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，以确定MT1-MMP潜伏性酶原细胞内激活的小分子抑制物。侵袭性、致瘤性MT1-14(MMP14)是癌细胞表面蛋白分解过程中的一种关键的蛋白水解酶。MT1-MMPs是一种很有前景的抗癌药物靶点。大范围的基质金属蛋白酶抑制剂在临床试验中表现不佳。这些抑制剂是针对MMPs高度同源的活性部位区域设计的，这就是为什么MT1-MMPs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-基质金属蛋白酶的激活机制，它是作为一种潜在的酶原合成的，带有N端抑制原域。因此，由于原结构域“诱饵区域”的同源性很低，所以基质金属蛋白酶酶原的激活机制是针对单个基质金属蛋白酶的。MT1-基质金属蛋白酶原结构域的这个独特的“诱饵区域”包含PGD？L50序列。为了研究MT1-MMPs在癌细胞中的激活途径，并监测MT1-MMP原在细胞内的PgD？L50位点的裂解情况，我们利用原结构域序列制备了荧光生物传感器(可裂解的RFP-Pro-GFP和不可裂解的RFP-L50D-GFP对照)。在与Sanford-Burnham NIH分子文库探针生产中心网络(MLPCN)筛选中心的合作下，我们将使用稳定转染生物传感器构建体的细胞来识别阻止MT1-MMP原加工的新分子，从而在酶原激活水平上灭活细胞内MT1-MMP活性。我们的两个特定目标是(1)通过表达RFP-Pro-GFP的细胞和突变的RFP-L50D-GFP生物传感器来筛选HT1080细胞中MT1-MMP原加工的抑制剂的MLSMR文库；(2)进行二次细胞检测，验证化学探针对癌细胞中ProMT1-MMP活性的特异性抑制活性，并探讨这些化合物调节ProMT1-MMP活性的机制(途径)。我们已经优化了分析方法，并对LOPAC1280化合物文库进行了中试筛选。对于我们的主屏幕，我们将使用在引导屏幕中优化的条件。为了确认初级筛查的初始命中，将进行细胞毒性和GFP光谱干扰反筛查。选定的化合物将在我们的实验室中使用多种基于细胞的和生化分析进行测试，包括使用FITC-明胶的原位明胶酶谱；通过明胶酶谱检测MT1-MMP2的激活；在Boyden小室中的细胞侵袭实验和酶分析的MMP活性。

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.