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。我们将实施一个真正的 变革性的，全新的，技术“蛋白质绘画”，用于快速直接测序隐藏的天然 蛋白质-蛋白质相互作用热点，其起源于NCI R21 IMAT CA 177535。我们的技术 采用以前未开发的小分子（12 μ m）芳烃染料或“油漆”切割，MS 序列，只有两个或更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。 蛋白质绘画采用了一种全新的原理，产生了更高的特异性和三倍 与化学足迹法相比，阳性命中数更高。油漆化学品有着极其 高亲和力（快速结合速率和非常慢的解离速率，其比大多数蛋白质-蛋白质高10至100倍 互动）。当与天然的预形成的蛋白质复合物混合仅5分钟时，涂料不需要添加剂。 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 获得对溶剂不可及的隐藏的蛋白质-蛋白质相互作用域的访问。每个油漆分子 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， 未结合的颜料被洗掉，蛋白质被解离。这使得油漆分子 不参与界面的曲面。解离后，将蛋白质线性化，用 油漆分子保持非共价结合后， 蛋白质是变性的。蛋白水解酶如胰蛋白酶将不切割蛋白质的区域， “画”。因此，在蛋白水解之后，从MS中出现的肽将仅从蛋白水解产物中产生。 蛋白质紧密接触的未修饰的相对点。所有原始R21目标和 军事目标完全实现了。在蛋白质涂敷和细胞周期之间发现非常高的相关性（p<0.0003）。 通过晶体结构预测接触点，对真阳性热点具有97%的特异性。我们发现 进化上保守的3向热点，并通过创建肽和 阻断相互作用并消除信号传导的mAb。在目标1下，我们将发现新的热点 与三种类型的相互作用蛋白质复合物具有广泛治疗相关性的序列：1）细胞表面 受体（PD 1/PDL-1，PDL-2）参与肿瘤免疫细胞抑制，2）Hippo癌抑制因子 途径蛋白，和3）最近阐明的癌基因滤泡蛋白。在目标2下，我们将验证功能 (drug候选人）的重要性，我们发现的相互作用热点，通过创建肽和单克隆 结合到相互作用蛋白质的相对热点面的抗体。我们将证实这些配体 会抑制蛋白质复合物的形成对于PD 1/PDL-1，PDL-2细胞表面复合物，我们将扩展 蛋白质绘画，第一次，MS序列细胞间相互作用的接触点，在培养的活细胞。