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。为了提供一种化学方法来鉴定目标，我们已经开发了p53的可光活化的α-螺旋（pSAH-p53 s），其能够通过用UV光激发而共价交联到其靶蛋白。为此，我们设计了我们的光活化化合物，使用SAH-p53-8序列作为模板，这是我们迄今为止最具活性的化合物。光化学交联是通过在肽序列中掺入对二苯甲酮苯丙氨酸残基来代替推定参与必要的蛋白质-蛋白质相互作用的氨基酸来实现的。作为原理研究的证明，我们将pSAH-p53与其靶蛋白HDM 2和旁观者蛋白的混合物以超化学计量比组合。我们发现，暴露于紫外线后，pSAH-p53选择性地与HDM 2交联，尽管其他蛋白质大量存在。有了这些数据，我们接着在细胞提取物的复杂环境中评估化合物。令人满意的是，我们已经发现pSAH-p53有效地交联来自裂解物的HDM 2和HDMX。更重要的是，我们还发现pSAH-p53也靶向其他蛋白。目前的努力旨在确定这些新的结合伙伴，并确定其意义的p53家族途径。鉴于我们成功地产生了选择性结合其靶标并在暴露于UV光时与其交联的pSAH-p53，我们已经通过固相肽合成基于其各自的TAD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，并且与p53非常相似，它们具有形成多聚体复合物并结合p53响应性启动子的能力。尽管存在这种冗余，但p63和p73在细胞中的功能是独特的，在细胞运动性、侵袭性和分化中发挥作用。此外，这两种蛋白质也具有它们自己的TAD，具有粗略的序列同源性。我们已经验证了这一战略在体外使用重组蛋白，我们现在正在研究其效用进行实验，使用细胞提取物和活细胞。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。

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.