A High Content Screen to Identify Inhibitors of MT1-MMP Activation

描述（由申请人提供）：我们的研究计划集中在MLSMR文库筛选上，以鉴定MT1-MMP潜伏酶原细胞内激活的小分子抑制剂。促侵袭性、致瘤性MT1-MMP （MMP-14）是癌细胞表面蛋白水解事件的关键蛋白酶。MT1-MMP是一种很有前景的抗癌药物靶点。广谱MMP抑制剂在临床试验中表现不佳。这些抑制剂旨在靶向MMPs的高度同源活性位点区域，这就是为什么MT1-MMP的选择性小分子抑制剂不可用的原因。我们的方法是不同的，因为我们将针对细胞内MT1-MMP的激活机制，MT1-MMP是由n端抑制原合成的潜伏酶原。因此，由于前域“诱饵区”的同源性较低，MMP酶原的激活机制对单个MMPs是特异性的。这个独特的“诱饵区域”的MT1-MMP原域包含PGD¿L50序列。为了研究MT1-MMP在癌细胞中的激活途径，并监测MT1-MMP前结构域在细胞内PGD -L50位点的切割情况，我们利用前结构域序列制备了荧光生物传感器（可切割的RFP-PRO-GFP和不可切割的RFP-L50D-GFP对照）。在与Sanford-Burnham NIH分子文库探针生产中心网络（MLPCN）筛选中心的合作中，我们将使用稳定转染了生物传感器结构的细胞来识别阻断MT1-MMP原结构域加工的新分子，从而在酶原激活水平上使细胞内MT1-MMP活性失活。我们的两个特定目标是：(1)通过使用表达RFP-PRO-GFP和突变RFP-L50D-GFP生物传感器的细胞，筛选MLSMR文库中癌症HT1080细胞中MT1-MMP前域加工抑制剂；(2)进行基于二级细胞的实验，验证化学探针对癌细胞中proMT1-MMP激活的特异性抑制活性，并研究这些化合物调节proMT1-MMP激活的机制（途径）。我们已经对实验进行了优化，并对LOPAC1280化合物文库进行了先导筛选。对于我们的主屏幕，我们将使用在试验屏幕中优化的条件。为了确认来自主屏的初始命中，将进行细胞毒性和GFP光谱干扰反屏。然后，选定的化合物将在我们的实验室使用多种基于细胞的生化分析进行测试，包括使用fitc -明胶的原位明胶酶谱法；明胶酶谱法测定mt1 -MMP依赖性MMP-2的激活，Boyden室细胞侵袭试验和MMP活性的酶分析。

DESCRIPTION (provided by applicant): Our research plan is focused on the MLSMR library screen to identify small molecule inhibitors of the intracellular activation of the MT1-MMP latent zymogen. Pro-invasive, pro- tumorigenic MT1-MMP (MMP-14) is a key proteinase in the proteolytic events at the cancer cell surface. MT1-MMP is a promising drug target in cancer. Broad-range MMP inhibitors did not perform well in clinical trials. These inhibitors are designed to target the highly homologous active site region of MMPs and this is why the selective small molecule inhibitors of MT1-MMP are not available. Our approach is different because we will target the activation mechanism of intracellular MT1-MMP which is synthesized as a latent zymogen with the N-terminal inhibitory prodomain. Thus, because of a low homology of the prodomain "bait region", the activation mechanism of the MMP zymogens is specific for the individual MMPs. This unique "bait region" of the MT1-MMP prodomain contains the PGD¿L50 sequence. To study the activation pathway of MT1-MMP in cancer cells and monitor the intracellular PGD¿L50 site cleavage of the MT1- MMP prodomain we used the prodomain sequence to generate the fluorescent biosensors (the cleavable RFP-PRO-GFP and the uncleavable RFP-L50D-GFP control). In a collaboration with the Sanford-Burnham NIH Molecular Libraries Probe Production Centers Network (MLPCN) screening center, we will use the cells stably transfected with the biosensor constructs to identify novel molecules that block the MT1-MMP prodomain processing, and as a result, inactivate the intracellular MT1-MMP activity at the zymogen activation level. Our two Specific Aims are (1) Screen the MLSMR library for inhibitors of the MT1-MMP prodomain processing in cancer HT1080 cells by employing the cells expressing the RFP-PRO-GFP and the mutant RFP-L50D-GFP biosensors, and (2) Perform secondary cell-based assays to validate the specific inhibitory activity of chemical probes toward the proMT1-MMP activation in cancer cells and to investigate the mechanism (pathway) by which these compounds modulate the proMT1- MMP activation. We have already optimized the assays and performed a pilot screen of the LOPAC1280 compound library. For our primary screen we will use conditions optimized in the pilot screen. To confirm the initial hits from the primary screen the cytotoxicity and GFP spectral interference counterscreens will be performed. The selected compounds will then be tested in our laboratory using multiple cell-based and biochemical assays including In Situ gelatin zymography using FITC-gelatin; MT1-MMP-dependent MMP-2 activation by gelatin zymography, cell invasion assay in Boyden chambers and enzymatic assays of MMP activity.