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.
Because of its importance in cancer, MT1-MMP is a promising drug target. However, there are no available small molecule inhibitors that are highly specific for MT1-MMP. By employing the MLSMR library screen and the cells expressing fluorescent biosensors developed in our laboratory, we will use a novel approach to target the activation mechanism rather than the enzyme activity to identify specific small molecule inhibitors of intracellular MT1- MMP activation.
|Golubkov, Vladislav S; Prigozhina, Natalie L; Zhang, Yong et al. (2014) Protein-tyrosine pseudokinase 7 (PTK7) directs cancer cell motility and metastasis. J Biol Chem 289:24238-49|