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

基本信息

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
项目类别：
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&apos 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

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&apos 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

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项目摘要

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（通过应用程序提供）：肽药物的益处包括比现有药物较低的毒性和更高的效率，在某些情况下，新的化合物本来是不可用的。但是，肽的开发导致商业药物仍然是一个非常挑战和耗时的过程。肽通常患有较差的体内稳定性，药代动力学差和生物利用度差。为了寻找更好的药物开发肽支架，研究人员最近专注于高度受约束的宠物，称为旋风，是极其稳定和多功能的脚手架，用于产生高亲和力配体。重要的是，循环剂支架表现出了广泛的生物学活性，在某些情况下是口服生物利用度。其他循环剂已显示出通过大型细胞增多症穿越细胞膜。最后，可以在细菌或动物细胞中编码和表达循环层结构，并且可以适合实质性序列变化。因此，我们认为，通过针对数千种无法通过当前治疗在功能上调节的细胞内蛋白质 - 蛋白质相互作用，可以为大量扩大“可药用”靶标的数量提供机会。我们建议将荧光蛋白用作颗粒蛋白，以筛选活细菌细胞内循环编码的循环编码库，以实现NF？B信号传导和炎症反应的潜在抑制剂。 NF？b在调节免疫和炎症反应中具有重要作用的转录因子。它介导了TNFA和异常在TNFA/NF？B信号中的生物学作用，在关节炎，动脉粥样硬化和克罗恩病等炎症性疾病中起关键作用。结果，阻止TNFA/NF？b信号传导的药物可能采取多种治疗作用。控制NF？b的作用并介导TNFA效应的关键因素是I？B激酶（IKK）。 IKK活动由变构激活剂Nemo调节。这是Nemo/Ikk¿相互作用的环肽抑制剂可能会阻止TNFA刺激NF？b信号传导，并且可能作为抗炎药有用。通过分子进化策略和高吞吐量的细胞筛选，使用荧光激活的细胞分选（FACS）来制备NEMO/IKK？结合的选择性环位抑制剂（如在此处提出的），以实现具有最佳结合和抑制特征的化合物的生成和选择。公共卫生相关性：数十年来研究了使用Petides调节细胞内过程的概念，因为Petides在体内每个细胞中都起着核心作用。这些策略在历史上失败了，因为大多数佩蒂斯都缺乏进入细胞的能力，而线性宠物在体内本质上不稳定。我们提出开发一类突破性的胡椒药物，称为旋风，它们具有稳定且具有生物活性的，具有良好的药物样特性，包括对蛋白水解降解的耐药性和跨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，已被确定为大多数人类疾病的关键控制点。具体而言，我们建议部署分子进化策略和高吞吐量细胞筛选的能力，以使能够抑制NF？b信号传导的循环剂的生成和选择。 TNFA/NF？B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关键作用。结果，阻止TNFA/NF？b信号传导的药物可能具有许多治疗作用作为抗炎药。具体而言，可以阻止Nemo与Ikk的结合相互作用的小分子药物抑制NF？B信号传导可以作为抗炎药发展。