Broadening the Utility of Stapled Peptides through Chemical Optimization

通过化学优化拓宽缝合肽的用途

• 批准号: 8763422
• 负责人: Federico Bernal
• 金额: $ 21.74万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763422
• 关键词: Amino Acids; Apoptotic; Benzophenones; Binding; Cell Extracts; Cell Nucleus; Cells; Chemicals; Complex; Data; Exposure to; Family; Family member; Hand; In Vitro; Life; MDM2 gene; Malignant Neoplasms; Methodology; Methods; Pathogenesis; Pathologic; Pathway interactions; Peptide Synthesis; Peptides; Phase; Phenylalanine; Play; Proteins; Recombinant Proteins; Research; Role; Sequence Homology; Signal Pathway; Solid; Subarachnoid Hemorrhage; Techniques; Technology; Ultraviolet Rays; alpha helix; base; cell motility; crosslink; design; member; novel; promoter; protein aminoacid sequence; protein protein interaction; research study; success

The stapled peptide technology has afforded a novel method for the stabilization of biologically relevant peptide helices. Thus far, it has created unique opportunities for targeting discrete components of complex signaling pathways relevant to the pathogenesis of cancer. The use of this methodology has enabled our study of the apoptotic signaling pathway and, more recently, the manipulation of transcriptional pathways restricted to the nucleus. We aim to significantly evolve the stapled peptide strategy through chemical refinement in order to expand our ability to target pathologic protein interactions implicated in cancer. Throughout the course of our research into the function of p53 family members in cancer (see ZIA BC 011376 project summary), we have found that while our HDM2/HDMX targeting compound SAH-p53-8 restores the transcriptional activity of inhibited p53, several other lines of activity have been found, some of which are entirely independent of p53. In order to provide a chemical approach to target identification, we have developed photactivatable alpha-helices of p53 (pSAH-p53s) which are capable of cross-linking covalently to their target proteins by excitation with UV light. To do this, we have designed our photoactivatable compounds using as a template the sequence of SAH-p53-8, our most active compound to date. Photochemical cross-linking is accomplished by incorporating into the sequence of the peptide a para-benzophenone phenylalanine residue in lieu of an amino acid putatively involved in the requisite protein-protein interaction. As a proof of principle study, we combined a pSAH-p53 with a mixture of its target protein HDM2 and a spectator protein in a superstoichiometric ratio. We found that after exposure to UV light, pSAH-p53 cross-links selectively to HDM2 despite the overwhelming presence of other proteins. With these data on hand, we then proceeded to evaluate the compounds in the complex setting of a cell extract. Gratifyingly, we have found that pSAH-p53s effectively cross-link HDM2 and HDMX from lysates. More importantly, we also found that the pSAH-p53s target other proteins as well. Current efforts are aimed at identifying these novel binding partners and determining their significance to p53 family pathways. Given our success in creating pSAH-p53s that selectively bind their targets and crosslink with them on exposure to UV light, we have carried out the synthesis of pSAH-p63 and pSAH-p73 peptides based on their respective TADs by solid phase peptide synthesis. As members of the same family, p63 and p73 share many structural similarities with p53, and much like p53, they have the ability to form multimeric complexes and bind to p53-responsive promoters. Despite this redundancy, there are functions of p63 and p73 in cells that are unique, playing roles in cellular motility, invasiveness and differentiation. Further, these two proteins also possess TADs of their own with cursory sequence homology. We have validated this strategy in vitro using both recombinant proteins and we are now investigating its utility in experiments carried out both using cell extracts and live cells. These experiments will provide a better understanding of the protein-protein interactions that drive p53, p63 and p73 function.

钉肽技术为生物学相关肽螺旋的稳定提供了一种新颖的方法。到目前为止，它为靶向与癌症发病机理相关的复杂信号通路的离散组件创造了独特的机会。这种方法的使用使我们能够研究凋亡信号通路，最近，对局部局部的转录途径进行操纵。我们的目标是通过化学改进来显着发展钉钉肽策略，以扩大我们靶向涉及癌症的病理蛋白相互作用的能力。在整个研究癌症中p53家族成员功能的整个过程中（请参见Zia BC 011376项目摘要），我们发现，尽管我们的HDM2/HDMX靶向化合物SAH-P53-8恢复了抑制P53的转录活性，但发现了其他几种活动行，其中一些完全独立于P53。为了提供一种化学方法的靶标识别方法，我们开发了可光活化的p53（PSAH-p53s）的α-螺旋，它们能够通过用紫外线激发与靶蛋白共价交联。为此，我们已经使用模板设计了我们的光活化化合物SAH-P53-8的序列，即我们迄今为止最活跃的化合物。光化学交联是通过将二苯二甲酮苯丙氨酸残基的序列纳入所必需蛋白质蛋白质相互作用的氨基酸来实现的。作为原则研究的证明，我们将PSAH-P53与其靶蛋白HDM2的混合物和超级计量比率的观众蛋白结合在一起。我们发现，在暴露于UV光之后，尽管存在压倒性的其他蛋白质，但PSAH-P53选择性地与HDM2有选择性。借助这些数据，我们开始在细胞提取物的复杂环境中评估化合物。令人满意的是，我们发现PSAH-P53有效地从裂解液中有效交叉链接HDM2和HDMX。更重要的是，我们还发现PSAH-P53也靶向其他蛋白质。当前的努力旨在确定这些新颖的结合伙伴并确定其对p53家庭途径的意义。鉴于我们成功地创建了PSAH-P53，这些PSAH​​-P53在暴露于紫外线的情况下选择性地结合其目标并与它们交联，因此我们通过固相肽的合成基于其各自的TADS进行了PSAH-P63和PSAH-P73肽的合成。作为同一家族的成员，p63和p73与p53具有许多结构相似性，就像p53一样，它们具有形成多聚体复合物并与p53响应启动子结合的能力。尽管有这种冗余，但在独特的细胞中仍然存在p63和p73的功能，在细胞运动，侵入性和分化中起着作用。此外，这两种蛋白质还具有粗略的序列同源性。我们已经使用重组蛋白在体外验证了该策略，现在我们正在研究其在使用细胞提取物和活细胞进行的实验中的实用性。这些实验将更好地了解驱动p53，p63和p73功能的蛋白质蛋白质相互作用。