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MT 1-MMP潜伏酶原细胞内活化的小分子抑制剂。促侵袭、促肿瘤发生的MT 1-MMP（MMP-14）是癌细胞表面蛋白水解事件中的关键蛋白酶。MT 1-MMP是一个很有前途的肿瘤药物靶点。广泛的MMP抑制剂在临床试验中表现不佳。这些抑制剂被设计为靶向MMP的高度同源活性位点区域，这就是MT 1-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，因为我们将靶向细胞内MT 1-MMP的活化机制，其作为具有N-末端抑制性前结构域的潜在酶原合成。因此，由于前结构域“诱饵区”的低同源性，MMP酶原的活化机制对单个MMP是特异性的。MT 1-MMP前结构域的这个独特的“诱饵区”包含PGD <$L50序列。为了研究MT 1-MMP在癌细胞中的活化途径并监测MT 1- MMP前结构域的细胞内PGD_L50位点切割，我们使用前结构域序列产生荧光生物传感器（可切割的RFP-PRO-GFP和不可切割的RFP-L50 D-GFP对照）。在与Sanford-Burnham NIH分子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生物传感器构建体的细胞来鉴定阻断MT 1-MMP前结构域加工的新型分子，从而在酶原活化水平上抑制细胞内MT 1-MMP活性。我们的两个具体目的是（1）通过使用表达RFP-PRO-GFP和突变体RFP-L50 D-GFP生物传感器的细胞来筛选癌症HT 1080细胞中MT 1-MMP前结构域加工的抑制剂的MLSMR文库，和（2）进行基于细胞的第二次测定以验证化学探针对癌细胞中proMT 1-MMP活化的特异性抑制活性并研究其机制（途径）这些化合物通过其调节proMT 1- MMP活化。我们已经优化了检测方法，并对LOPAC 1280化合物文库进行了中试筛选。对于我们的主屏幕，我们将使用在试点屏幕中优化的条件。为了确认初步筛选的初始命中，将进行细胞毒性和GFP光谱干扰反筛选。然后在我们的实验室中使用多种基于细胞的生化测定法对所选化合物进行测试，包括使用FITC-明胶的原位明胶酶谱法;通过明胶酶谱法进行的MT 1-MMP依赖性MMP-2活化，Boyden室中的细胞侵袭测定法和MMP活性的酶测定法。

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.