UNIVERSITY OF PISA
School of Graduate Studies
“Drug Sciences and Bioactive substances”
PhD THESIS 2014-2017
“Design, Synthesis and Development of Molecules able to Modulate the
Bio-Pharmacological Activity of Metalloenzymes Involved in Degenerative
Diseases .”
SSD: CHIM/08
XXX CYCLE
Student:
Doretta Cuffaro
DIRECTOR OF THE SCHOOL SUPERVISOR
Prof. Adriano Martinelli Prof. Armando Rossello
SUMMARY
Chapter 1. Introduction 1
1. The extracellular matrix 1
2 Metzincines 5 3 Matrix Metalloproteinases 6 3.1 Classification of MMPs 6 3.2 MMP structure 7 3.3 MMPs Mechanism of action 10 3.4 MMPs regulation 11 3.5 Metzincins inhibitors 15 3.5.1 MMPs inhibitors 15 4. ADAM metalloproteinases 24
4.1 The ADAM multidomain structure 25
4.2 The ADAM ectodomain shedding 29
4.3 The ADAM regulation and synthesis 31
4.4 ADAM inhibitors : general guidelines. 34
Bibliography 35
Chapter 2. Development of novel selective inhibitors of Matrix Metalloproteinase 12
38
1.Introduction 38
1.1 MMP-12 Biological activity 39
1.2 MMP-12 inhibitors: the importance of ZBG 40
1.3 MMP-12 selective inhibitors 42
2.Sugar-based arylsulfonamide carboxylates as MMP-12 inhibitors 45 2.1 Sugar- arylsulfonamide carboxylates as selective and water soluble
MMP-12 inhibitors 45
2.1.1 Aim of the work 45
2.1.2 Chemistry 46
2.1.3 MMP inhibition and crystallographic studies 51
2.1.4 Conclusions 57
2.2 Further optimization of glycoconjugates MMP-12 inhibitors 59
2.2.1 Aim of the work 59
2.2.2 Chemistry 60
2.2.3 MMP inhibition studies 64
2.2.4 Conclusions 68
2.3 Experimental section 68
3.Development of thioaryl-based MMP-12 inhibitors with alternative
zinc binding groups 96
3.1 Aim of the work 96
3.2 Chemistry 98 3.3 MMP inhibition studies 104 3.4 Crystallographic studies 107 3.5 Conclusions 110 3.6 Experimental section 110 Bibliography 121
Chapter 3. Desing, synthesis and biological evaluation of bifunctional ligands of MT1MMP.125
1.Introduction 125
1.2 MT1-MMP biological role: Collagen degradation, proMMP-2 and pro-MMP13
activation and cell adhesion molecule cleavage 126
1.3 Synthetic inhibitors for MT1-MMP 129
1.3.1 Small molecules 129
1.3.2 Antibodies 130
2. Desing of novel MT1-MMP inhibitors 131
3. Results and Discussions 134
3.1 Chemistry 134
3.2 Biological activity on isolated enzymes 138
3.3 MTS cytotoxicity assay 138
3.4 Effect of inhibitors (2 and 6) on MT1MMP activity: F-gelatin film degradation
assay on HT1080 cells 139
4. Conclusions 142
5. Experimental section 142
5.1 Chemistry 142
5.2 Matherials and Methods of biological experiments 148
Bibliography 151
Chapter 4. Biological evaluation of selective ADAM-10 inhibitors able to inhibit DDR1 and DDR2 shedding 154
1.Introduction 154
1.1 ADAM-10 154
1.2 ADAM-10 biological function in cell physiology and its role in cancer progression
and inflammatory diseases 156
1.3 ADAM-10 selective inhibitors 157
2. Results and Discussions 163
2.1 Biological evaluation of previously synthetized 4.1 and 4.2 163
2.2 MTS cytotoxicity assay 164
2.3 Effect of inhibitors (4.1 and 4.2) on Collagen induced DDR1 shedding by ADAM-10
on A431 cells 165
2.4 Effect of inhibitors (4.1 and 4.2) on Collagen induced DDR1 and DDR2 shedding
by ADAM-10 on HT1080 cells 166
2.5 Effect of inhibitors (4.1 and 4.2) on MT1MMP activity: F-gelatin film degradation
assay on A431 cells 168
2.6 Effect of inhibitors (4.1 and 4.2) on MT1MMP activity: F-gelatin film degradation
assay on HT1080 cells 170
3. Conclusions 173
4. Experimental section 173
Bibliography 180
