Mechanism & Inhibition of Collagenolytic Activity

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基本信息

批准号：
7185769
负责人：
GREGG B FIELDS
金额：
$ 29.52万
依托单位：
FLORIDA ATLANTIC UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-05 至 2008-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7185769
关键词：
Adamts5 protein Affect Amino Acid Sequence Amino Acids Arthritis Back Binding Biological Assay Cells Circular Dichroism Class Classification Cleaved cell Collagen Connective Tissue Discrimination Disease Disintegrins Drug Delivery Systems Endopeptidases Enzyme Interaction Enzymes Esters Family Gelatinase A Gelatinase B Generations Goals Hydrolysis Individual Interstitial Collagenase Investigation Kinetics Knowledge Label Laboratories Matrix Metalloproteinase Inhibitor Matrix Metalloproteinases Metalloproteases Methodology Methods Modeling Molecular Conformation NMR Spectroscopy Nature Neoplasm Metastasis Neutrophil Collagenase Object Attachment Peptide Hydrolases Peptides Phase Phase III Clinical Trials Play Pliability Range Relative (related person)Reporting Research Role Series Site Solid Specificity Stromelysin 1 Structure Testing Thermolysin Thinking Tissues Trypsin Tumor Cell Invasion Vertebral column aggrecanase aggrecanase-1 analog base cathepsin K collagenase 3 design enzyme activity feeding human MMP14 protein inhibitor/antagonist matrix metalloproteinase 26 member metalloenzyme metastatic process mutant novel novel strategies phosphonate programs protein aminoacid sequence research study synthetic construct synthetic protein thermostability triple helix two-dimensional

Adamts5 protein Affect Amino Acid Sequence Amino Acids Arthritis Back Binding Biological Assay Cells Circular Dichroism Class Classification Cleaved cell Collagen Connective Tissue Discrimination Disease Disintegrins Drug Delivery Systems Endopeptidases Enzyme Interaction Enzymes Esters Family Gelatinase A Gelatinase B Generations Goals Hydrolysis Individual Interstitial Collagenase Investigation Kinetics Knowledge Label Laboratories Matrix Metalloproteinase Inhibitor Matrix Metalloproteinases Metalloproteases Methodology Methods Modeling Molecular Conformation NMR Spectroscopy Nature Neoplasm Metastasis Neutrophil Collagenase Object Attachment Peptide Hydrolases Peptides Phase Phase III Clinical Trials Play Pliability Range Relative (related person)Reporting Research Role Series Site Solid Specificity Stromelysin 1 Structure Testing Thermolysin Thinking Tissues Trypsin Tumor Cell Invasion Vertebral column aggrecanase aggrecanase-1 analog base cathepsin K collagenase 3 design enzyme activity feeding human MMP14 protein inhibitor/antagonist matrix metalloproteinase 26 member metalloenzyme metastatic process mutant novel novel strategies phosphonate programs protein aminoacid sequence research study synthetic construct synthetic protein thermostability triple helix two-dimensional

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项目摘要

DESCRIPTION (provided by applicant): The dissolution of the collagen triple-helix has been implicated in a variety of diseases that effect the structural integrity of various components of the body. Collagen also provides a barrier between tissues and cells; destruction of this barrier plays a role in tumor cell invasion and metastasis. The matrix metalloproteinase (MMP) family has been recognized for their collagenolytic activities, and has thus been the subject of intense research efforts, in order to elucidate their mechanisms of action and allow for rational design of inhibitors. We have developed methodology for constructing triple-helical peptides (THPs) and have applied these synthetic proteins for the study of enzyme interactions with collagens. "Triple-helical peptidase" activity was found to be more widespread amongst proteases than previously believed. It appears that the unique aspect of collagenolytic activity may not be the ability to cleave a triple-helix, but rather the ability to bind and orient the native collagen molecule properly. This paradigm shift could have significant effects on the design of inhibitors against collagenolytic activity. To further explore the nature of triple-helical peptidase activity, a series of THP substrates will be assembled, incorporating known sites of collagen hydrolysis and varying over a range of Tm values. Substrate thermal stability will be correlated to enzyme activity. In tandem, 2D NMR experiments using 15N-labeled amino acids will examine the mobilities of the peptide backbone in these substrates. Individual kinetic parameters and activation energies will be evaluated for MMP, trypsin, thermolysin, cathepsin K, and aggrecanase hydrolysis of THPs. MMP-1, MMP-2, MMP-8, and cathepsin K mutants will be utilized to determine the regions within these enzymes critical for triple-helical peptidase activity. Finally, selective THP substrates and novel THP inhibitors will be designed and tested. Overall, triple-helical peptidase activity will be systematically evaluated for a variety of proteases as a function of substrate sequence and conformational flexibility, and the mechanism of collagenolytic activity will better understood.

描述（由申请人提供）：胶原蛋白三螺旋的溶解与各种疾病有关，这些疾病影响了身体各个成分的结构完整性。胶原蛋白还提供了组织和细胞之间的障碍。这种障碍的破坏在肿瘤细胞侵袭和转移中起作用。基质金属蛋白酶（MMP）家族因其胶原式活性而被认可，因此成为强烈的研究工作的主题，以阐明其作用机理并允许对抑制剂的合理设计。我们已经开发了构建三螺旋肽（THP）的方法，并将这些合成蛋白应用于研究酶相互作用与胶原蛋白的研究。发现“三螺旋肽酶”活性在蛋白酶中比以前认为的更为普遍。看来胶原式活性的独特方面可能不是裂解三螺旋的能力，而是能够正确结合和定向天然胶原蛋白分子的能力。这种范式转移可能会对抑制剂对胶原性活性的设计产生重大影响。为了进一步探索三螺旋肽酶活性的性质，将组装一系列THP底物，结合已知的胶原蛋白水解位点，并在一系列TM值中变化。底物热稳定性将与酶活性相关。在串联中，使用15N标记氨基酸的2D NMR实验将检查这些底物中肽主链的迁移率。将评估单个动力学参数和激活能量的MMP，胰蛋白酶，热溶胶蛋白，组织蛋白酶K和Aggrecanase酶水解THP。将利用MMP-1，MMP-2，MMP-8和组织蛋白酶K突变体来确定这些酶中对三螺旋肽酶活性至关重要的区域。最后，将设计和测试选择性THP底物和新型THP抑制剂。总体而言，将系统地评估三螺旋肽酶活性的各种蛋白酶，这是底物序列和构象柔韧性的函数，并且胶原式活性的机制将更好地理解。