Broadening the Utility of Stapled Peptides through Chemical Optimization

钉合肽技术为生物相关肽螺旋的稳定化提供了一种新方法。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细胞凋亡信号通路，最近，限制到细胞核的转录途径的操纵。我们的目标是通过化学精制显着发展钉合肽策略，以扩大我们靶向与癌症有关的病理性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整个研究过程中（参见ZIA BC 011376项目摘要），我们发现，虽然我们的HDM 2/HDMX靶向化合物SAH-p53-8恢复了受抑制的p53的转录活性，但已经发现了几种其他活性线，其中一些完全不依赖于p53。我们假设，SAH-p53-8中的功能基团的掺入，可以触发创建一个共价键与附近的结合蛋白将允许选择性标记，捕获和随后识别这些新的蛋白伴侣。我们选择包括光反应性化学基团，苯甲酰苯丙氨酸（Bpa）暴露于紫外线照射触发，到SAH-p53-8的骨架结构。我们合成了亲本肽SAH-p53-8的衍生物，其在三个关键位置掺入Bpa部分，这三个关键位置与已知的HDM 2靶蛋白直接疏水接触。与HDM 2结合的直接测量显示，三种衍生物中的两种（W23 Bpa和L26 Bpa）具有与亲本肽相当的结合亲和力，而一种（F19 Bpa）在该生物化学测定中未表现出任何结合亲和力。我们还证明了结合亲和力与光反应性肽在溶液中共价交联至重组HDM 2的能力相关，因为使Bpa紧密接近靶标的正确取向和选择性结合是成功的光化学反应的先决条件。特别地，发现含有W23和L26 Bpa的肽在暴露于UV光时形成共价连接的肽-HDM 2产物，而非结合的F19 Bpa肽不产生任何交联产物。利用更强结合的L26 Bpa肽进行另外的实验，以观察UV反应动力学以及在其他非靶标存在下的选择性。将含有1：1比例的肽和HDM 2蛋白（各5 μ M）的反应混合物暴露于长达两小时的连续UV照射，并监测反应蛋白与未反应蛋白的外观。我们发现，在该浓度下在两小时内形成的交联产物的最大产率为80%，反应在短短30分钟内完成60%。还研究了在牛血清白蛋白（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1小时的UV暴露后，没有与BSA的交联产物是明显的，靶蛋白质HDM 2的反应产率也不受影响，表明这种光反应性肽的特异性。此外，我们已经表明，反应混合物中等摩尔浓度的亲本肽的存在足以胜过W23-和L26 Bpa肽的结合，并且不产生交联产物。该性质是一种有用的工具，通过能够潜在地消除假阳性结果，仅鉴定SAH-p53-8特异性靶点。我们还表明，肽-HDM 2共价产物可以使用L26 Bpa的生物素封端形式从溶液中分离。由此，我们证明了光反应性SAH-p53肽可用于在生物化学系统中结合、反应和分离（通过生物素-链霉亲和素下拉）靶蛋白。我们目前正在将我们的工作扩展到内源性含有HDM 2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，即使在裂解物存在下，肽与重组HDM 2（掺入裂解物中）的光反应也是成功的。我们目前正在探索什么样的肽浓度与细胞蛋白质浓度是鉴定条带和促进下拉分离所必需的。

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.