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

使用基于环肽的分子支架来选择特定的蛋白质-蛋白质inh

• 批准号: 7996667
• 负责人: Mark W Nowak
• 金额: $ 20.07万
• 依托单位: ENCODE BIO, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7996667
• 关键词: Adoption; Adverse effects; Affect; Affinity; Anabolism; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Arthritis; Atherosclerosis; Attention; Bacteria; Base Sequence; Binding; Binding Sites; Bioavailable; Biological; Biological Assay; Biological Availability; Blood; California; Cell Nucleus; Cell Separation; Cell Survival; Cell membrane; Cells; Characteristics; Chemicals; Chronic; Cloning Vectors; Code; Complementary DNA; Complex; Consensus; Crohn' s disease; Cyclization; Development; Disease; Drug Kinetics; Escherichia coli; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescence-Activated Cell Sorting; Gene Expression; Generations; Hormones; Hot Spot; Human Resources; Immune; In Vitro; Induction of Apoptosis; Inflammatory; Inflammatory Response; Kidney; Label; Laboratories; Lead; Length; Libraries; Licensing; Life; Ligands; Ligation; Link; Mammalian Cell; Measures; Mediating; Molecular; Molecular Evolution; Monitor; NMR Spectroscopy; Normal Cell; Oral; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Preparations; Phosphotransferases; Plasmids; Play; Process; Production; Property; Protein Kinase; Proteins; Randomized; Reaction; Reporter; Research Personnel; Resistance; Role; Screening procedure; Signal Transduction; Site; Solutions; Stream; Structure; Surface; System; TNF gene; Techniques; Technology Transfer; Testing; Therapeutic; Time; Toxic effect; Universities; Variant; anthrax lethal factor; antimicrobial; base; chemical synthesis; cost; cytokine; cytotoxic; drug development; drug discovery; extracellular; fluorophore; human disease; in vivo; inhibitor/antagonist; intein; member; mutant; preclinical study; prevent; protein protein interaction; public health relevance; receptor; scaffold; small molecule; transcription factor

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.

描述（由申请人提供）：肽类药物的益处包括比现有药物更低的毒性和更大的功效，并且在某些情况下，是另外不可用的新类别的化合物。然而，将肽开发成商业药物仍然是一个非常具有挑战性和耗时的过程。肽通常具有差的体内稳定性、差的药代动力学和差的生物利用度。为了寻找更好的肽支架用于药物开发，研究人员最近专注于高度约束的肽，称为环肽，作为用于生产高亲和力配体的极其稳定和通用的支架。重要的是，环肽支架已经显示出广泛的生物活性，并且在某些情况下具有口服生物利用度。其他环肽类化合物已被证明通过巨胞饮作用穿过细胞膜。最后，环肽结构可以在细菌或动物细胞中编码和表达，并且可以进行大量的序列变异。因此，我们认为，环肽药物可以提供机会，大大扩大了“药物”的目标，通过靶向细胞内的蛋白质-蛋白质相互作用，不能通过目前的治疗功能调节的数千个数量。我们建议使用荧光蛋白作为FRET夫妇筛选基因编码库内的活细菌细胞的潜在抑制剂NF？B信号传导和炎症反应。NF？B是在调节免疫和炎症反应中具有重要作用的转录因子。它介导TNF α的生物学作用和TNF α/NF？B信号传导在炎性疾病如关节炎、动脉粥样硬化和克罗恩病中起关键作用。因此，阻断TNF α/NF？B信号传导可能具有许多治疗作用。控制NF作用的关键因素？B介导TNF α的作用是I？B激酶（IKK）。IKK活性由别构激活剂NEMO调节。因此，NEMO/IKK相互作用的环肽抑制剂可能会阻断TNF α刺激NF？B信号，可能是有用的抗炎药物。NEMO/IKK结合的选择性环肽抑制剂的发现将通过分子进化策略的力量和使用荧光激活细胞分选（FACS）的基于高通量细胞的筛选来促进（如本文所提出的），以能够产生和选择具有最佳结合和抑制特性的化合物。 公共卫生相关性：使用肽来调节细胞内过程的概念已经研究了几十年，因为肽在体内的每个细胞中都起着核心作用。这些策略在历史上失败了，因为大多数肽缺乏进入细胞的能力，并且线性肽在体内固有地不稳定。 我们建议开发一类突破性的肽类药物，称为cyclotides，它们相当稳定，具有生物活性，具有良好的药物样特性，包括抗蛋白水解降解和穿越哺乳动物细胞膜的能力。这类化合物代表了调节细胞内蛋白质-蛋白质相互作用的解决方案，其已被确定为大多数人类疾病的关键控制点。具体而言，我们建议部署的权力，分子进化策略和高通量细胞为基础的筛选，使生成和选择的环肽，可以抑制NF？B信令。 TNF α/NF？B信号传导在炎性疾病如关节炎、动脉粥样硬化和克罗恩病中起关键作用。因此，阻断TNF α/NF？B信号传导可以作为抗炎剂具有许多治疗作用。具体来说，小分子药物，阻断结合相互作用的NEMO IKK？抑制NF？B信号传导可被开发为抗炎药物。