr）超家族，是精神分裂症甚至癌症等疾病的重要药物靶点。NTS1和许多肽激活gpcr设计的复杂性部分源于结构信息的缺乏，这些信息可以为合理的药物设计提供重要信息。膜结合神经紧张素受体进行结构和生物物理研究的障碍主要是由于以下三个挑战：1。获得足够水平的表达哺乳动物受体；功能上重组成膜模拟物和3。最重要的是，由于gpcr的构象灵活性，稳定了特定的非活性或活性状态构象。虽然步骤(1)和(2)是可以克服的，但步骤(3)gpcr的构象灵活性严重阻碍了进一步结晶或gpcr的任何其他生物物理研究的蛋白质纯化过程。因此，解决挑战(3)对于解决大分子结构和配体与gpcr的结合至关重要，需要预测计算模型与实验技术相结合。我们建议开发一种计算方法“litcondesign”，用于设计大鼠神经紧张素受体1 （NTS1）的热稳定突变体（一般为a类gpcr）。litcondesign将基于“Liticon”，这是一种预测方法，可以精确建模gpcr的各种功能配体稳定构象状态。预测的NTS1突变体将被表达并进行热稳定性测试，并获得足够数量的纯功能受体用于结构研究。本研究结果将为获得稳定、功能纯正的NTS1蛋白提供一种突破性的方法，该方法可扩展到其他a类gpcr中。拟议的跨学科合作是由希望之城的Nagarajan Vaidehi博士（PI）， Reinhard Grisshammer博士和Christopher Tate博士（英国剑桥大学）进行的。Vaidehi博士在开发gpcr建模技术方面拥有丰富的经验，而Tate博士是gpcr耐热突变体的专家。格里斯沙默博士是神经紧张素受体及其药理学方面的专家。预算只分配给Vaidehi博士，因为Grisshammer博士是由研究所的内部资金资助的，Tate博士有足够的资金进行这项工作。他的实验室正在进行这项工作。