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: 8938032
• 负责人: Federico Bernal
• 金额: $ 20.91万
• 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/8938032
• 关键词: Affect; Affinity; Am 80; Apoptotic; Appearance; Binding; Binding Proteins; Biochemical; Biological Assay; Biotin; Bovine Serum Albumin; Cell Nucleus; Chemicals; Complex; Cytoskeleton; Exhibits; Exposure to; Family member; Hour; Kinetics; Link; MDM2 gene; Malignant Neoplasms; Measurement; Methodology; Methods; Monitor; Parents; Pathogenesis; Pathologic; Pathway interactions; Peptides; Positioning Attribute; Property; Proteins; Reaction; Recombinants; Research; Serum Proteins; Signal Pathway; Solutions; Specificity; Streptavidin; Structure; Subarachnoid Hemorrhage; System; Techniques; Technology; UV Radiation Exposure; Ultraviolet Rays; Vertebral column; Work; base; benzoylphenylalanine; chemical group; covalent bond; crosslink; design; functional group; novel; protein protein interaction; research study; tool; ultraviolet irradiation

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. We hypothesize that incorporation of a functional group into SAH-p53-8 that could be triggered to create a covalent bond with a nearby bound protein would allow for the selective tagging, capture and subsequent identification of these new protein partners. We chose to include the photo-reactive chemical group, benzoylphenylalanine (Bpa) triggered by exposure to UV irradiation, into the backbone structure of SAH-p53-8. We synthesized derivatives of the parent peptide, SAH-p53-8, which incorporated the Bpa moiety at three key positions which make direct hydrophobic contact with the known HDM2 target protein. Direct measurement of binding to HDM2 showed that two of the three derivatives (W23Bpa and L26Bpa) had binding affinities comparable to the parent peptide, while one (F19Bpa) did not exhibit any binding affinity in this biochemical assay. We also demonstrated that binding affinity correlated with the ability of the photo-reactive peptides to covalently crosslink to recombinant HDM2 in solution, as correct orientation and selective binding which puts Bpa in close proximity to the target is a prerequisite for a successful photochemical reaction. In particular, the W23- and L26Bpa-containing peptides were found to form covalently-linked peptide-HDM2 products upon exposure to UV light, while the non-binding F19Bpa peptide did not result in any cross-linked product. Additional experiments utilizing the stronger binding L26Bpa peptide were done to look at UV reaction kinetics as well as selectivity in the presence of other non-targets. Reaction mixtures containing a 1:1 ratio of peptide and HDM2 protein (5 uM each) were exposed to up to two hours of continuous UV irradiation, and monitored for the appearance of reacted vs. unreacted protein. We found that the maximum yield of cross-linked product formed at this concentration in two hours was 80%, with the reaction reaching 60% completion in as little as thirty minutes. Reaction completion and off-target reaction in the presence of bovine serum albumin (BSA), a serum protein that can result in non-specific binding, was also investigated. Despite the presence of 0.5, 1.0 and 3.0 equivalents of BSA, after one hour of UV exposure, no cross-linked product with BSA was apparent nor was the reaction yield of the target protein, HDM2, affected, indicting the specificity of this photo-reactive peptide. Additionally, we have shown that the presence of an equimolar concentration of parent peptide in the reaction mixture is sufficient to out-compete both W23- and L26Bpa peptides for binding and results in no cross-linked product. This property is a useful tool in terms of identifying only targets specific to SAH-p53-8, by being able to potentially eliminate false positive results. We have also shown that a peptide-HDM2 covalent product can be isolated from solution using a biotin-capped version of the L26Bpa. With this, we have demonstrated that the photo-reactive SAH-p53 peptides can be used to bind to, react with and isolate (via biotin-streptavidin pull down) target proteins in a biochemical system. We are currently expanding our work into cellular extracts that endogenously contain HDM2 and HDMX, known protein targets of the SAH-p53 peptides, as proof-of-principle of this peptide-based bind/react/capture methodology in a more complex cellular matrix. We have demonstrated that the photo-reaction of peptide to recombinant HDM2 (spiked into lysate) is successful even in the presence of lysate. We are currently exploring what concentration of peptide to cellular protein concentration is necessary to both identify bands and facilitate the pull down isolation.

钉合肽技术为稳定生物学相关的肽螺旋提供了一种新方法。迄今为止，它为靶向与癌症发病机制相关的复杂信号通路的离散成分创造了独特的机会。这种方法的使用使我们能够研究细胞凋亡信号通路，以及最近限制于细胞核的转录通路的操作。我们的目标是通过化学精炼显着发展钉合肽策略，以扩大我们针对癌症中涉及的病理蛋白相互作用的能力。在我们对 p53 家族成员在癌症中的功能的研究过程中（参见 ZIA BC 011376 项目摘要），我们发现，虽然我们的 HDM2/HDMX 靶向化合物 SAH-p53-8 恢复了受抑制的 p53 的转录活性，但还发现了其他几种活性线，其中一些完全独立于 p53。我们假设，将一个功能基团掺入 SAH-p53-8 中，可以触发该功能基团与附近结合的蛋白质形成共价键，从而可以选择性标记、捕获和随后识别这些新的蛋白质伙伴。我们选择将光反应化学基团苯甲酰基苯丙氨酸 (Bpa) 纳入 SAH-p53-8 的主链结构中。我们合成了母肽 SAH-p53-8 的衍生物，它在三个关键位置掺入了 Bpa 部分，与已知的 HDM2 靶蛋白进行直接疏水接触。与 HDM2 结合的直接测量表明，三种衍生物中的两种（W23Bpa 和 L26Bpa）具有与母体肽相当的结合亲和力，而一种（F19Bpa）在此生化测定中没有表现出任何结合亲和力。我们还证明，结合亲和力与光反应肽在溶液中与重组 HDM2 共价交联的能力相关，因为正确的方向和选择性结合使 Bpa 非常接近靶标，这是光化学反应成功的先决条件。特别地，发现含有W23和L26Bpa的肽在暴露于UV光时形成共价连接的肽-HDM2产物，而非结合的F19Bpa肽不产生任何交联产物。利用结合力更强的 L26Bpa 肽进行了其他实验，以观察 UV 反应动力学以及其他非靶标存在下的选择性。将含有 1:1 比例的肽和 HDM2 蛋白（各 5 uM）的反应混合物暴露于长达两小时的连续紫外线照射下，并监测已反应蛋白与未反应蛋白的出现情况。我们发现，在此浓度下，两小时内形成的交联产物的最大产率为 80%，反应在短短 30 分钟内即可完成 60%。还研究了牛血清白蛋白（BSA）（一种可导致非特异性结合的血清蛋白）存在下的反应完成和脱靶反应。尽管存在 0.5、1.0 和 3.0 当量的 BSA，但在紫外线照射一小时后，没有明显与 BSA 的交联产物，目标蛋白 HDM2 的反应产率也没有受到影响，表明这种光反应肽的特异性。此外，我们还表明，反应混合物中存在等摩尔浓度的母体肽足以在结合方面胜过 W23- 和 L26Bpa 肽，并且不会产生交联产物。该特性是一种有用的工具，能够潜在地消除假阳性结果，从而仅识别 SAH-p53-8 特有的目标。我们还表明，可以使用生物素封端的 L26Bpa 从溶液中分离肽-HDM2 共价产物。由此，我们证明了光反应性 SAH-p53 肽可用于在生化系统中与目标蛋白结合、反应并分离（通过生物素-链霉亲和素下拉）目标蛋白。我们目前正在将我们的工作扩展到内源性含有 HDM2 和 HDMX（SAH-p53 肽的已知蛋白质靶标）的细胞提取物中，作为这种基于肽的结合/反应/捕获方法在更复杂的细胞基质中的原理证明。我们已经证明，即使在存在裂解物的情况下，肽与重组 HDM2（掺入裂解物中）的光反应也能成功。我们目前正在探索什么浓度的肽与细胞蛋白浓度对于识别条带和促进下拉分离是必要的。