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用小分子药物完成， 结合在蛋白质的深层疏水口袋中。对于蛋白质-蛋白质相互作用， 在蛋白质的表面区域，使小分子结合到表面区域并从而影响蛋白质功能是具有挑战性的。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，因此开发抑制KRas与Sos相互作用的技术至关重要。 致癌突变KRas与Sos选择性结合。其中一种有前途的技术是化学表位靶向 技术，开发用于设计对免疫球蛋白的特定区域具有高亲和力和特异性的肽配体。 小分子或抗体可能无法接近细胞内蛋白质。这项技术涉及使用 用于筛选靶蛋白的特定区域以分离肽配体的邻近催化反应。 PI建议简化这项技术，使其更容易获得和用户友好，然后定制 该技术成功靶向突变KRas（G12 V）蛋白，特别是在Ras-Sos界面。的 将通过开发和表征合成大环一珠一化合物来简化技术 （OBOC）肽文库，其可通过一步光暴露切割，用于通过串联质谱进行无缝测序。 光谱法（MS/MS）（目的1）。研究了四嗪与四氢呋喃的反电子需求Diels-Alder（IEDDA）反应。 烯烃将用于筛选过程，而不是目前使用的叠氮化物-炔环加成。 PI应证明，作为概念验证，取代的四嗪和 烯烃可以被蛋白质邻位催化。具有一个Cys的KRas（G12 C）或KRas突变体将在Cys处被修饰 用四嗪。用已知结合在Ras-16处的含烯烃的Sos-螺旋肽标记复合物， 将使用串联质谱法（Aim 2）监测Sos界面。最低温度 将鉴定四嗪和烯烃之间的背景IEDDA反应，以使筛选的背景最小化 下一个步骤。 针对与GDP-I复合的化学表位KRas（G12 V）的化学表位靶向筛选 将使用S，S-四嗪环化的OBOC肽文库（Aim 3）进行烯烃小分子的测定。筛查 随后使用分裂荧光素酶平台抑制KRas（G12 V）-Sos相互作用。目标1和2应是 实现，并在资助期内启动目标3。该项目是多学科的，将使PI能够 培养本科生和研究生在克拉克大学在各种化学和生物化学技术。

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.