Molecular Interactions that Drive p63 and p73 Function

作为我们实验室新安装的TETRAS肽合成器的一部分，我们合成了一小块碳氢化合物钉接的p63和p73肽（SAH-p63和SAH-p73）。设计主要集中在三个方面。首先，我们保留了与HDM2和HDMX结合所必需的氨基酸残基（FxxxWxxL），就像最初设计的SAH-p53s一样。其次，制备了i， i + 4和i， i + 7化合物，以评估短纤维长度的影响。第三，每一种变体都被制造出来，以增加肽在生理pH下的电荷，并增强细胞的渗透性。采用基于fmoc的固相肽自动合成技术合成了这些化合物。合成的化合物纯度高（>95%），产率高（每个合成步骤&gt；95%）。我们对SAH-p63和SAH-p73肽的生物化学进行了初步研究。我们发现FITC-SAH-p63s和FITC-SAH-p73s以高亲和力结合重组HDM2，我们正在确定控制结合的热力学参数，并将这一分析扩展到HDMX。圆二色光谱显示这些化合物的二级结构有很大的变化。此外，我们已经能够通过共免疫沉淀确定只有中性或带正电的sah -p63和sah -p73具有细胞渗透性。这些初步研究表明，我们已经构建了可用于研究细胞内过程的化合物。我们已经开始设置生物实验，这些实验将在生化表征完成后进行。某些SAH-p63和SAH-p73肽穿透完整细胞的能力为开发针对细胞内蛋白-蛋白相互作用的新试剂提供了机会。许多蛋白质之间的相互作用很弱，通常很难用常用的生化手段检测出来。结合我们在化学优化项目中的努力，我们正在开发具有细胞渗透性的SAH-p63和SAH-p73肽，其氨基酸能够将赖氨酸残基的e-胺转化为叠氮化物。这些肽将被测试其特异性结合细胞内伴侣的能力，并通过产生化学反应性和生物正交的官能团在结合位点标记它们。我们目前正在评估这种方法在含水生理条件下的应用。同样，我们也在构建含有光反应性二苯甲酮侧链的SAH-p63和SAH-p73肽的变体。该官能团已被用作芳香替代物，能够与位于蛋白质-蛋白质相互作用位点的残基进行光化学共价反应。

As part of our trial runs using the newly installed TETRAS peptide synthesizer in our laboratory, we synthesized a small panel of hydrocarbon stapled p63 and p73 peptides (SAH-p63 and SAH-p73). The designs focused on three specific aspects. Firstly, we retained the amino acid residues essential for binding to HDM2 and HDMX (FxxxWxxL) just as in the original design of SAH-p53s. Secondly, both i, i + 4 and i, i + 7 compounds were made to assess the effects of staple length. Thirdly, a variant of each was made to increase the charge of the peptide at physiological pH and enforce cell permeability. The compounds were synthesized by automated Fmoc-based solid phase peptide synthesis. The syntheses yielded compounds in high purity (>95%) and yield (>95% per synthetic step). We have conducted several preliminary studies into the biochemistry of the SAH-p63 and SAH-p73 peptides. We have found that FITC-SAH-p63s and FITC-SAH-p73s bind to recombinant HDM2 with high affinity, and we are in the process of determining the thermodynamic parameters that govern the binding as well extending this analysis to HDMX. Circular dichroism spectroscopy has shown large variability in the secondary structure of these compounds. Additionally, we have been able to determine by co-immunoprecipitation that only neutral or positively charged SAH-p63s and SAH-p73s are cell permeable. These preliminary studies demonstrate that we have constructed compounds that can be used to study intracellular processes. We have begun the setup of the biological experiments that will ensue after the biochemical characterization has been completed. The ability of certain SAH-p63 and SAH-p73 peptides to penetrate intact cells provides an opportunity for the development of novel reagents to target intracellular protein-protein interaction. Many protein-protein interactions are weak and usually very difficult to detect by commonly-used biochemical means. In conjunction with our efforts in the chemical optimization projects, we are developing cell-permeable SAH-p63 and SAH-p73 peptides with an amino acid capable of converting the e-amine of lysine residues to an azide. The peptides will be tested for their ability to bind their intracellular partners with specificity and tag them at the binding site by producing a chemically reactive and bioorthogonal functional group. We are currently evaluating the use of this methodology under aqueous, physiological conditions. In the same vein, we are also constructing variants of the SAH-p63 and SAH-p73 peptides containing the photoreactive benzophenone side chain. This functional group has been used as an aromatic surrogate capable of covalently reacting photochemically with residues located at protein-protein interaction sites.