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：
8476236
负责人：
Nagarajan Vaidehi
金额：
$ 30.78万
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 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8476236
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 Design Drug Targeting 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）：神经降压素受体属于肽激活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 和许多肽激活的 GPCR 药物设计的复杂性部分源于结构信息的缺乏，而结构信息可以为合理的药物设计提供重要信息。进行膜结合神经降压素受体的结构和生物物理研究的障碍主要是由于三个挑战：1.获得足够水平的表达哺乳动物受体，2.在功能上将它们重组为膜模拟物，3.最重要的是，稳定特定的非活性或由于 GPCR 的构象灵活性而形成的活性状态构象。虽然步骤 (1) 和 (2) 是可以克服的，但步骤 (3) GPCR 中的构象灵活性严重阻碍了 GPCR 进一步结晶或任何其他生物物理研究的蛋白质纯化过程。因此，满足挑战（3）对于解决大分子结构和配体与 GPCR 的结合至关重要，并且需要预测计算模型与实验技术的融合。我们建议开发一种计算方法“LiticonDesign”，该方法将能够设计大鼠神经降压素受体1（NTS1）（一般为A类GPCR）的热稳定突变体。 LiticonDesign 将基于“Liticon”，这是一种预测方法，可以对 GPCR 的各种功能性配体稳定构象状态进行精确建模。预测的 NTS1 突变体将被表达并测试热稳定性，并获得足够数量的纯功能受体用于结构研究。这项工作的结果将为获得稳定且功能性的 NTS1 纯蛋白提供突破性方法，并可推广到其他 A 类 GPCR。拟议的跨学科合作由 City of Hope 的 Nagarajan Vaidehi 博士（PI）、NINDS/NIH 的 Reinhard Grisshammer 博士和 Christopher Tate 博士（英国剑桥大学）共同参与。 Vaidehi 博士在开发 GPCR 建模技术方面拥有丰富的经验，而 Tate 博士是衍生 GPCR 热稳定突变体的专家。 Grissammer 博士是神经降压素受体及其药理学方面的专家。预算仅分配给 Vaidehi 博士，因为 Grisshammer 博士由 NINDS 的校内资金资助，而 Tate 博士有足够的资金来开展这项工作。拟议的工作正在他的实验室进行。