Co(III) Schiff base complexes as selective and irreversible inhibitors of MMP-2

Co(III) 希夫碱配合物作为 MMP-2 的选择性和不可逆抑制剂

• 批准号: 8512054
• 负责人: Thomas J Meade
• 金额: $ 7.73万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8512054
• 关键词: 3-Dimensional; Active Sites; Adverse effects; Antimetastatic Agent; Antineoplastic Agents; Behavior; Binding; Biological; Biological Assay; Cancer cell line; Cells; Cleaved cell; Clinical Trials; Cobalt; Complex; Development; Disseminated Malignant Neoplasm; Effectiveness; Electron Transport Complex III; Endopeptidases; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Fluorescence Microscopy; Gelatinase A; Goals; Histidine; Human; Imidazole; Immunofluorescence Immunologic; In Situ; In Vitro; Inhibition of Matrix Metalloproteinases Pathway; Kinetics; Ligands; Marimastat; Matrix Metalloproteinase Inhibitor; Matrix Metalloproteinases; Modeling; Monitor; Musculoskeletal; Neoplasm Metastasis; Peptide Hydrolases; Peptides; Proteins; Sampling; Schiff Bases; Specificity; Testing; Thermodynamics; Tissues; Transition Elements; Vertebral column; Zinc; absorption; analog; biological systems; cancer cell; cytotoxicity; improved; in vivo; inhibitor/antagonist; loss of function; metastasis prevention; migration; monolayer; novel; peptide A; protein structure; public health relevance; screening; small molecule; tumor; tumor microenvironment

DESCRIPTION (provided by applicant): Our goal is to develop a transition metal agent that specifically inhibits matrix metalloproteinase (MMP)-2 by a unique mechanism. Such an agent may have potential use in antimetastatic therapy. A MMP-2 specific peptide substrate will be tethered to a Co(III)-Schiff base (Co(III)-sb) complex that has been previously shown to irreversibly inhibit histidine (His)-containing enzymes. The MMP-2 targeting peptide will impart specificity for MMP-2 over other enzymes and bring the Co(III)-sb complex to close proximity of the target protein such that selective inhibition of MMP-2 occurs. The peptide conjugate is expected to show low levels of off-target effects while maintaining effective inhibition of the target enzyme. Peptides known to be selectively cleaved by MMP-2 over other MMPs will be synthesized and tethered to Co(III)-sb (Co(III)-pep), and fluorescent analogues (Co(III)-flupep) will be synthesized to allow monitoring of their action and localization in biological systems usin fluorescence microscopy. The effectiveness of Co(III)-pep and Co(III)-flupep as a MMP-2-specific inhibitor will be evaluated by investigating kinetic and thermodynamic interactions between the inhibitor and enzyme. In addition, the specificity of Co(III)-pep and Co(III)-flupep fo MMP-2 over other enzymes will be investigated by screening them against a range of MMPs and non-MMP enzymes using inhibition assays and zymography. The binding and inhibitory activity of Co(III)-pep and Co(III)-flupep will be compared to the non-targeted Co(III)-sb complex to evaluate the suitability of targeted Co(III) complexes as MMP-2 specific inhibitors. Further, the effect of Co(III)-pep on the invasion and migration of various cancer cell lines will be validated by testing in biological systems. Migration and invasion assays on cell monolayers and 3-dimensional tumor models such as spheroids will be carried out to evaluate the effect of the peptide conjugates in inhibiting metastatic activity of the cell. Immunofluorescence assays will be performed to ascertain the extent of MMP-2 inhibition in spheroid sections. Upon completion of the proposed in vitro biological studies and if successful results are obtained, we believe this novel inhibitor can be utilized as an antimetastatic agent in vivo.

描述（由申请人提供）：我们的目标是开发一种通过独特机制特异性抑制基质金属蛋白酶（MMP）-2的过渡金属制剂。这种药物可能在抗转移治疗中具有潜在用途。 MMP-2 特异性肽底物将与 Co(III)-希夫碱 (Co(III)-sb) 复合物连接，该复合物先前已被证明可不可逆地抑制含组氨酸 (His) 的酶。 MMP-2 靶向肽将赋予 MMP-2 优于其他酶的特异性，并使 Co(III)-sb 复合物非常接近靶蛋白，从而发生 MMP-2 的选择性抑制。肽缀合物预计会表现出低水平的脱靶效应，同时保持对靶酶的有效抑制。将合成已知被 MMP-2 而非其他 MMP 选择性裂解的肽，并将其连接到 Co(III)-sb (Co(III)-pep)，并且将合成荧光类似物 (Co(III)-flupep)，以允许使用荧光显微镜监测它们在生物系统中的作用和定位。 Co(III)-pep 和 Co(III)-flupep 作为 MMP-2 特异性抑制剂的有效性将通过研究抑制剂和酶之间的动力学和热力学相互作用来评估。此外，将通过使用抑制测定和酶谱法针对一系列 MMP 和非 MMP 酶进行筛选，研究 Co(III)-pep 和 Co(III)-flupep 对 MMP-2 相对于其他酶的特异性。 Co(III)-pep 和 Co(III)-flupep 的结合和抑制活性将与非靶向 Co(III)-sb 复合物进行比较，以评估靶向 Co(III) 复合物作为 MMP-2 特异性的适用性抑制剂。 此外，Co(III)-pep对各种癌细胞系侵袭和迁移的影响将通过生物系统测试得到验证。将进行单层细胞和球体等3维肿瘤模型的迁移和侵袭测定，以评估肽缀合物抑制细胞转移活性的效果。将进行免疫荧光测定以确定球体切片中 MMP-2 抑制的程度。在完成拟议的体外生物学研究并获得成功结果后，我们相信 新型抑制剂可用作体内抗转移剂。