Mechanism and Inhibition of Collagenolytic Activity

胶原蛋白分解活性的机制和抑制

• 批准号: 9242588
• 负责人: GREGG B FIELDS
• 金额: $ 27.01万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-05 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9242588
• 关键词: Adverse effects; Amino Acids; Arterial Fatty Streak; Behavior; Binding; Binding Sites; Breast Carcinoma; Breast Melanoma; Catabolism; Collagen; Collagen Type I; Data; Development; Enzymes; Family; Fluorescence Resonance Energy Transfer; Funding; Gelatinase A; Gelatinase B; Goals; Hemopexin; Hydrolysis; Impairment; Individual; Inhibition of Matrix Metalloproteinases Pathway; Interstitial Collagenase; Laboratories; Lead; Libraries; MMP14 gene; Malignant Neoplasms; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Modeling; Musculoskeletal; NMR Spectroscopy; Neutrophil Collagenase; Pathologic; Peptide Hydrolases; Peptides; Physiology; Play; Process; Progress Reports; Property; Proteins; Research; Role; Rupture; Scanning; Site; Specificity; Stromelysin 1; Structure; Substrate Specificity; Syndrome; Techniques; Testing; Therapeutic Agents; Tissues; Tumor Cell Invasion; Zinc; analog; base; cartilage degradation; collagenase 3; combinatorial; design; in vivo; inhibitor/antagonist; interest; interstitial; matrix metalloproteinase 18; melanoma; member; metalloenzyme; molecular imaging; mouse model; non-Native; novel; preference; protein aminoacid sequence; public health relevance; scaffold; triple helix; tumor; tumor growth; tumor progression

DESCRIPTION (provided by applicant): Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Only a small number of proteases have been identified capable of efficient processing of triple-helical regions of collagens. Several members of the zinc metalloenzyme family, specifically matrix metalloproteinases (MMPs), possess collagenolytic activity. A mechanistic understanding of the cleavage of intact collagens has been pursued for many years; the results of such studies could lead to the development of truly selective MMP inhibitors. Our laboratory developed triple-helical peptides (THPs) as MMP substrates, with the goal of using these models to dissect collagenolytic behavior and develop selective MMP inhibitors. In the most recent funding period of the present project (04/01/08-present), we have experimentally derived the initial steps of collagenolysis and identified specific residues involved in this process, quantified the roles of specific collagen residues in MMP substrate specificity, and developed selective MMP inhibitors based on secondary binding sites (exosites), and demonstrated the in vivo use of such inhibitors. Our studies also revealed intriguing, unexpected behaviors of collagenolytic MMPs, such as MMPs bind the triple-helix using slightly different orientations, one can use exosite binding THPs to inhibit some, but not all, proteolytic activities for a given enzyme, which could significantly reduce side effects, and substrate selectivity seen for single-stranded peptides is not observed in the triple-helix. The research plan described herein focuses on further development of triple-helical probes for teasing out collagenolytic MMP sequence specificities, identifying selective MMP inhibitors, and advancing the mechanism of collagenolysis. To achieve these goals we propose to (a) identify THP sequence preferences for the MMP family utilizing positional scanning combinatorial libraries, (b) design and characterize MMP selective, homotrimeric and heterotrimeric triple-helical transition-state analog inhibitors, and (c) explore the latter steps of collagenolysis usin state-of-the-art NMR spectroscopic techniques and design exosite binding THPs. Select inhibitors will be tested in mouse models of breast carcinoma and melanoma. Ultimately, we would like to obtain inhibitors that target those proteases implicated in cancers such as melanoma and breast carcinoma (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) while sparing proteases with host-beneficial functions (MMP-3 and MMP-8).

描述（由申请人提供）：胶原蛋白充当结构支架和组织之间的屏障，因此胶原蛋白分解代谢（胶原蛋白分解）被要求是正常生理学中严格调节的过程。反过来，病理状态下胶原蛋白的破坏或损伤在肿瘤生长和侵袭、软骨退化或动脉粥样硬化斑块形成和破裂中发挥作用。只有少数蛋白酶已被鉴定出能够有效加工胶原蛋白的三螺旋区域。锌金属酶家族的几个成员，特别是基质金属蛋白酶（MMP），具有胶原溶解活性。多年来，人们一直在追求对完整胶原蛋白裂解的机制理解。此类研究的结果可能会导致真正选择性 MMP 抑制剂的开发。 我们的实验室开发了三螺旋肽（THP）作为 MMP 底物，目的是利用这些模型来剖析胶原蛋白溶解行为并开发选择性 MMP 抑制剂。在本项目最近的资助期间（04/01/08至今），我们通过实验导出了胶原蛋白溶解的初始步骤，并鉴定了参与该过程的特定残基，量化了特定胶原蛋白残基在MMP底物特异性中的作用，并开发了基于次级结合位点（exosites）的选择性MMP抑制剂，并证明了此类抑制剂的体内使用。我们的研究还揭示了胶原蛋白水解 MMP 的有趣且意想不到的行为，例如 MMP 使用稍微不同的方向结合三螺旋，可以使用外部位点结合 THP 来抑制给定酶的部分（但不是全部）蛋白水解活性，这可以显着降低在三螺旋中没有观察到单链肽的副作用和底物选择性。 本文描述的研究计划侧重于进一步开发三螺旋探针，用于梳理胶原蛋白溶解 MMP 序列特异性、识别选择性 MMP 抑制剂并推进胶原蛋白溶解机制。为了实现这些目标，我们建议（a）利用位置扫描组合文库确定 MMP 家族的 THP 序列偏好，（b）设计和表征 MMP 选择性、同源三聚体和异源三聚体三螺旋过渡态类似物抑制剂，以及（c）探索使用最先进的核磁共振波谱技术进行胶原溶解的后期步骤，并设计外位点结合 THP。选定的抑制剂将在乳腺癌和黑色素瘤的小鼠模型中进行测试。最终，我们希望获得针对与黑色素瘤和乳腺癌等癌症有关的蛋白酶（MMP-1、MMP-2、MMP-9、MMP-13 和 MT1-MMP）的抑制剂，同时保留对宿主有益的蛋白酶功能（MMP-3 和 MMP-8）。

项目成果

Identification of specific hemopexin-like domain residues that facilitate matrix metalloproteinase collagenolytic activity.

鉴定促进基质金属蛋白酶溶胶原活性的特定血红素样结构域残基。

• 发表时间: 2009-09-04
• 作者: Lauer;Chalmers, Michael J;Busby, Scott A;Minond, Dmitriy;Griffin, Patrick R;Fields, Gregg B
• 通讯作者: Fields, Gregg B

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis.

基质金属蛋白酶 1 催化胶原蛋白分解的结构基础。

• 发表时间: 2012-02-01
• 影响因子: 15
• 作者: Bertini, Ivano;Fragai, Marco;Luchinat, Claudio;Melikian, Maxime;Toccafondi, Mirco;Lauer, Janelle L;Fields, Gregg B
• 通讯作者: Fields, Gregg B

Unraveling hidden regulatory sites in structurally homologous metalloproteases.

揭示结构同源金属蛋白酶中隐藏的调控位点。

• 发表时间: 2013-07-10
• 影响因子: 5.6
• 作者: Udi, Yael;Fragai, Marco;Grossman, Moran;Mitternacht, Simon;Arad;Calderone, Vito;Melikian, Maxime;Toccafondi, Mirco;Berezovsky, Igor N;Luchinat, Claudio;Sagi, Irit
• 通讯作者: Sagi, Irit

Monitoring and Inhibiting MT1-MMP during Cancer Initiation and Progression.

在癌症发生和进展过程中监测和抑制 MT1-MMP。

• 发表时间: 2014-02-17
• 影响因子: 5.2
• 作者: Pahwa, Sonia;Stawikowski, Maciej J;Fields, Gregg B
• 通讯作者: Fields, Gregg B

Development of high throughput screening assays and pilot screen for inhibitors of metalloproteases meprin α and β.

金属蛋白酶 meprin α 和 β 抑制剂的高通量筛选测定和中试筛选的开发。

• 发表时间: 2014-09
• 影响因子: 2.9
• 作者: Madoux, Franck;Tredup, Claudia;Spicer, Timothy P;Scampavia, Louis;Chase, Peter S;Hodder, Peter S;Fields, Gregg B;Becker;Minond, Dmitriy
• 通讯作者: Minond, Dmitriy