Protein painting identifies therapeutic targets at protein-protein interfaces

蛋白质绘画识别蛋白质-蛋白质界面的治疗靶点

• 批准号: 9392299
• 负责人: Lance Allen Liotta
• 金额: $ 9.45万
• 依托单位: GEORGE MASON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9392299
• 关键词: Affinity; Amino Acids; Binding; Biological Assay; Categories; Cell Communication; Cell Surface Receptors; Cell surface; Cells; Chemicals; Chemistry; Complex; Consensus; Crystallization; Crystallography; Data; Deuterium; Dissociation; Dyes; Face; Folliculin; Hot Spot; Hydrogen; Immune; Immune Cell Suppression; Immunotherapy; In Vitro; Ligands; Malignant Neoplasms; Maps; Methods; Molecular; Molecular Conformation; Monoclonal Antibodies; Oncogenes; PDCD1LG1 gene; Paint; Pathway interactions; Peptide Hydrolases; Peptide antibodies; Peptides; Production; Protein Binding Domain; Protein Region; Proteins; Proteolysis; Protocols documentation; Receptor Cell; Receptor Signaling; Resolution; Side; Signal Transduction; Signaling Protein; Site; Solvents; Specificity; Structure; Suppressor Genes; Surface; Technology; Therapeutic; Time; Trypsin; Tumor Suppressor Proteins; Validation; Western Blotting; aryl hydrocarbons; chymotrypsin; crosslink; drug candidate; monoclonal antibody production; neoplastic cell; new technology; next generation; novel; pointed protein; protein complex; protein protein interaction; small molecule; surface coating; synthetic peptide; therapeutic target; tumor

The next generation of molecular cancer therapeutics will target pivotal protein-protein interaction interfaces participating in immune cell receptor signaling, oncogenes, and suppressor genes. We will implement a truly transformative, wholly novel, technology “protein painting” for the rapid direct sequencing of hidden native protein-protein interaction hot spots, that was originated under NCI R21 IMAT CA177535. Our technology employs previously unexplored small molecule (12 Å) aryl hydrocarbon dyes or “paints” to cut out, and MS sequence, only the hidden unmodified contact interfaces between two or more interacting native proteins. Protein painting employs a completely new principle that yields a much higher specificity and three times higher number of positive hits compared to chemical footprinting methods. Paint chemistries have extremely high affinities (rapid on-rates, and very slow off-rates that are ten to 100 times higher than most protein-protein interactions). When mixed with a native pre-formed protein complex for only 5 minutes, the paints non- covalently coat all external sites on the protein without altering the 3D conformation of the complex, but cannot gain access to the solvent inaccessible hidden protein-protein interaction domains. Each paint molecule spans 3 amino acids or less, and has high affinity for protease cleavage consensus sites. Following painting, the unbound paints are washed away and the proteins are dissociated. This leaves the paint molecules coating surfaces not participating in the interface. Following dissociation, the proteins are linearized, digested with proteolytic enzymes, and sequenced by MS. The paint molecules remain non-covalently bound after the proteins are denatured. Proteolytic enzymes such as trypsin will not cleave the regions of the protein that are “painted”. Following proteolysis, therefore, peptides emerging from MS will exclusively be generated from the unmodified opposing points where the proteins were in intimate contact. All of the original R21 Aims and Milestones were fully met. A very high correlation (p<0.0003) was found between protein painting and the contact points predicted by crystal structure, with a 97% specificity for true positive hot spots. We discovered evolutionarily conserved 3-way hotspots and verified their functional importance by creating peptides and mAbs that block the interaction and extinguish signaling. Under Aim1, we will discover novel hotspot sequences of broad therapeutic relevance for interacting protein complexes of three types: 1) cell surface receptors (PD1/PDL-1,PDL-2) involved in tumor immune cell suppression , 2) Hippo cancer suppressor pathway proteins, and 3) the recently elucidated oncogene Folliculin.Under Aim 2 we will validate the functional (drug candidate) importance of the interaction hotspots we discover, by creating peptides and monoclonal antibodies that bind to the opposing hotspot faces of the interacting proteins. We will verify that these ligands will suppress protein complex formation. For the PD1/PDL-1,PDL-2 cell surface complexes, we will extend protein painting, for the first time, to MS sequence cell-cell interaction contact points in cultured live cells.

下一代分子癌症疗法将针对关键的蛋白质-蛋白质相互作用界面 参与免疫细胞受体信号传导、癌基因和抑制基因。我们将真正实施 革命性的、全新的“蛋白质绘画”技术，用于快速直接测序隐藏的天然基因 蛋白质-蛋白质相互作用热点，起源于 NCI R21 IMAT CA177535。我们的技术 采用以前未开发的小分子 (12 Å) 芳基烃染料或“涂料”进行剪切，并且 MS 序列，仅是两个或多个相互作用的天然蛋白质之间隐藏的未修饰的接触界面。 蛋白质涂装采用全新原理，可产生更高的特异性和三倍 与化学足迹法相比，阳性命中数更高。涂料化学具有极其 高亲和力（快速结合速率和非常慢的解离速率，比大多数蛋白质-蛋白质高 10 到 100 倍） 互动）。当与天然预形成蛋白质复合物混合仅 5 分钟时，该涂料就不会发生变化。 共价包被蛋白质上的所有外部位点而不改变复合物的 3D 构象，但不能 获得溶剂无法进入的隐藏的蛋白质-蛋白质相互作用域。每个油漆分子跨越 3个氨基酸或更少，并且对蛋白酶切割共有位点具有高亲和力。绘画之后， 未结合的油漆被洗掉，蛋白质被解离。这使得油漆分子涂层 不参与界面的表面。解离后，蛋白质被线性化，用 蛋白水解酶，并通过 MS 测序。涂料分子在反应后仍保持非共价键合 蛋白质变性。胰蛋白酶等蛋白水解酶不会切割蛋白质的区域。 “绘”。因此，在蛋白水解后，从 MS 中产生的肽将完全由 蛋白质紧密接触的未修饰的相对点。所有原始 R21 目标和 里程碑已全部实现。发现蛋白质涂色和 通过晶体结构预测接触点，对真正的阳性热点具有 97% 的特异性。我们发现 进化上保守的三向热点并通过创建肽和验证其功能重要性 阻断相互作用并消除信号传导的单克隆抗体。在Aim1下，我们会发现新奇的热点 与三种类型的相互作用蛋白复合物具有广泛治疗相关性的序列：1) 细胞表面 参与肿瘤免疫细胞抑制的受体 (PD1/PDL-1,PDL-2) , 2) Hippo 癌症抑制因子 途径蛋白，以及 3) 最近阐明的癌基因 Folliculin。在目标 2 下，我们将验证其功能 （候选药物）我们通过创建肽和单克隆抗体发现的相互作用热点的重要性 与相互作用蛋白质的相对热点面结合的抗体。我们将验证这些配体 会抑制蛋白质复合物的形成。对于PD1/PDL-1,PDL-2细胞表面复合物，我们将扩展 首次对培养的活细胞中的细胞间相互作用接触点进行蛋白质绘制。