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.

钉接肽技术为生物相关肽螺旋的稳定提供了一种新的方法。到目前为止，它为靶向与癌症发病机制相关的复杂信号通路的离散组分创造了独特的机会。这种方法的使用使我们能够研究凋亡信号通路，以及最近限制在细胞核内的转录通路的操纵。我们的目标是通过化学改进来显著发展钉接肽策略，以扩大我们靶向与癌症有关的病理蛋白质相互作用的能力。在我们对p53家族成员在癌症中的功能的研究过程中（见ZIA BC 011376项目总结），我们发现我们的HDM2/HDMX靶向化合物SAH-p53-8在恢复被抑制p53的转录活性的同时，还发现了其他一些活性线，其中一些活性线完全独立于p53。为了提供一种化学方法来鉴定靶标，我们开发了p53的光激活α -螺旋（pSAH-p53s），它能够通过紫外光激发与其靶蛋白交联共价。为了做到这一点，我们设计了光活化化合物，使用SAH-p53-8的序列作为模板，这是我们迄今为止最活跃的化合物。光化学交联是通过在肽序列中加入对二苯甲酮苯丙氨酸残基来代替被认为参与必要的蛋白质-蛋白质相互作用的氨基酸来完成的。作为一项原理验证研究，我们将pSAH-p53与其靶蛋白HDM2和旁观者蛋白的混合物以超化学计量比结合。我们发现，暴露在紫外线下后，pSAH-p53选择性地与HDM2交联，尽管其他蛋白质大量存在。有了这些数据在手，我们接着在细胞提取物的复杂设置中评估化合物。令人欣慰的是，我们发现pSAH-p53s可以有效地交联HDM2和HDMX。更重要的是，我们还发现pSAH-p53s也靶向其他蛋白质。目前的工作旨在识别这些新的结合伙伴，并确定它们对p53家族通路的重要性。鉴于我们成功地创建了pSAH-p53s，该pSAH-p53s在紫外线照射下选择性地结合其靶标并与它们交联，我们已经根据pSAH-p63和pSAH-p73各自的TADs通过固相肽合成方法合成了pSAH-p63和pSAH-p73肽。作为同一家族的成员，p63和p73与p53在结构上有许多相似之处，并且与p53非常相似，它们具有形成多聚体复合物并结合p53应答启动子的能力。尽管存在这种冗余，但p63和p73在细胞中的功能是独特的，在细胞运动、侵袭性和分化中发挥作用。此外，这两种蛋白还具有各自的tad序列，具有粗略的序列同源性。我们已经用两种重组蛋白在体外验证了这一策略，我们现在正在研究它在细胞提取物和活细胞实验中的效用。这些实验将更好地理解驱动p53、p63和p73功能的蛋白质-蛋白质相互作用。