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

开发一种新型邻近催化化学表位靶向技术，用于分离 KRas(G12V)-Sos 相互作用的大环肽抑制剂

• 批准号: 10046966
• 负责人: Arundhati Nag
• 金额: $ 45.09万
• 依托单位: CLARK UNIVERSITY (WORCESTER, MA)
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-10 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046966
• 关键词: Affect; Affinity; Alder plant; Alkenes; Alkynes; Antibodies; Area; Azides; Binding; Biochemical; Biology; Biomedical Research; Chemicals; Cleaved cell; Complex; Consumption; Cyclic Peptides; Cyclization; Cysteine; Development; Diels Alder reaction; Dimerization; Electrons; Epitopes; Grant; Hydrophobicity; Label; Libraries; Ligand Binding; Ligands; Light; Luciferases; Membrane Proteins; Methods; Modification; Monitor; Oncogenic; Peptide Library; Peptides; Pharmaceutical Preparations; Positioning Attribute; Process; Protein Region; Proteins; Reaction; Specificity; Structure; Surface; Techniques; Technology; Temperature; Therapeutic; Thermodynamics; Time; Training; Triazoles; Universities; combinatorial; cycloaddition; design; graduate student; improved; inhibitor/antagonist; multidisciplinary; mutant; new technology; novel; protein function; protein protein interaction; public health relevance; screening; small molecule; tandem mass spectrometry; tool; undergraduate student; user-friendly

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.

抽象的 用于治疗目的的蛋白质功能修饰通常是用小分子药物来完成的 结合蛋白质深疏水口袋。对于在延长的浅浅上发生的蛋白质蛋白相互作用 区域，将小分子与表面积结合并影响蛋白质功能是一项挑战。拉斯 -SOS是广泛蛋白质蛋白表面相互作用的经典例子，因此抑制仍然具有挑战性 Krassos相互作用。 KRAS被SOS激活，开发技术以抑制相互作用的技术至关重要 与SOS有选择性的致癌突变体Kras。一种有希望的技术是化学表位靶向 技术，用于设计具有高亲和力和特异性针对特定区域的肽配体 细胞内蛋白可能无法访问小分子或抗体。该技术涉及使用 接近催化反应，用于筛选靶蛋白的特定区域以分离肽配体。 PI建议简化这项技术，以使其更容易访问和用户友好，然后量身定制 这项技术成功地靶向突变体KRAS（G12V）蛋白，特别是在RAS-SOS界面上。这 技术将通过开发和表征合成宏观晶体一孔对一体化合物来简化技术 （OBOC）可以通过一步光曝光来裂解的肽库，以通过串联质量进行无缝测序 光谱法（MS/MS）（AIM 1）。逆电子需求DIELS-ALDER（IEDDA）在四嗪和 烯烃将用于筛选过程，而不是当前使用的叠氮化物环加成。 PI应证明，作为概念验证，IEDDA在取代四嗪和A之间的反应 烯烃可以通过蛋白质接近诱导。 KRAS（G12C）或具有一个CYS的KRAS突变体将在CYS处进行修改 与四嗪。用含有SOS-螺旋肽的烯烃标记复合物的标记，已知在Ras-上结合 SOS界面将使用串联质谱法监测（AIM 2）。最低温度的最佳温度 将确定四嗪和烯烃之间的背景IEDDA反应，以最大程度地减少屏幕的背景 在下一步。 针对化学表位的化学表位靶向化学表位，KRAS（G12V）与GDP-复合 烯烃小分子将使用S-三嗪环化的OBOC肽库进行（AIM 3）。筛选 将随后使用分裂荧光素酶平台抑制KRAS（G12V）-SOS相互作用。目标1和2应该是 实现并在赠款期内启动目标3。这个项目是多学科的，将允许PI 克拉克大学的各种化学和生化技术培训本科生和研究生。