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

说明(申请人提供)：与现有药物相比，多肽药物的好处包括毒性更低，疗效更好，在某些情况下，还包括以其他方式无法获得的新类别的化合物。然而，开发一种导致商业药物的多肽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等问题。为了寻找更好的用于药物开发的多肽支架，研究人员最近将重点放在了被称为环肽的高度受限的多肽上，这些多肽是非常稳定和多功能的支架，用于生产高亲和力配体。重要的是，环肽支架显示了广泛的生物活性，在某些情况下还显示了口服生物利用度。其他环类化合物已被证明通过巨噬细胞吞噬作用穿过细胞膜。最后，环肽结构可以在细菌或动物细胞中编码和表达，并易于发生实质性的序列变化。因此，我们认为，环肽药物可以提供机会，通过靶向数以千计的细胞内蛋白质-蛋白质相互作用来极大地扩大可用药靶点的数量，这些相互作用不能被目前的疗法调节。我们建议使用荧光蛋白作为FRET偶联来筛选活细菌细胞内的环肽的遗传编码文库，以寻找潜在的核因子？B信号和炎症反应的抑制物。核因子？B是一种转录因子，在调节免疫和炎症反应中起重要作用。它介导了TNFa的生物学作用，而TNFa/NF？B信号的异常在关节炎、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信号转导的药物可能具有多种治疗作用。控制NF？B的作用和介导TNFa作用的一个关键因素是I？B激酶(IKK)。IKK活性受变构激活剂NEMO的调节。因此，NEMO/IKK相互作用的环肽抑制剂可能会阻断TNFa对NF？B信号的刺激，并可能作为抗炎药物使用。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，可以生成和选择具有最佳结合和抑制特性的化合物，从而促进NEMO/IKK？结合的选择性环肽抑制剂的发现(如本文所建议的)。 与公共卫生相关：使用多肽来调节细胞内过程的概念已经被研究了几十年，因为多肽在身体的每个细胞中都发挥着核心作用。这些策略历来都是失败的，因为大多数多肽缺乏进入细胞的能力，而线性多肽在体内天生就不稳定。我们建议开发一类突破性的多肽药物，称为环肽，这些药物相当稳定，具有生物活性，具有良好的类药物性质，包括抵抗蛋白质降解和穿越哺乳动物细胞膜的能力。这类化合物代表了一种调节细胞内蛋白质-蛋白质相互作用的解决方案，这些相互作用已被确定为大多数人类疾病的关键控制点。具体地说，我们建议利用分子进化策略和基于高通量细胞的筛选来产生和选择能够抑制核因子？B信号的环肽。TNFa/NF？B信号的异常在关节炎、动脉粥样硬化和克罗恩病等炎症性疾病中起重要作用。因此，阻断TNFa/NF？B信号转导的药物可以作为抗炎药物发挥多种治疗作用。具体地说，阻断NEMO与IKK结合的小分子药物可抑制核因子B信号转导，可作为抗炎药物开发。

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.