Directed Evolution of Peptide Inhibitors of Myc-Max Dimerization (PQ18)

描述(申请人提供)：解除myc基因家族成员的表达是肿瘤进展和维持所必需的，并与许多高侵袭性和低分化的人类癌症有关。在一些肿瘤中，即使是短暂的Myc下调也会导致肿瘤的消退。MYC的功能是相互作用的转录因子网络的一部分，每个转录因子都具有参与蛋白质相互作用和DNA结合的相关但不同的结构域。通过这个基本-螺旋-环-螺旋拉链(BHLHZip)结构域，Myc与Max形成高度特异的异源二聚体。MYC-MAX异源二聚体结合了约15%的基因组位点，通常导致参与细胞生长、增殖、新陈代谢、 细胞凋亡与分化。相反，MXD转录因子竞争性地与MAX结合并抑制Myc基因靶点，作为Myc拮抗剂。在这里，我们建议使用一种新开发的技术来筛选MAX拉链结构域的~600,000个变异体的文库，并识别出能特异性抑制Myc-Max二聚化的多肽，但不能抑制MXD-Max。由于MXD-Max异源二聚体抑制由Myc-Max激活的基因，我们预测单独阻断Myc-Max将导致抑制Myc驱动的细胞增殖。从我们的筛选中提取的多肽抑制剂将在生化分析和肿瘤细胞中进行测试。利用计算模型、定向进化和生物学验证，我们将评估抑制的分子和功能机制，以确保在降低激活的Myc水平和抑制肿瘤生长方面的蛋白质特异性和治疗效果。 公共卫生相关性：Myc蛋白是正常细胞增殖的主要调节因子，但当myc基因被破坏时，其失控的表达会导致肿瘤的形成。在这项研究中，我们建议使用新的基因组技术来产生一种Myc功能的多肽抑制物，它将抑制依赖Myc的肿瘤。我们预计，这种方法将成为开发除Myc以外的其他基因调控因子的抑制剂的模型，这些基因调控因子对肿瘤进展至关重要。

DESCRIPTION (provided by applicant): Deregulation of the expression of myc gene family members is required for tumor progression and maintenance, and is associated with many highly aggressive and poorly differentiated human cancers. In some tumors even transient downregulation of Myc results in tumor regression. Myc functions as part of a network of interacting transcription factors each possessing related but distinct domains involved in protein interaction and DNA binding. Through this basic-helix-loop-helix-zipper (bHLHZip) domain, Myc forms highly specific heterodimers with Max. Myc-Max heterodimers bind ~15% of genomic loci, often resulting in transactivation of genes involved in cellular growth, proliferation, metabolism, apoptosis and differentiation. Conversely, Mxd transcription factors competitively bind to Max and repress Myc gene targets, acting as Myc antagonists. Here we propose to employ a newly developed technology to screen a library of ~600,000 variants of the Max zipper domain and identify peptides that will specifically inhibit Myc-Max dimerization, but not Mxd-Max. Because Mxd-Max heterodimers repress genes activated by Myc-Max we predict that disruption of Myc-Max alone will result in inhibition of Myc driven cell proliferation. Peptide inhibitors derived frm our screen will be tested in biochemical assays and tumor cells. Using computational modeling, directed evolution and biological validation, we will assess the molecular and functional mechanisms of inhibition to ensure protein specificity and therapeutic efficacy in reducing levels of activated Myc and attenuating tumor growth PUBLIC HEALTH RELEVANCE: The Myc protein is a major regulator of normal cell proliferation, but when the myc gene is disrupted its out-of-control expression drives tumor formation. In this study we propose to employ new genomic technology to generate a peptide inhibitor of Myc function that will arrest tumors dependent on Myc. We expect that this approach will serve as a model for developing inhibitors against other gene regulatory factors, in addition to Myc, that are critical for tumor progression.