Using a cyclotide-based molecular scaffold to select specific protein-protein inh

描述（由申报人提供）：多肽药物的好处包括比现有药物毒性更低、疗效更好，在某些情况下，可以获得其他药物无法获得的新类别化合物。然而，从多肽到商业药物的开发仍然是一个非常具有挑战性和耗时的过程。多肽通常存在体内稳定性差、药代动力学差和生物利用度差的问题。为了寻找更好的肽支架用于药物开发，研究人员最近将重点放在高度受限的肽上，即环核苷酸，作为生产高亲和力配体的极其稳定和通用的支架。重要的是，环肽支架具有广泛的生物活性，在某些情况下具有口服生物利用度。其他环聚糖已被证明通过巨噬细胞作用穿过细胞膜。最后，环肽结构可以在细菌或动物细胞中编码和表达，并且可以适应大量的序列变化。因此，我们相信环肽药物可以通过靶向当前治疗方法无法功能调节的数千种细胞内蛋白质-蛋白质相互作用，提供极大地扩大“可药物”靶点数量的机会。我们建议使用荧光蛋白作为fret偶联来筛选活细菌细胞内遗传编码的环核苷酸文库，以寻找潜在的NF？B信号和炎症反应。NF吗?B是一种转录因子，在调节免疫和炎症反应中起重要作用。它介导TNFa的生物学作用和TNFa/NF的异常。B信号在关节炎、动脉粥样硬化和克罗恩病等炎症性疾病中起着关键作用。因此，阻断TNFa/NF？B信号传导可能有许多治疗作用。控制NF作用的关键因素？B和介导TNFa的作用是I？B激酶（IKK）。IKK活性由一种变构活化剂NEMO调节。因此，NEMO/IKK¿相互作用的环肽抑制剂可能会阻断TNFa对NF？B信号，可能作为消炎药有用。通过分子进化策略的力量和使用荧光激活细胞分类（FACS）的高通量细胞筛选，可以促进NEMO/IKK¿结合选择性环肽抑制剂的发现，从而产生和选择具有最佳结合和抑制特性的化合物。

DESCRIPTION (provided by applicant): Benefits of peptide drugs include lower toxicity and greater efficacy than existing drugs and in some cases, new classes of compounds that are otherwise unavailable. However, the development of a peptide lead to a commercial drug continues to be a very challenging and time consuming process. Peptides generally suffer from poor in vivo stability, poor pharmacokinetics, and poor bioavailability. In search of better peptide scaffolds for drug development, researchers have recently focused on highly constrained peptides known as cyclotides as extremely stable and versatile scaffolds for the production of high affinity ligands. Importantly, cyclotide scaffolds have demonstrated a wide spectrum of biological activities and in some cases oral bioavailability. Other cyclotides have been shown to cross the cell membrane through macropinocytosis. Finally, cyclotide structures can be encoded and expressed in bacteria or animal cells, and are amenable to substantial sequence variation. Thus, we believe that cyclotide drugs could provide the opportunity to greatly expand the number of "druggable" targets by targeting the thousands of intracellular protein-protein interactions that cannot be functionally modulated by current therapeutics. We propose to use fluorescent proteins as a FRET-couple to screen genetically-encoded libraries of cyclotides inside living bacterial cells for potential inhibitors of NF?B signaling and inflammatory responses. NF?B is a transcription factor with an important role in regulating immune and inflammatory responses. It mediates the biological actions of TNFa and abnormalities in TNFa/NF?B signaling play critical roles in inflammatory diseases such as arthritis, atherosclerosis and Crohn's disease. As a consequence, drugs blocking TNFa/NF?B signaling could have a number of therapeutic actions. A key factor controlling the actions of NF?B and mediating the effects of TNFa is I?B kinase (IKK). IKK activity is modulated by an allosteric activator, NEMO. Thus, cyclotide inhibitors of the NEMO/IKK¿ interaction will likely block TNFa stimulation of NF?B signaling and might be useful as anti-inflammatory drugs. Discovery of selective cyclotide inhibitors of NEMO/IKK¿ binding would be facilitated (as proposed here) through the power of molecular evolution strategies and high throughput cell based screening using fluorescence-activated cell sorting (FACS) to enable generation and selection of compounds with optimal binding and inhibitory characteristics. PUBLIC HEALTH RELEVANCE: The concept of using peptides to modulate intracellular processes has been investigated for decades, as peptides play a central role in every cell in the body. These strategies have historically failed because most peptides lack the ability to enter cells, and linear peptides are inherently unstable within the body. We propose development of a breakthrough class of peptide drugs called cyclotides that are rather stable and biologically active, with good drug-like properties, including resistance to proteolytic degradation and the ability to cross mammalian cell membranes. This class of compounds represents a solution for modulating intracellular protein-protein interactions, which have been identified as critical control points for most human diseases. Specifically, we propose to deploy the power of molecular evolution strategies and high throughput cell based screening to enable generation and selection of cyclotides that could inhibit NF?B signaling. Abnormalities in TNFa/NF?B signaling play critical roles in inflammatory diseases such as arthritis, atherosclerosis and Crohn's disease. As a consequence, drugs blocking TNFa/NF?B signaling could have a number of therapeutic actions as anti-inflammatory agents. Specifically, small molecule drugs that block the binding interaction of NEMO to IKK¿ inhibit NF?B signaling could be developed as anti-inflammatory drugs.