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 FDA批准的所有药物中约有三分之一针对G蛋白偶联受体(GPCRs)，而这种蛋白质 家庭拥有无数尚未发现的治疗机会。GPCRs信号通过变构的变化 蛋白质结构，以及对有效的、高度通用的构象分析工具的巨大需求。 对这些受体的控制。​这个项目有两个特定的目标，都使用了计算蛋白质 用罗塞塔设计构建结合和调节gpcr构象的蛋白质​de nevo。关于配基结合 和激活，GPCRs与G蛋白结合并通过G蛋白传递信号。在第一个目标中，我们设计了蛋白质​ NOVO模拟内源Gα蛋白亚基的相互作用结构域。因为这种互动 受体和G-α之间高度保守，我们建议开发的蛋白质工具具有广泛的应用前景。 表达了编码​de novo Gα模拟物的合成基因，并从 ​与几种不同洗涤剂增溶的GPCR结合的​通过下拉和 Western blotting。功能性G-α模拟蛋白的结构将被表征并用作模板 重新设计了一套适用于所有Gα亚型的模拟程序。此外，我们建议利用Gα 促进GPCRs的表达和纯化的模拟物。为了演示此系统，我们将尝试 首次产生三种不同的GPCRs：在大脑中表达的超保守受体1-3。在 第二个目标，我们将设计二硫键受限制的多肽，作为所有人的激动剂和拮抗剂。 结构使能的B族GPCRs。这个受体家族识别内源性多肽配体，它们是 很难用小分子药物。B家族中60%的人有详细的配基识别结构 受体；我们将利用这些作为​从头设计高亲和力拮抗剂配体的基础 二硫键结合的多肽。然后我们将筛选出成千上万的合理设计的​de nevo 多肽采用酵母展示、荧光激活的细胞分选和深度测序。我们独特的设计 方法使我们能够定制多肽拓扑，以便我们可以很容易地将我们的对手转换为 通过延伸多肽N-末端的高亲和力激动剂。设计的配体通过​识别的有效性 高通量筛选将使用荧光素酶报告试验对GPCR信号进行单独评估。这个 这里提出的研究代表了计算性​从头开始蛋白质设计将首次应用于 控制gpr的构象。这项工作有可能创造出有价值的工具来增强GPCR的能力 研究社区并加速发现针对许多疾病的铅疗法。

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