Protein painting identifies therapeutic targets at protein-protein interfaces

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、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、用于快速直接对隐藏的原生生物进行测序的“蛋白质涂抹”技术 蛋白质相互作用热点，起源于NCI R21 IMAT CA177535。我们的技术 使用以前未曾探索过的小分子(12？)芳基碳氢化合物染料或“涂料”来裁剪，以及MS 序列，只有两个或更多相互作用的天然蛋白质之间隐藏的未修改的接触界面。 蛋白质涂色采用了一种全新的原理，它产生了更高的特异性和三倍 与化学足迹方法相比，阳性命中率更高。油漆的化学成分具有极高的 高亲和力(快速开启和非常缓慢的关闭速度，比大多数蛋白质-蛋白质高10到100倍 交互)。当与天然预形成的蛋白质复合体混合仅5分钟时，涂料不会 在不改变复合体3D构象的情况下共价覆盖蛋白质上的所有外部位点，但不能 获得溶剂无法访问的隐藏蛋白质-蛋白质相互作用域。每种油漆分子都跨越了 3个或更少的氨基酸，并与蛋白酶裂解共识部位有很高的亲和力。在绘画之后， 未结合的油漆被冲走，蛋白质被解离。这就留下了油漆分子的涂层 不参与界面的曲面。解离后，蛋白质线性化，用 蛋白水解酶，并经MS测序后，油漆分子保持非共价结合 蛋白质被变性。蛋白水解酶，如胰酶，不会切割蛋白质的区域， “画好了”。因此，在蛋白质分解之后，从MS中产生的多肽将专门从 未经修饰的蛋白质密切接触的对立点。所有最初的R21目标和 里程碑的目标完全实现了。研究发现，蛋白质涂抹与蛋白质含量之间存在很高的相关性(p&lt；0.0003)。 通过晶体结构预测接触点，对真阳性热点的特异性为97%。我们发现 进化上保守的三向热点，并通过产生多肽和 阻断相互作用并阻断信号传递的单抗。在Aim1下，我们将发现新的热点 与三种类型相互作用的蛋白质复合体具有广泛治疗意义的序列：1)细胞表面 参与肿瘤免疫细胞抑制的受体(PD1/PDL-1，PDL-2)，2)河马抑癌基因 途径蛋白，以及3)最近阐明的癌基因卵泡蛋白。在目标2下，我们将验证其功能 (候选药物)我们通过创造多肽和克隆发现的相互作用热点的重要性 结合在相互作用蛋白质的相反热点表面上的抗体。我们会核实这些配体 会抑制蛋白质复合体的形成。对于PD1/PDL-1，PDL-2细胞表面复合体，我们将扩展 蛋白质涂抹，首次将MS序列中的细胞-细胞相互作用接触点在培养的活细胞中。

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.