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Tesi etd-09222014-110854

Thesis type
Tesi di laurea specialistica LC5
Synthesis and inhibitory properties of potent and selective inhibitors of ADAM-17 and MMP-8
Corso di studi
relatore Prof. Rossello, Armando
relatore Prof. Nuti, Elisa
Parole chiave
  • Metalloendopeptidase
  • Matrix Metalloproteinase
  • Adamalysin
  • MMP-8
  • ADAM-17
  • Selective Inhibitor
Data inizio appello
Riassunto analitico
My Thesis project was focused on the study of new ADAMs and MMPs synthetic inhibitors. ADAMs and MMPs are two subfamilies of the Metzincyns group belonging to the superfamily of Metalloendopeptidases. Metallopeptidases are involved in the regulation of the human metabolism and are distributed among all kingdoms of life. These are zinc-dependent endopeptidases, and hence are named zincins; their general structure shows i) a short consensus sequence, HEXXH; ii) two histidines that are linked with the catalityc zinc and iii) the glutamate wich acts as a general base. Metzincins include adamalysins (ADAMs), matrix metalloproteinases (MMPs), serralysins, snapalysins, and leishmanolysins [Xavier Gomis-Rüth, 2003].
MMPs are involved in tissue remodeling and extracellular matrix (ECM) degradation . They are secreted as proenzymes and processed to their active forms.
Under normal conditions, their activity is fine-regulated by several mechanisms, but if this control fails, it can give rise to pathologies such as inflammation, tissue destruction, neurological diseases, cardiovascular disorders and even cancer. In particular MMP-8, also known as neutrophil collagenase, is considered as an antitarget for cancer therapy, but it has been claimed to have a key role in heart disease, osteoarthritis, and various inflammatory conditions such as hepatitis and ulcerative colitis.
For these reasons, several research groups have focused their attention on the development of low-molecular-weight inhibitors of MMP-8 as new therapeutic agents for the above diseases.
During my thesis project I have synthesized two new sulfone-based hydroxamates (CAM12 and CAM13) as analogues of a nanomolar inhibitor of MMP-8 previously described (FC7). It was changed the previous scaffold to improve the selectivity of the inhibitors for MMP-8 over the other MMPs. The new derivatives were tested in vitro on human recombinant MMPs by fluorometric assay, using a FRET peptide as substrate and the results were compared with those of FC 7.

Table 1. IC50 [nM]
Compd MMP-1 MMP-3 MMP-8 MMP-9 MMP-14
FC7 28000 4000 87 930 1300
CAM13 >5000 2400 98 1200 670
CAM12 167000 12000 57 2300 1140

As shown in Table 1, the introduction of a different substituent caused just a small increase in inhibitory activity against MMP-8 but an interesting increase in selectivity over MMP-1, MMP-3, MMP-9 and -14 with respect to FC7. These compounds will be tested in a mice model of corneal perforation by Dr. Cintia de Paiva (Baylor College of Medicine, Huston, USA).

In the second part of my thesis, my studies have focused on another subclass of Metzincins, the adamalysins. The ADAMs (A Disintregrin And Metalloproteinases) are transmembrane and secreted proteins with a complex multi-domain structure, consisting of an N-terminal prodomain, a metalloprotease domain, a disintegrin domain with cysteine-rich region, an EGF domain, a transmembrane domain and a Cytoplasmatic tail. To date 21 ADAMs have been described in the human genome and only 12 of these have the typical metalloprotease Zn-binding active site.
The majority of these 12 have proteolytic and adhesive properties. They are involved in “ectodomain shedding” (proteolytic ectodomain releases) of several growth factors, cytokines, receptors and adhesion molecules. The ADAMs partecipate in diverse (patho)physiologic processes, such as fertilisation, neurogenesis, inflammatory disease and cancer.
Specifically, two ADAMs, ADAM-10 and ADAM-17, have been implicated in cancer development and progression. ADAM-17, also named TACE (tumor necrosis factor-α convertase), is the principal protease necessary for the activation of pro-TNF-α that has been described both as a mutagen and as a tumor promoter. Moreover this metalloprotease is involved in the formation of active forms of Epidermal Growth Factor Receptor (EGFR) ligands. A dysregulation of ADAM activity and EGFR activation might also contribute to human diseases. In recent years, great care has been taken to create specific and selective inhibitors of ADAMs for use in anti-cancer therapy. The majority of them use hydroxamate as the zinc-binding group, for example INCB3619 is a highly investigated selective ADAM inhibitor.
Currently, Prof. Rossello’s group is studying the properties of a new class of ADAM-17 inhibitors. In the second part of my Thesis project I have synthesized a compound (CAM1) as analogue of a previously obtained inhibitor (JG34) with a good selectivity for ADAM-17. The new compound was tested vitro on human recombinant ADAM-17 and MMPs by fluorometric assay, in comparison with the reference compound (Table 2). The introduction of a key substituent was able to increase the inhibitory activity on ADAM-17 (IC50= 24nM) and also the selectivity over the tested MMPs with respect to JG34. Further studies will be conducted with this inhibitor in order to study its activity in cancer cell lines.

Table 2. IC50 [nM]

Compd ADAM-17 MMP-1 MMP-2 MMP-9 MMP-14
JG34 164 415000 4660 3320 57000
CAM1 24 >200000 3000 8800 50000