Tesi di laurea magistrale LM5
Development of specific ligands able to inhibit the shedding of Discoidin Domain Receptors (DDRs) by ADAM10
Corso di studi
CHIMICA E TECNOLOGIA FARMACEUTICHE
relatore Prof. Rossello, Armando
relatore Dott.ssa Nuti, Elisa
relatore Dott.ssa Nuti, Elisa
- selective inhibitors
- cell migration
- ADAM metalloproteinases
Data inizio appello
Data di rilascio
Eukaryotic cells of multicellular organisms organize themselves into tissues where most of the volume is made up of extracellular space; this is filled with a complex meshwork of macromolecules called the Extracellular Matrix (ECM). ECM is something more than a mechanical and a structural support, because it can directly influence cell activities like cell growth, division and survival, cell migration, cell differentiation and apoptosis. Matrix Metalloproteinases (MMPs) have been recognized as the main responsible for degradation of ECM components and general tissue remodeling but recently also ADAM metalloproteinases, in the metzincins superfamily, have been associated with those processes; consequently they are attracting growing interest as potential therapeutic targets, as they have been implicated in cancer, neurological and cardiovascular diseases, asthma, infection and inflammatory diseases1.<br>ADAM metalloproteinases are a family of Integral membrane glycoproteins, usually localized on cell surface, with a multi-domain structure; they are characterized by A unique Disintegrin receptor binding integrin domain and A Metalloproteinase Domain that requires a divalent zinc ion to carry out the catalytic process. The typical substrates are transmembrane proteins such as differentiation factors, cell adhesion molecules, cytokines, chemokines, growth factors, so these enzymes strongly influence cell physiology2. Their expression is finely controlled at the level of transcription and activation and in addition they can be regulated by endogenous inhibitors (TIMPs).<br>Several potential approaches have been carried out to selectively inhibit ADAM protease activity: the use of purified or synthetic ADAM pro-domains, TIMPs or monoclonal antibody and synthetic small molecule inhibitors; of these possible approaches only the last one has been subjected to a detailed investigation. Low molecular weight ADAM inhibitors are designed to bind the MMP-like catalytic site and most of them have a hydroxamate group as zinc binding group; since the design and the synthesis of selective inhibitors are not easy to carry out, only a small number of compounds supposed to be active only on specific ADAMs have reached the first phases of clinical trials3.<br>Two promising selective ADAM-10 inhibitors, LT4 and MN8, have been developed by Prof. Rossello's research group in recent years; these synthesized compounds have been initially tested in vitro by fluorimetric assay, on human recombinant ADAM-10 and ADAM-17 and on other human recombinant MMPs (-1, -2, -9, -14) showing a high (MN8 IC50 9.2nM, LT4, IC50 40nM) and<br>selective (LT4 IC50 40nM vs 1500nM on ADAM17) activity on ADAM-10. Recently, they have been<br>tested on Hodgking Linfoma cell lines showing to increase their sensitivity to NKG2D-dependent cell killing exerted by natural killer and T cells. Moreover the biological activity of LT4 and MN8 appears to be more potent than that of the commercial inhibitor GI254023X4.<br>On the basis of these recent findings, in my thesis project I was involved in the synthesis of three new synthetic selective inhibitors of ADAM-10; our goal was to further investigate about the<br>￼￼￼￼￼<br>sulfonamide hydroxamate structure of LT4 and MN8, trying to improve both the activity and the selectivity against ADAM-10.<br>In the second part of my thesis project I further analyzed the biologic activity of LT4 and MN8 carrying out several assays on A431 cell line and HT1080 cell line. I went to the Kennedy Institute of Rheumatology, University of Oxford, where in Dr. Yoshi’s research laboratory I’ve demonstrated their inhibitory activity on ADAM-10 dependent DDR1 ectodomain shedding; then I’ve investigated about their selectivity analyzing their influence on CD44 shedding and gelatin degradation by MT1- MMP. Lastly, through some wound-closure assays, I’ve observed that LT4 and MN8 have both a good trend of inhibition on cell migration, better than that of marimastat (a well known broad- spectrum zinc metalloproteinase inhibitor used as reference compound).
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