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

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的过渡金属试剂。这样的药剂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）-席夫碱（Co（III）-sb）复合物连接，该复合物先前已显示出不可逆地抑制含组氨酸（His）的酶。MMP-2靶向肽将赋予MMP-2超过其它酶的特异性，并使Co（III）-sb复合物与靶蛋白紧密接近，从而发生MMP-2的选择性抑制。预期肽缀合物显示低水平的脱靶效应，同时保持对靶酶的有效抑制。将合成已知被MMP-2选择性切割而不是被其他MMP切割的肽，并将其连接到Co（III）-sb（Co（III）-pep），并合成荧光类似物（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，Co（III）-pep和Co（III）-flupep对MMP-2相对于其他酶的特异性将通过使用抑制测定和酶谱法针对一系列MMP和非MMP酶筛选它们来研究。将Co（III）-pep和Co（III）-flupep的结合和抑制活性与非靶向Co（III）-sb络合物进行比较，以评价靶向Co（III）络合物作为MMP-2特异性抑制剂的适用性。 此外，Co（III）-pep对各种癌细胞系的侵袭和迁移的作用将通过在生物系统中的测试来验证。将对细胞单层和三维肿瘤模型如球状体进行迁移和侵袭测定，以评价肽缀合物在抑制细胞转移活性中的作用。将进行免疫荧光测定以确定球状体切片中MMP-2抑制的程度。在完成拟定的体外生物学研究后，如果获得成功的结果，我们认为 新的抑制剂可用作体内抗转移剂。

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.