Molecule-Guided Investigations into p53 Biology

伯纳尔实验室的建立于2010年秋末完成。实验室的设置包括安装用于表征和纯化生物活性分子的液相色谱质谱计，以及安装异步、高通量肽合成器。这些仪器构成了伯纳尔实验室的核心基础设施，它们是我们合成工作的重要组成部分。在三个月的时间里，我们能够合成数毫克数量的各种化合物，不仅涵盖了p53项目，还包括其他新的研究项目。在这一点上，我们有能力生产高纯度的材料，每个合成步骤的化学收率超过95%。这使我们能够最大限度地提高我们综合努力的效率。我们还获得了NCI-Frederick艾滋病和癌症病毒项目蛋白质化学核心成员的协助，对我们合成的所有化合物进行了完整的表征和定量。以前，用于生物学研究的化合物的可用性是我们工作的最大瓶颈。我们已经完全克服了这个障碍，合成化合物现在是我们小组日常工作的一部分。伯纳尔实验室的p53项目涵盖了广泛的研究工作。这项工作的灵感来自于碳氢化合物钉接肽SAH-p53-8的使用。这个肽的序列是基于p53蛋白的转激活结构域。该序列已经过优化，包括两个碳氢化合物交联氨基酸，与癌蛋白HDM2和HDMX结合所需的三个基本残基，以及使其能够穿过细胞膜的其他修饰。我们已经证明，这种化合物能够在过表达HDM2或HDMX的细胞中重新激活p53通路。虽然这一发现具有重要意义，因为SAH-p53-8是迄今为止唯一一种能够破坏p53-HDM2和p53-HDMX蛋白复合物的化合物，但这种能力使我们能够阐明一种机制框架，以确定哪些癌细胞容易受到单药HDM2或HDMX的抑制，并通过HDM2和HDMX的协同靶向来克服耐药细胞中的p53抑制。这些结果发表于2010年11月（见：Bernal等）。癌症杂志，2010,18,411)。作为发表在《癌细胞》杂志上的研究成果的一部分，我们开始与来自比利时鲁汶天主教大学分子癌症生物学实验室的Jean-Christophe Marine博士进行研究合作。marine博士的研究需要确定导致黑色素细胞转化为黑色素瘤的致病因子。marine博士的研究已经确定，HDMX的黑素细胞特异性过表达与Ras的突变合作，促进了黑色素瘤的表现。鉴于SAH-p53-8在研究p53- hdm2 - hdmx轴中的作用，我们提出了一项计划，以确定在黑色素瘤中破坏p53功能的关键机制。根据我们的合作计划，我们合成了大量的SAH-p53-8用于海洋实验室的qPCR和动物研究。同时，我们评估了SAH-p53-8单独使用以及与Nutlin-3联合使用对来自患者的多种黑色素瘤细胞的疗效。令人欣慰的是，我们发现我们在之前的工作中发现的分子蓝图确实适用于黑色素瘤。这些结果直接转化为p53转录研究和海洋实验室研究的黑色素瘤动物模型。这个项目的最终实验目前正在进行中，我们希望在2011年底之前提交一份手稿发表。虽然我们对将SAH-p53-8转化为临床的前景感到鼓舞，但我们发现，尽管其具有坚实的药效学效果，但其药代动力学参数使其无法有效地发挥治疗作用。它未能通过NCI-60的第一层筛选就证明了这一点。为了规避这个问题，我们提出了一项建议，利用发表在《癌细胞》杂志手稿上的技术开发一种小分子屏幕。该计划的重点是使用高通量荧光偏振测定来寻找p53-HDMX相互作用的选择性抑制剂。我们目前正在验证该分析的高通量，我们正在与NCGC成员讨论执行该项目。上述研究与p53的转录调控研究直接相关；然而，已知p53具有许多其他功能，这些功能独立于其作为转录因子的功能。这些非转录功能包括各种各样的细胞过程，从线粒体凋亡和应激反应到细胞运动和细胞结构完整性的维持。鉴于大多数癌症都含有p53蛋白序列的突变，利用其细胞质功能为挽救突变p53的肿瘤抑制功能提供了一种新的途径，尽管它无法激活转录机制。我们的第一个观察来自于一个实验，该实验显示，在一个表达显性阴性p53-DD迷你蛋白的细胞系中，凋亡机制被重新激活。经SAH-p53-8处理的表达p53-DD （SJSA-DD）的骨肉瘤细胞显示caspase-3/7活化显著增加，caspase-3/7活化是细胞凋亡的标志。这一结果也在内源性p53蛋白被截断且缺乏核定位序列的淋巴瘤细胞系（OCI-Ly3）中得到了复制。为了研究在有缺陷的p53细胞中控制细胞凋亡诱导的蛋白-蛋白相互作用，我们在分离的线粒体中用几种变体的SAH-p53肽进行了实验。数据显示，确实，SAH-p53s诱导细胞色素c释放，而已知的无活性的钉接p53肽SAH-p53-8F19A是完全惰性的，表明p53依赖于这一过程。除了我们发现细胞凋亡的再激活外，我们还注意到SAH-p53处理的细胞结构完整性发生了显著变化，这些细胞也具有内源性p53缺陷。我们在一系列不同严重程度的乳腺癌细胞系中进行了p53功能损伤的细胞迁移研究。这些实验表明，虽然用SAH-p53-8再激活p53可以诱导野生型p53（如MCF-7）乳腺癌细胞系的凋亡细胞死亡，但同样的刺激会损害p53突变的侵袭性细胞系（如MDA-MB-231）的细胞运动性。此外，免疫组织化学证据显示有丝分裂突变的痕迹和肌动蛋白丝的残余，表明这些影响是由细胞结构完整性的崩溃介导的。这个项目包含了伯纳尔实验室目前正在进行的大部分工作。我们相信，使用化学合成和化学生物学的工具将使我们能够研究蛋白质之间的相互作用和功能，到目前为止，这些都是难以捉摸的。

The setup of the Bernal Laboratory was completed late in the fall of 2010. The setup of the laboratory included the installation of a liquid chromatography mass spectrometer for the characterization and purification of biologically active molecules and the setup of an asynchronous, high throughput peptide synthesizer. These instruments form the core infrastructure of the Bernal laboratory, and they are essential components in our synthesis work. Within a span of three months, we were able to synthesize multi-milligram quantities of a wide variety compounds covering not only the p53 project, but other newer research endeavors. At this point, we have the capability to produce materials in high purity with chemical yields exceeding 95% per synthetic step. This has allowed us to maximize the efficiency of our synthetic efforts. We have also secured the assistance of the members of the Protein Chemistry Core in the AIDS and Cancer Virus Program at NCI-Frederick for the complete characterization and quantification of all of the compounds we have synthesized. Previously, the availability of compounds for biological studies was the biggest bottleneck in our work. We have fully surmounted that obstacle, and the synthesis of compounds is now part of the daily routine in our group. The p53 project in the Bernal Lab encompasses a broad spectrum of research endeavors. The inspiration for this work arose from the use of the hydrocarbon-stapled peptide SAH-p53-8. The sequence of this peptide is based on the transactivation domain of the p53 protein. The sequence has been optimized to include two hydrocarbon cross-linking amino acids, the three essential residues required for binding to the oncoproteins HDM2 and HDMX, and other modifications that enable it to traverse cell membranes. We have shown that this compound is capable of reactivating the p53 pathway in cells that overexpress either HDM2 or HDMX. While this finding is significant in the context that SAH-p53-8 is the only compound disclosed to date that is capable of disrupting p53-HDM2 and p53-HDMX protein complexes, this capability enabled us to elucidate a mechanistic framework for determining which cancer cells will be susceptible to single agent HDM2 or HDMX inhibition and overcoming p53 suppression in a resistant cell through synergistic targeting of HDM2 and HDMX. These results were published in November of 2010 (see: Bernal, et al. Cancer Cell 2010, 18, 411). As a segway to the research published in Cancer Cell, we embarked on a research collaboration with the group of Dr. Jean-Christophe Marine from the Laboratory for Molecular Cancer Biology at the Catholic University of Leuven in Belgium. Dr. Marines research entails the determination of the causative agents responsible for the transformation of melanocytes into melanoma. Dr. Marines research has determined melanocyte specific overexpression of HDMX cooperates with mutations in Ras to promote the manifestation of melanoma. Given the utility of SAH-p53-8 in studying the p53-HDM2-HDMX axis, we set forth a plan to identify the key mechanisms that disrupt the function of p53 in melanoma. Per our collaborative plan, we synthesized a large batch of SAH-p53-8 for use in the Marine laboratory for qPCR and animal studies. At the same time, we evaluated the efficacy of SAH-p53-8 alone and in combination with Nutlin-3 in a wide variety of melanoma cells derived from patients. Gratifyingly, we have found that the molecular blueprint we uncovered in our previous work indeed holds for melanoma. The results translate directly to the p53 transcription studies and the melanoma animal models studied in the Marine lab. The final experiments in this project are currently in progress, and we expect to have a manuscript submitted for publication before the end of 2011. While we are encouraged by the prospect of translating SAH-p53-8 to the clinic, we have found that despite its solid pharmacodynamic effects, its pharmacokinetic parameters preclude it from functioning effectively as a therapeutic. This was evidenced by its failure to pass the first layer of screening in the NCI-60. In order to circumvent this problem, we have developed a proposal to develop a small molecule screen using the techniques that were published in the Cancer Cell manuscript. The plan focuses on the use of high throughput fluorescence polarization assays to find selective inhibitors of the p53-HDMX interaction. We are currently in process of validating the assay for high throughput, and we are involved in discussions with members of NCGC to execute this project. The aforementioned study is directly linked to the study of the transcriptional modulation of p53; however, p53 is known to have many other functions that are independent of its ability to function as a transcription factor. These non-transcriptional functions comprise a wide variety cellular processes ranging from mitochondrial apoptosis and stress responses to cell motility and the maintenance of the structural integrity of the cell. Given that the majority of all cancers contain mutations in the sequence of the p53 protein, exploiting its cytoplasmic functions provides a novel avenue to rescue the tumor suppressor functions of mutant p53 despite its inability to activate the transcriptional machinery. Our first observation came about from an experiment which showed reactivation of the apoptosis machinery in a cell line engineered to express the dominant-negative p53-DD mini-protein. Osteosarcoma cells expressing p53-DD (SJSA-DD) treated with SAH-p53-8 displayed a marked increase in caspase-3/7 activation, a hallmark of apoptosis. This result was also replicated in a lymphoma cell line (OCI-Ly3) whose endogenous p53 protein is truncated and lacks a nuclear localization sequence. In order to study the protein-protein interactions that govern the induction of apoptosis in cells with defective p53, we have performed experiments in isolated mitochondria treated with several variants of the SAH-p53 peptides. The data show that, indeed, SAH-p53s induce cytochrome c release while the known inactive stapled p53 peptide SAH-p53-8F19A is completely inert, demonstrating the p53 dependence on this process. In addition to our findings on the reactivation of apoptosis, we have also noticed significant changes in the structural integrity of cells treated with SAH-p53 which also possess defects in endogenous p53. We have conducted cell migration studies in a series of breast cancer cell lines with different degrees of severity in the impairment of p53 function. These experiments have shown that while reactivation of p53 with SAH-p53-8 can induce apoptotic cell death in the breast cancer lines with wild type p53 (such as MCF-7), the same stimulus impairs cell motility in invasive cell lines with mutations in p53 (i.e. MDA-MB-231). Moreover, immunohistochemical evidence shows vestiges of mitotic catastrophe with remnants of actin filaments, suggesting that these effects are mediated by the collapse in cellular structural integrity. This project comprises the majority of the work currently being done in the Bernal Laboratory. We believe that using the tools of chemical synthesis and chemical biology will allow us to investigate protein-protein interactions and functions that, to this date have been elusive.