De novo design of generalizable allosteric modulators and peptide ligands for G protein coupled receptors

G 蛋白偶联受体的通用变构调节剂和肽配体的从头设计

• 批准号: 10160902
• 负责人: Christopher David Bahl
• 金额: $ 17.98万
• 依托单位: INSTITUTE FOR PROTEIN INNOVATION, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-07 至 2022-01-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10160902
• 关键词: Address; Affinity; Agonist; Binding; Binding Proteins; Biological; Biological Assay; Brain; C-terminal; Cell surface; Communities; Custom; DNA; Detergents; Disease; Disulfides; Drug Design; Drug Targeting; Engineering; Escherichia coli; Extracellular Domain; FDA approved; Family; Fluorescence-Activated Cell Sorting; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Genetic; Goals; Heterotrimeric GTP-Binding Proteins; Individual; Investigation; Lead; Ligand Binding; Ligands; Luciferases; Mediating; Methods; Modeling; Molecular; Molecular Conformation; N-terminal; Oligonucleotides; Peptides; Pharmaceutical Preparations; Process; Production; Protein Engineering; Protein Family; Protein Subunits; Proteins; Receptor Activation; Reporter; Research; Signal Transduction; Signaling Protein; Site; Source; Specificity; Structure; Surface; Synthetic Genes; System; Technology; Testing; Therapeutic; Time; Transmembrane Domain; Western Blotting; Work; Yeasts; base; deep sequencing; design; drug discovery; experimental study; high throughput screening; improved; in vivo; mimetics; nanobodies; overexpression; peptide structure; protein expression; protein protein interaction; protein purification; protein structure; receptor; receptor binding; receptor function; screening; small molecule; structural biology; thermostability; tool

Project Summary Roughly one third of all FDA approved drugs target G protein coupled receptors (GPCRs), and this protein family holds myriad therapeutic opportunities yet to be discovered. GPCRs signal through allosteric changes in protein structure, and there is a large unmet need for effective, highly generalizable tools for conformational control of these receptors. ​This project has two Specific Aims, both of which employ computational protein design with Rosetta to build proteins ​de novo that bind and modulate GPCR conformation. Upon ligand binding and activation, GPCRs associate with and signal through G proteins. In the first Aim, we design proteins ​de novo which mimic the interaction domain of the endogenous Gα protein subunit. Because the interaction between receptor and Gα is highly conserved, the protein tools we propose to develop can be applied broadly. Synthetic genes encoding ​de novo Gα mimetics have been expressed, and protein was purified from Escherichia coli.​ Binding to several different detergent-solubilized GPCRs was demonstrated ​via pull-down and Western blotting. Functional Gα mimetic proteins will be structurally characterized and used as templates for re-engineering a small suite of mimetics for all Gα subtypes. Additionally, we propose to leverage the Gα mimetics to enhance the expression and purification of GPCRs. To demonstrate this system, we will attempt to produce three different GPCRs for the first time: super conserved receptor expressed in brain 1-3. In the second aim, we will design disulfide constrained peptides to serve as agonists and antagonists for all structure-enabled family B GPCRs. This receptor family recognizes endogenous peptide ligands, and they are difficult to drug with small molecules. Structures that detail ligand recognition are available for 60% of family B receptors; we will use these as the basis for ​de novo design of high-affinity antagonist ligands based on disulfide-constrained peptides. We will then screen hundreds of thousands of rationally designed ​de novo peptides using yeast display, fluorescence activated cell sorting, and deep sequencing. Our unique design approach enables us to custom tailor the peptide topology so that we can readily convert our antagonists into high affinity agonists by extending the peptide N-terminus. The efficacy of designed ligands identified ​via the high-throughput screen will be assessed individually using a luciferase reporter assay for GPCR signaling. The research proposed here represents the first time that computational ​de novo protein design will be applied to control GPCR conformation. This work has the potential to create valuable tools to empower the GPCR research community and accelerate discovery of lead therapeutics for many diseases.

项目摘要 大约三分之一的FDA批准的药物靶向G蛋白偶联受体（GPCR），这种蛋白质 家庭拥有无数有待发现的治疗机会。GPCR信号通过变构变化， 蛋白质结构，并且存在对用于构象分析的有效的、高度可推广的工具的大量未满足的需求。 控制这些受体。 这个项目有两个特定的目标，这两个都采用计算蛋白质 设计与Rosetta从头构建蛋白质结合和调节GPCR构象。在配体结合后 和激活，GPCR与G蛋白结合并通过G蛋白发出信号。在第一个目标中，我们设计了蛋白质， novo，其模拟内源性Gα蛋白亚基的相互作用结构域。由于交互 由于受体与Gα之间存在高度保守性，因此我们提出的蛋白质工具具有广泛的应用前景。 已经表达了编码从头Gα模拟物的合成基因，并从大肠杆菌中纯化了蛋白质。 通过拉下和电泳证明了与几种不同的洗涤剂溶解的GPCR的结合。 蛋白质印迹法。功能性Gα模拟蛋白将在结构上表征并用作模板， 为所有Gα亚型重新设计一小套模拟物。此外，我们建议利用Gα 模拟物以增强GPCR的表达和纯化。为了演示这个系统，我们将尝试 首次产生三种不同的GPCR：脑中表达的超保守受体1-3。在 第二个目标，我们将设计二硫键限制肽作为所有的激动剂和拮抗剂， 结构激活的家族B GPCR。该受体家族识别内源性肽配体，并且它们是 很难用小分子药物。详细描述配体识别的结构可用于60%的家族B 受体;我们将使用这些作为从头设计高亲和力拮抗剂配体的基础， 二硫键约束肽。然后我们会筛选出数十万个合理设计的重新设计的 使用酵母展示、荧光激活细胞分选和深度测序的肽。我们独特的设计 这种方法使我们能够定制肽的拓扑结构，这样我们就可以很容易地将我们的拮抗剂转化为 通过延伸肽的N-末端来制备高亲和力激动剂。设计的配体的功效通过 高通量筛选将使用用于GPCR信号传导荧光素酶报道基因测定单独评估。的 这里提出的研究代表了第一次计算从头蛋白质设计将应用于 对照GPCR构象。这项工作有可能创造有价值的工具，增强气相化学还原的能力。 研究社区和加速发现许多疾病的领先疗法。

项目成果

Toward complete rational control over protein structure and function through computational design.

• DOI: 10.1016/j.sbi.2020.10.015
• 发表时间: 2021-03
• 期刊: Current opinion in structural biology
• 影响因子: 6.8
• 作者: Adolf-Bryfogle J;Teets FD;Bahl CD
• 通讯作者: Bahl CD

Complete Combinatorial Mutational Enumeration of a protein functional site enables sequence-landscape mapping and identifies highly-mutated variants that retain activity.

蛋白质功能位点的完整组合突变枚举可实现序列景观图谱并识别保留活性的高度突变变体。

• DOI: 10.21203/rs.3.rs-2248327/v1
• 发表时间: 2023
• 期刊: Research square
• 作者: Colom,MireiaSolà;Vucinic,Jelena;Adolf-Bryfogle,Jared;Bowman,JamesW;Verel,Sébastien;Moczygemba,Isabelle;Schiex,Thomas;Simoncini,David;Bahl,ChristopherD
• 通讯作者: Bahl,ChristopherD