Development of a novel proximity catalyzed Chemical Epitope Targeting technology for isolating macrocyclic peptide inhibitors of KRas(G12V)-Sos interaction

摘要 用于治疗目的的蛋白质功能修饰通常是通过小分子药物完成的 结合在蛋白质的深层疏水口袋中。对于在延伸的浅层上发生的蛋白质-蛋白质相互作用 在蛋白质的表面积上，小分子与表面积结合，从而影响蛋白质的功能，这是具有挑战性的。RAS -SOS是广泛的蛋白质-蛋白质表面相互作用的经典例子，因此抑制它仍然具有挑战性 KRAS-SOS相互作用。KRAS是由SOS激活的，开发技术来抑制 癌基因突变体KRAS与SOS选择性结合。其中一项很有前途的技术是化学表位靶向 为设计针对特定区域的高亲和力和特异性的多肽配体而开发的技术 小分子或抗体可能无法接触到的细胞内蛋白质。这项技术涉及使用 邻近催化反应，用于筛选目标蛋白的特定区域以分离多肽配体。 PI建议简化这项技术，使其更易于访问和用户友好，然后定制 这项技术成功地靶向突变的KRAS(G12V)蛋白，特别是在RAS-SOS界面。这个 将通过开发和表征合成大环一珠一酮化合物来简化技术 (OBOC)多肽库，可通过一步光曝光切割，实现串联质谱仪的无缝测序 光谱分析(MS/MS)(目标1)。四氮杂环上的反电子需求Diels-Alder(IEDDA)反应 烯烃将用于筛选过程，而不是目前使用的叠氮-炔环加成反应。 作为概念验证，PI应证明取代四嗪与一种 烯烃可以被一种蛋白质近邻催化。KRAS(G12C)或带有一个Cys的KRAS突变体将在Cys进行修改 加了四氢呋喃。用含有SOS-螺旋多肽的烯烃标记该络合物，已知该多肽结合在RAS- SOS界面，将使用串联质谱仪(AIM 2)进行监测。最低温度的最适温度 背景将确定四嗪和烯烃之间的IEDDA反应，以最小化屏幕的背景 在下一步。 针对化学表位KRAS(G12V)的化学表位靶向筛选 烯烃小分子，将用S、S-四嗪环化的OBOC多肽文库(目标3)进行合成。筛选 随后将使用分裂的荧光素酶平台抑制KRAS(G12V)-SOS相互作用。目标1和目标2应该是 目标3将在授权期内启动。这个项目是多学科的，将使PI能够 在克拉克大学培训本科生和研究生，掌握各种化学和生化技术。

ABSTRACT Protein function modifications for therapeutic purposes are typically accomplished with small molecule drugs that bind in deep hydrophobic pockets of proteins. For protein-protein interactions occurring over an extended shallow area, it is challenging to have small molecules binding to the surface area and thereby affecting protein functions. Ras -Sos is a classic example of extensive protein-protein surface interaction, and therefore it remains challenging to inhibit KRas- Sos interactions. KRas is activated by Sos, and it is critical to develop technologies to inhibit interactions of oncogenic mutant KRas with Sos selectively. One such promising technology is the Chemical Epitope Targeting technology, developed for designing peptide ligands with high affinity and specificity against specific regions of an intracellular protein that may be inaccessible to small molecules or antibodies. This technology involves using proximity-catalyzed reaction for screening a specific region of the target protein to isolate peptide ligands. The PI proposes to streamline this technology to make it more accessible and user-friendly, and then to tailor this technology to successfully target mutant KRas(G12V) protein, specifically at the Ras-Sos interface. The technology will be streamlined by developing and characterizing synthetic macrocyclic one-bead-one compound (OBOC) peptide libraries that can be cleaved by one-step light exposure for seamless sequencing by tandem Mass Spectrometry (MS/MS) (Aim 1). Inverse Electron Demand Diels-Alder (IEDDA) reaction between tetrazine and alkene will be used for the screening process rather than the currently used azide-alkyne cycloaddition. The PI shall demonstrate, as a proof-of-concept, that IEDDA reaction between substituted tetrazine and an alkene can be proximity-catalyzed by a protein. KRas(G12C) or KRas mutants with one Cys will be modified at Cys with tetrazine. The labeling of the complex with an alkene containing Sos-helix peptide, known to bind at the Ras- Sos interface, will be monitored using tandem Mass Spectrometry (Aim 2). The optimal temperature for minimal background IEDDA reaction between tetrazine and alkene will be identified, to minimize background for the screen in the next step. A Chemical Epitope Targeting screen against the Chemical Epitope, KRas(G12V) complexed to a GDP- alkene small molecule, will be performed using S,S-tetrazine cyclized OBOC peptide libraries (Aim 3). Screening for inhibition of KRas(G12V)-Sos interaction using a split luciferase platform will follow. Aims 1 and 2 should be achieved, and Aim 3 be initiated within the grant period. This project, being multidisciplinary, will allow the PI to train undergraduate and graduate students at Clark University in a variety of chemical and biochemical techniques.