UNIVERSITÀ DI PISA
Dipartimento di Farmacia
Corso di Laurea Magistrale in Chimica e
Tecnologia Farmaceutiche
Tesi di Laurea
Synthesis and inhibitory properties of
carboxylate-based inhibitors and their
fluorescence-labeled analogues with high affinity
for some Metzincins
Relatori:
Candidata:
Prof. Armando Rossello
Elena De Vita
___________________
___________
Dott.ssa Elisa Nuti
___________________
Settore Scientifico Disciplinare: CHIM/08
Anno Accademico 2013 – 2014
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SUMMARY
ABSTRACT 6 1. INTRODUCTION 7 1.1METZINCINS 7 1.2MATRIX METALLOPROTEINASES 8 1.2.1THE EXTRACELLULAR MATRIX 9 1.2.2MMPS STRUCTURE 11 1.2.3MMPS MECHANISM OF ACTION 14 1.2.4MMPS REGULATION 161.2.5PHYSIOLOGICAL AND PATHOLOGICAL ACTIVITY OF MMPS 19
1.3MMP-9 19
1.3.1MMP-9 STRUCTURE AND ACTIVITY 20
1.3.2MMP-9 HOMODIMERISATION 21 1.4MMP-12 22 1.5CELLS MIGRATION 23 1.6ADAMTS 25 1.7ADAMTS-5 27 1.8METZINCINS INHIBITORS 28 1.8.1MMPS INHIBITORS 28 1.8.2ADAMTS INHIBITORS 36
2. INTRODUCTION TO THE EXPERIMENTAL PART 39
2.1DESIGN AND SYNTHESIS OF MMP INHIBITORS 39
2.2BIOLOGICAL RESULTS OF MMP INHIBITORS 48
2.3DESIGN AND SYNTHESIS OF ADAMTS INHIBITORS 49
2.4BIOLOGICAL RESULTS OF ADAMTS INHIBITORS 53
3. EXPERIMENTAL PART 54
3.1MATERIALS AND METHODS 54
3.2SYNTHESIS PROCEDURES 54
3.2.1SYNTHESIS OF FINAL COMPOUND 3 54
3.2.3SYNTHESIS OF FINAL COMPOUND 2 60
3.2.4SYNTHESIS OF FINAL COMPOUND 5 61
3.2.5SYNTHESIS OF FINAL COMPOUND 6 61
3.2.6SYNTHESIS OF FINAL COMPOUND 8 62
3.2.7SYNTHESIS OF FINAL COMPOUND 23 64 3.2.8SYNTHESIS OF FINAL COMPOUND 31 66 3.2.9SYNTHESIS OF FINAL COMPOUND 32 68
4. BIBLIOGRAPHY 70
To whom I love and to those who love me.
Abstract
Matrix Metalloproteinases (MMPs) are a family of enzymes that are attracting growing interest as therapeutic targets. They are proteases whose fundamental role has been recognised in the degradation of the Extracellular Matrix (ECM) components. Their expression is finely regulated at many levels (transcription, activation, inhibition) while unregulated profiles have been found in many pathological conditions such as cancer, arthritis, atherosclerosis, and inflammatory diseases.
Since the use of endogenous inhibitors is not easy to accomplish, the design and synthesis of new small molecules that could allow the regulation of these proteins is a vanguard in nowadays research of new anticancer drugs, as well as for other therapies.
Some promising MMP inhibitors have been developed by Prof. Rossello’s group in recent years. The best results were achieved with an inhibitor particularly selective for MMP-9 and MMP-12 that was able to arrest human glioblastoma cancer cell invasiveness in models studied.
On the basis of Prof. Rossello's group recent findings, in my Thesis project I was involved in the synthesis of a series of novel MMP inhibitors, derivatives of the previously discovered compounds. The newly synthesised entities have been tested on the principal targets, which are MMP-9 and MMP-12. The assays have been conducted
in vitro on human recombinant MMPs by fluorometric HTS methods, using a
fluorogenic peptide as substrate.
All compounds are more active on MMP-12 than on MMP-9, with a lower inhibitory activity compared to previously achieved results. However, selectivity of the new inhibitors over other MMPs should be further investigated.
In the second part of my Thesis, I carried out the synthesis of two derivatives of an ADAMTS-5 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs) selective inhibitor previously described by Prof. Rossello’s group, [0]. This specific enzyme inhibition has given promising results in the protection against osteoarthritis in mice models studied.
The arylsulfonamidic structure of this inhibitor was slightly modified to improve potency and the synthesis of two new compounds was accomplished. The newly synthesised compounds have been tested for their inhibitory activity on ADAMTS-5 by Prof. Hideaki Nagase’s group (University of Oxford, UK).
1. INTRODUCTION
1.1 MetzincinsTo grow and spread, a tumour needs to encounter a favourable environment, which includes receiving help by extracellular components for its migration in the different districts of our body. To study the responsible agents of this cellular event has always been a main focus in the development of a cure against this pathology. Also, the remodelling activities are implied in other important pathologies such as arthritis, periodontal diseases, atherosclerosis, aneurysms, nephritis, tissue ulcers, and fibrosis. In these circumstances, great importance is to be conferred on the family of the Metzincins and other correlated enzymes that use a metal ion, generally zinc, for their catalytic activity.
These proteinases belong to one of the five major catalytic classes, which are: Aspartic proteinases
Cysteine proteinases Metalloproteinases Serine proteinases Threonine proteinases
The Metalloproteinases one is the largest among these classes and includes the superfamily of the Metzincins, which are characterised by a typical structure feature, the Met-turn. This consists in a tight 1,4-turn that is found directly below the zinc-binding site of these enzymes. The Met-turn is structurally and spatially conserved and always shows a methionine at position 3 that is the reason for the “Metzincins” name.
Fig. 1 The Zinc Metalloproteinases and the Metzincins superfamily 1
In my Thesis work, I focused my studies towards two subfamilies that belong to the Metzincins superfamily (see Figure 1): Matrix MetalloProteinases (MMPs), and A
Disintegrin And Metalloproteinase with Thrombospondin motifs (ADAMTSs).
Both families are deeply involved in tissue remodelling processes, which represent an essential physiologic activity. Unfortunately, these enzymes have been demonstrated to often show altered profiles in many pathological conditions. [1]
1.2 Matrix Metalloproteinases
Matrix Metalloproteinases, or Matrixins, are a family of 24 zinc-dependent endopeptidases homogeneous for structure, function and localisation. They belong to the class of the Metalloproteinases, the most abundant catalytic proteases, involved in the turnover of the connective tissue. Twenty-three of these proteins are found in humans.
MMPs accomplish their remodelling role amongst the extracellular matrix environment, where they can virtually degrade all components.
1
[Fig.1] Murphy, G., The ADAMs: signalling scissors in the tumour microenvironment, Nature
1.2.1 The extracellular matrix
The extracellular matrix (ECM, i.e. basement membrane and interstitial stroma) is the jelly mixture that occupies the extracellular volume of tissues. It is fundamentally composed by water, proteins and polysaccharides, its constituting macromolecules being proteoglycans (PGs) and fibrous proteins (see Figure 2). Each tissue displays a peculiar ensemble of these elements.
Fig. 2 General composition of the ECM 2
The proteoglycans are made of glycosaminoglycan (GAG) chains covalently linked to a protein core (with the exception of Hyaluronan, which is only composed by disaccharidic repeats chains). They are grouped into three main families: small leucine-rich proteoglycans (SLRPs), modular proteoglycans and cell-surface proteoglycans. These elements usually exist in the form of a hydrated gel, which confers high compressive-force resistance on the ECM.
2
[Fig.2] Frantz, C., Stewart, K. M., and Weaver, V. M., The extracellular matrix at a glance, J.
The fibrous proteins mainly found in ECM are collagens, elastins, fibronectins and
laminins.
Collagen constitutes the 30% of the total protein mass of a multi-cellular animal, as it
represents the main structural element of the ECM. Each tissue is usually characterised by a predominant type of collagen. This protein is generally secreted by fibroblasts in a triple-stranded helix and by them organised into sheets and cables that furnish tensile strength, regulation of cell adhesion, direction to tissue development, and support to chemotaxis and migration.
Elastin fibres, which provide recoil after stretching, are proteins highly cross-linked by
their Lys-residues (thanks to the action of the Lysyl Oxidases) and covered by glycoprotein microfibrils.
Fibronectins (FN) are the major directors of the ECM organisation: they can resist to
the several stretches caused by cellular traction forces, can mediate cellular adhesion and functions, and likewise they mediate cell-migration. They are secreted as dimers and present several binding sites (to other fibronectins, collagens, heparins and cell-surface integrin receptors), which the soluble FN uses to anchor the cell-cell-surface. Once fixed to the cell-surface the FN can assemble into longer fibrils. [2]
Laminins are found principally in the basement membrane of ECM. They are trimeric
proteins that contain several binding sites (to other laminin, cell membranes and further ECM components). Their biological importance is shown by their influence on cell differentiation, migration, and adhesion. [3]
For the homeostasis of all these elements, the role of Matrix Metalloproteinases is crucial as well as the counterbalancing action of the Tissue Inhibitors of Metalloproteinases (TIMPs).
1.2.2 MMPs structure
Fig. 3 Schematic structure of Matrix Metalloproteinases 3
3
[Fig.3] Page-McCaw, A., Ewald, A. J., and Werb, Z., Matrix metalloproteinases and the
Table 1 MMPs domains composition: resuming table 4
4
[Tab.1] Nuti, E., Tuccinardi, T., and Rossello, A., Matrix Metalloproteinase Inhibitors: New
Challenges in the Era of Post Broad-Spectrum Inhibitors, Curr. Pharm. Design, (2007), 13:
The family of MMPs mostly shares a common three domain-based structure that consists of a propeptide domain (about 80 residues) at the amino-terminal, a central
catalytic domain (about 160-170 residues) and a hemopexin domain at the
carboxylic-terminal. Two exceptions to this characteristic structure are represented by MMP-7 and MMP-26, the Matrilysins, which do not contain the hemopexin domain. The propeptide domain contains the highly conserved ‘cysteine switch motif’ (Pro-Arg-Cys-Gly-X-Pro-Asp, where “X” can represent any aminoacid) that only lacks in MMP-23. This pro-domain is preceded by a signal peptide domain (about 20 residues) that is removed during translation. Another conserved sequence is contained in the catalytic
domain (His-Glu-X-X-His-X-X-Gly-X-X-His). Its three His together with the Cys of the
‘switch motif’ tetra-coordinate the catalytic zinc ion in a stable, inactive form (zymogen), the active site resulting thus hidden. A hinge (about 60-70 residues) connects the catalytic domain to the hemopexin one.
As shown in Figure 3, membrane-bound MMPs (also called Membrane-Type, MT-MMPs) present additional features that permit their anchoring to the plasma membrane. These are a trans-membrane linker, a trans-membrane domain and a cytoplasmatic domain for MMP-14, MMP-15, MMP-16 and MMP-24. Alternatively, a glycosylphosphatidylinositol anchoring sequence is found right after the trans-membrane linker for MMP-17 and MMP-25.
The Gelatinases, a two-member sub-class of MMPs formed by MMP-2 and MMP-9 (respectively Gelatinase A and B), add to the basic structure described above three repetitions of fibronectin type II, interspersed in the catalytic domain. [4]
An exhaustive overview of each matrixin domains composition can be found in Table 1.
X-ray crystallography nowadays produced very important results so as to get a more precise idea of the 3D structure of the MMPs domains.
The prodomain, which has been studied at least in MMP-1, MMP-2, MMP-3, MMP-9, and MMP-12, generally consists of 3 α-helices connected by loops. Of these loops, the one connecting first and second helices is protease-sensitive, while the loop-sequence following the third helix is the portion that contains part of the conserved ‘cysteine switch motif’. This region is found close to the substrate-binding portion of the catalytic domain. In the proMMPs the cysteine switch motif forms the fourth coordination binding of the zinc ion and so maintains the enzyme in its inactive form due to the steric obstruction of the metal ion.
The catalytic site structure is strictly conserved amongst MMPs and also shows similarity for all Metalloproteinases, leading to difficulties in site-targeted selective inhibitors design. This domain consists of a 5-stranded β-sheet, three α-helices (A, B
and C), and the related connective loops, which contain the conserved “Met-turn” that supports the structure around the catalytic zinc ion. This can be accompanied by another structural zinc ion, plus some calcium ions (generally three).
The three fibronectin repeats, typical of the Gelatinases, are found between the fifth β-strand and the successive α-helix of the catalytic site. Each repeat is constituted by two antiparallel β-sheets and a central short α-helix, stabilized by two disulfide bonds. Finally, the hemopexin domain, which has been fully studied at least in MMP-9, since it promotes this protein homodimerisation, shows a peculiar structure. It has a 4-bladed β-propeller fold with blades disposed thoroidally to a central axis. Each blade consists of a 4-stranded antiparallel β-sheet. This folding is typically found in regions (of also other protein than MMPs) that mediate protein-protein interactions The tertiary structure is given by a disulfide bond that connects blade I to blade IV, which result almost perpendicular to each other. [5]
1.2.3 MMPs mechanism of action
A more in detail study of the catalytic sequence and structure is needed to comprehend MMPs mechanism of action.
The substrate binding cleft (see Figure 4) is represented by the IV strand of the β-sheet, the B helix and the successive loop region of the catalytic domain. In these portions we find a hydrophobic pocket, namely S1’, which confers the substrate-specificity varying amongst the different classes of MMPs in its amminoacidic sequence and depth, so that MMPs are classified on the basis of S1’ depth in shallow (MMP-1, MMP-7), intermediate (MMP-2, MMP-8, MMP-9), and deep (MMP-3, MMP-11, MMP-12, MMP-13, MMP-14) pocket MMPs. Pocket S1’ accommodates the side chain of the substrate that will become the new N-terminus. It is the best known, yet not the only subsite that is present in the catalytic site. The nomenclature generally accepted to identify all of these subsites consists of S accompanied by a number for the sites present on the left side of the zinc ion, while S’ accompanied by a number is used to recognise the subsites on the right of the zinc ion. The binding groups that interact with these sites are consequently named P accompanied by a number and P’ accompanied by a number, respectively. The unprimed sites are the most exposed to solvent. S2 and S2’ are the pockets adjacent to the zinc ion. S2’ and S3’ do participate in the bonding together with S1’, but they are shallower pockets. [6]
Fig. 4 MMPs substrate pockets 5
The catalytic mechanism, after the substrate binding, involves principally the zinc ion, which, in the active form of the enzyme, is coordinated by the three conserved His from the catalytic domain and a water molecule that replaces the Cys coordination of the prodomain, lessened after the proteolytic activation. Also a glutamic acid, the one adjacent to the first coordinating His, is essential (see Figure 5).
The carbonyl group of the peptide bond to be hydrolysed coordinates the zinc, displacing the water molecule. The Glu residue then serves as a base to deprotonate this water molecule, so that it becomes a better nucleophile to attack the scissile bond. Then, the tetrahedral intermediate is formed as an sp3-hybridized gem-diolate.
Now the Glu serves as an acid, and the resulting protonation of the ammonium permits the separation of the two products. The release of the carboxylic group is the speed-limiting step, since it has to be replaced by a new water molecule. After the new solvent coordination, there are repulsing forces that help to remove the side chains from their respective pockets. [5],[6]
Fig. 5 Binding and cleavage of substrate by MMPs 6
5
[Fig.4] Gupta, S. P., and Patil, V., M., Specificity of Binding with Matrix Metalloproteinases, Ex.
S., (2012), 103: 35-56.
6
[fig.5] Tallant, C., Marrero, A., Gomis-Ruth, F. X., Matrix metalloproteinases: Fold and function
1.2.4 MMPs regulation
The action of matrixins can be regulated at three main levels, which are the
expression of the proteins, their activation, since they are produced as zymogens,
and their inhibition promoted by endogenous molecules.
The gene expression is mainly mediated by an AP-1 site that exists near the promoter regions of MMPs genes, although nowadays other upstream regions have been described too. The MMPs genes are generally not expressed constitutively, but in response of external signals mediated by cytokines (such as IL-4, IL-10) and growth factors (such as EGF, bFGF, TGF-α, TGF-β-1) or by the interaction between the cell and the matrix or the adjacent cells. These extracellular signals interact with cell-surface receptors leading to the MAP kinases cascade activation, which eventually causes the AP-1 transcription factor activation. [4]
The zymogen activation, which consists in the propeptide proteolytic cleavage, is then required to obtain active MMPs. These are stored in Golgi apparatus in their inactive form, to be secreted out of the cell where generally the proteolytic activation takes place. Sometimes, localised activation can be obtained in specific zones by starters like plasminogen and urokinase plasminogen activator, which are membrane-associated precursors of plasmin.
Plasmin for example has been reported as an activator of proMMP-1, proMMP-3, proMMP-7, proMMP-9, proMMP-10, and proMMP-13. The activation, though, takes actually place in a multi-step way in the majority of the cases. Initially, a protease cleaves the proMMP in the loop region between the first and the second helix of the prodomain that is exposed to the attack. The removal of this part of the prodomain probably leads to the destabilisation of the entire domain, so that the Cys-zinc coordination lessens. This conducts the partially activated MMP (which is already capable of cleaving some substrates) to be finally processed by the proteolytic cleavage of another MMP already activated.
In vitro experiments with chemicals such as thiol-modifying agents also led to activation
of MMPs, but then required the final intermolecular proteolysis to give completely activated proteins, confirming the mechanism proposed above (see Figure 6). [5]
Fig. 6 Activation of MMPs achieved naturally or chemically. The red circle shows the
loop region exposed to the first cleavage 7
Lastly, activated MMPs are regulated by endogenous inhibitors. Many proteins have been reported to inhibit MMPs, but more efforts are still needed to reveal their interaction mechanisms. However, the main endogenous inhibitors of MMPs have been recognised in TIMPs, which are the Tissue Inhibitors of MetalloProteinases.
Four of these proteins have been identified so far, namely TIMP-1, TIMP-2, TIMP-3 and TIMP-4. They are principally expressed during development and tissue remodelling. Their structure has been revealed thanks to X-ray crystallographic studies. It goes from 21 to 29 kDa, and has been characterised as two domains, the N-terminal domain (~125 AA) and the C-terminal domain (~65 AA). The structure is stabilised by conserved disulfide bonds. TIMPs overall present a wedge shape, which is composed by 10 β-strands (A to J) and 4 α-helices (1 to 4).
Mimicking the backbone contacts of the substrate, TIMPs slot in the active-site cleft of MMPs and expel the water molecule from the catalytic zinc coordination (see Figure 7). This can be achieved thanks to the 4 conserved Cys and the loop region between strand C and D in the N-terminal domain.
7
[Fig.6] Visse, R.,et al., Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases -
Tests so far demonstrated that TIMPs can inhibit all MMPs, but in vivo specificity has shown that TIMP-2 preferably binds to proMMP-2, and TIMP-1 preferably binds proMMP-9 (and for the gelatinases, the interaction has been observed at the hemopexin domain instead of the catalytic one), while other MMPs are inhibited in their active form. TIMP-3, on the other hand, has shown peculiar specificity. It has been recognised as a strong inhibitor of ADAM-17, ADAM-10, ADAM-12 and aggrecanases (ADAMTS-4, ADAMTS-5).
Fig. 7 PDB structure of TIMP-2, catalytic domain of MT1-MMP and their interaction 8
Studies for direct applications of TIMPs as MMPs inhibitors (both wild types and engineered types) are being conducted with better results than the small molecules tested so far. However, other biological functions have been recognised and more are to be discovered for TIMPs, so the need for small molecules with new inhibition approaches will not cease. [5]
8
[Fig.7] Visse, R.,et al., Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases -
1.2.5 Physiological and pathological activity of MMPs
The reducing role of ECM degraders is nowadays outdated for the MMPs family. Their proteolytic activity is now considered as a major responsible for many physiological
processes. MMPs action can lead to cell migration during reproduction, growth and
development of tissues, permits leukocyte mobilization and is strongly involved during inflammation and wound healing. These activities are not only achieved by space-creation and tissue architecture control. MMPs proteolysis in fact can produce specific substrate-cleavage fragments that generally show independent biological functions. Already biologically active precursors can be cleaved in still active fragments that mediate completely different effects. These elements bring the MMPs role to a wide range of possibilities in physiological processes. [7]
However, increased MMPs activity has been observed in a variety of pathological
conditions as well, including cancer, inflammation, infective diseases, neurodegenerative diseases and vascular diseases, while loss of MMPs generally leads to development problems of different entities depending on the specific MMP involved.
In cancer, for example, tumour cells overexpress MMPs in order to degrade the basement membrane and invade the surrounding tissues. They are indispensable for intravasation and extravasation phenomena. Previous to the migration process, MMPs have also been implicated in the epithelial to mesenchymal transition (EMT), a fundamental step in the metastasis, with which tumour cells acquire the migratory characteristics. Proposed mechanisms for this action include growth factor activation and cell-cell adhesion cleavage.
A variety of cytokines, chemokines and their respective receptors have also been found to undergo MMPs mediated cleavage. This fact, united to growth factor and surface receptors bioavailability modulation can regulate cell proliferation. Studies have further demonstrated that MMPs are involved in the angiogenic switch not only for its initiation but also for the maintenance of the growing vascular bed.
The regulation of these pathological processes has been underlined for MMPs, which are nowadays recognised as both positive and negative regulators. [8]
1.3 MMP-9
Amongst MMPs, MMP-9 represents a particularly relevant target to be studied because its over-expression is widely observed in pathological models, especially for cancer and osteoarthritis.
1.3.1 MMP-9 structure and activity
Fig. 8 Gelatinase B, X-ray crystallography of prodomain, catalytic domain and three
Fn II (fibronectin type II) domains 9
Matrix metalloproteinase-9 (92kDa), or gelatinase B, is one of the most studied members of the MMPs family. Its structure (see Figure 8) has been fully revealed and has been described as the one typical of the gelatinases, which presents three fibronectin type II repeats in addition to the general domains structure of the MMPs. For MMP-9 however, the linking region connecting the catalytic domain to the hemopexin one has been well described and has shown to be different from other MMPs. It counts approximately 64 AA (while for other MMPs it is usually ~10 AA) so that it has sometimes been considered as an independent domain. The high Pro content provides the linker homology with collagen type V. Also, it has been found to be widely glycosilated, approximately 85% O-linked and 15% N-linked. This suggests that a function may be provided by this domain, which is still unknown today. However, the high flexibility of this linker must be implied at least in the correct orientation of the hemopexin domain and in its binding to TIMP-1, because deletion of this region in recombinant MMP-9 studies led to decreased affinity of the protein for the inhibitor. [9]
9
[Fig.8] Elkins, P.A., Ho, Y.S., Smith, W.W., Janson, C.A., D'Alessio, K.J., McQueney, M.S., Cummings, M.D., Romanic, A.M., Structure of the C-terminally truncated human ProMMP9, a
MMP-9 presents the proteolytic profile of the gelatinases, which means that it is able to degrade gelatins, denatured collagens, and a great number of ECM molecules including native type IV, V and XI collagens, laminin and aggrecan core protein. However, it does not have a collagenases-like proteolytic profile as it has been reported for MMP-2 (Gelatinase A). Other substrates comprehend chemotactic molecules, latent growth factors and growth factor binding proteins, cell surface receptors, adhesion molecules, other proteinases and even intracellular substances. [10]
1.3.2 MMP-9 homodimerisation
Recent studies have completely revealed the homodimerisation pattern of MMP-9 (215kDa). This occurs at the hemopexin domain, which binds with the IV blade and some portions of the III blade of the β-propeller. However, the dimeric structure of this domain results asymmetric, so that an hemopexin domain A can be distinguished from a different hemopexin domain B (see Figure 9).
Fig. 9 Left, dimeric structure of hemopexin domains. Right, interacting residues detail 10
10
[Fig.9] Cha, H., Kopetzki, E., Huber, R., Lanzendorfer, M., Brandstetter, H., Structural basis of
Although first studies hypothesised a covalent bond in the dimer formation due to its reduction sensitiveness, today the hydrophobic nature of the interaction is recognised except for a salt-bridge between the C-terminal of one chain (A) and the R677 of the other (B). The reduction probably destabilises the whole hemopexin domain structure by cleaving the disulfide bond between blade I and IV. Even if the two interacting blades are almost parallel, the β-sheet does not extend.
As shown in Figure 9, on the left, the axes of the two hemopexin domains present an anti-parallel orientation, so that the top of PEX-A looks up, while the top of PEX-B looks down. Also, the free form of the hemopexin domain contains a sodium ion, which here is only maintained in PEX-A. The ion is coordinated with Asp522 (blade I), Asp568 (II) and His622 (IV), while the fourth coordination is given by Gly615 (III) in the monomer. The dimerisation occurs precisely at this level with what is called the glycine switch. The coordination of the Gly, which gave high stability and rigidity to the structure, is substituted by a water molecule. This change provides the III blade with higher movement possibility and promotes the dimerisation. [11]
1.4 MMP-12
Fig. 10 Crystal structure of full-length human MMP-12 11
11
[Fig.10] Bertini, I., Calderone, V., Fragai, M., Jaiswal, R., Luchinat, C., Melikian, M., Mylonas, E., Svergun, D.I., Evidence of reciprocal reorientation of the catalytic and hemopexin-like
Little is known about this specific MMP compared to some other members of the family. Also known as Macrophage Metalloelastase (54kDa), it was first identified in smokers' alveolar macrophages. Its structure (see Figure 10) is the one typical of MMPs, and consists of three main domains: a prodomain (preceded by a signal peptide), a catalytic domain (which contains the catalytic zinc, the structural zinc and three calcium ions) connected to a hemopexin domain by a linker region (16 AA). The tertiary structure of these domains reflects the general description already given for MMPs. The hemopexin domain can be removed by maturation processes to give a final protein of 22kDa. What distinguishes this proteinase from the other members of the family so far is the fact that it has been found to be expressed only in macrophage cells, hypertrophic chondrocytes and osteoclasts, while no expression has been detected in normal tissues.
MMP-12 has a broad substrate specificity. It is able to degrade elastin, type IV collagen, laminin, fibronectin, entactin, vitronectin, and heparan/chondroitin sulfates. MMP-12 contribution has been reported for different processes. For example, it can degrade elastic fibres in the atherosclerosis pathogenesis, while in inflammatory bowel diseases it supports the basement membrane degradation. Its role is significant in airway inflammation and remodelling, due to the release of this enzyme by macrophages, which are recognised to over-express proteinases for example in Chronic Obstructive Pulmonary Disease (COPD).
Also, MMP-12 has been shown to be fundamental for macrophage migration in other tissues. This probably occurs by the degradation of the basement membrane. [12]
1.5 Cells migration
One of the most interesting activity of MMPs is to be found in their contribution to cellular migration. This is a complex process (see Figure 13) that involves mainly ECM components as well as intracellular ones. Its start is due to a series of signalling events, the migratory signals. These signals can derive for example from the ECM degradation products.
To these signals a cell can react with non-directed migration (chemokinesis) or following the signal inducers concentration gradient (chemotaxis). The first reactions of the cells consist in membrane polarisation and in the increase of the membrane front phenomena. These are mainly lamellipodia, which are broad, sheet-like structures the membrane front can assume, and filopodia, which are more thin and cylindrical, needle-like structures and can lead to invasion processes. The formation of these structures proceeds by the assembly of actin core structures, followed by the stockpile
of proteolytic elements that can process not only the ECM degradation but also the cell-cell/cell-matrix interactions disruption. [10]
Fig. 11 Schematic steps of cell migration 12
The cell migration phenomenon is correlated to many physiological processes (e.g. macrophage migration, as well as tissue remodelling), but at the same time can lead to drastic pathological conditions, one above all the tumour metastasis.
It may seem intuitive to correlate the degradation of the ECM and of the cell-cell adhesion means to the possibility for a cell to migrate from a tissue to another. Yet, the precise mechanism that describes how this actually happens still needs to be fully studied and understood.
In this sense, great importance is to be conferred on the research of Dr. Antoine Dufour's group, which was the first to correlate the MMP-9 hemopexin domain to the cell migration phenomenon (proteolytic activity resulting not relevant). This discovery led the group to further investigate the role of this matrixin in the process, and finally a study in 2010, [13], proved that the homodimerisation of MMP-9 is indispensable for enhanced cell migration given by this protein.
Mutant proteins in which the hemopexin domain was replaced with MMP-2 corresponding domain (not able to form homodimers) were studied in cell cultures. The migration activity of these mutants was compared to that of cells that contained wild type MMP-9. MMP-9 mediated enhancement of cell migration failed in the mutant cells, demonstrating the role of homodimerisation in this process. TIMP-1 binding to MMP-9
12
[Fig.11] Bjorklund, M., Koivunen, E., Gelatinase-mediated migration and invasion of cancer
was also correlated with similar tests to reduced cell migration by its inhibition of homodimers formation.
Moreover, additional studies moved from the MMP-9 possibility of binding membrane glycoprotein CD44, which is involved in the cellular migration processes. Blade I of the hemopexin domain was demonstrated to be responsible of this interaction. Comparing the migration process between cells containing CD44 and wild MMP-9 and cells only containing wild MMP-9 also led to observe a strongly reduced phenomenon in the second case. So the MMP-9/CD44 interaction is also responsible of the tumour invasiveness. [13]
Further studies of Dr. Dufour's group confirmed the hypotheses previously presented. A series of pyrimidinonic compounds were synthesised and showed specificity for MMP-9, which they bound at the hemopexin domain. The catalytic activity was not modified, but the proteins were no longer able to homodimerise. In tumour model cells expressing MMP-9 treated with these inhibitors, retarded tumour growth was observed together with metastasis inhibition. [14]
1.6 ADAMTS
The A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) family is a recently discovered family of enzymes closely related to the ADAM family.
ADAMTSs maintain the general structure of the Metzincins (characterised by the Met-turn), which consists of a prodomain at the N-terminal followed by a highly conserved
catalytic domain that contains at least one zinc ion. At the C-terminal though the
structure starts to differ (see figure 11). Right after the catalytic domain in ADAMTSs, a
disintegrin-like domain is found, which presents a 14 aminoacids loop that is
supposed to confer on these proteins the capability of interacting with integrins. Following this domain, a thrombospondin type I motif, a cysteine-rich domain and a cytoplasmatic tail preceded by a spacer complete the common structure of all ADAMTSs. A varying number of thrombospondin repeats as well as one or two additional specific domains, depending on the specific enzyme, are then found at the C-terminal. The thrombospondin repetitions are fundamental for substrate recognition and cleavage (they are believed to function as a binding domain for sulfated glycosaminoglycans present on proteoglycans) and they replace the trans-membrane sequence, which is generally found in the closely related ADAMs, so that ADAMTS are primarily secreted proteins.
Fig. 12 Comparison of closely related Metzincins 13
(SVMP=snake venom metalloproteinase)
19 ADAMTSs have been reported in humans, which share common activities such as aggrecanases, or better ‘hyalectanase’ (degraders of hyalectans: aggrecan, versican and brevican), procollagen N-proteinases and vonWillebrand factor (vWF) cleaving proteinases.
The characteristics of the prodomain and of the catalytic domain, as well as the general mechanism of protein activation (mostly intracellular), catalytic action and substrate recognition, can be considered quite similar to those seen for MMPs. However, the presence of the cysteine-rich domain, which is bound to the disintegrin-like domain by disulfide bonds, confers on the protein a characteristic “C” shape that exposes an hypervariable region (probably implied in substrate recognition) comprised in the cysteine-rich sequence. This shape is stabilised by both the disulfide bonds and the calcium ions that are present in the disintegrin-like domain.
Considerable attention has been reserved to the ADAMTS family based on the different roles exerted in pathological conditions. ADAMTS-13 autoimmune inhibition of vonWillebrand factor cleavage causes thrombotic thrombocytopenic purpura, while many disorders in ADAMTS activities have been related to arthritis and other connective tissue diseases. Recently, ADAMTSs have also been studied for their
13
[Fig.12] Edwards, D. R., Handsley, M. M., Pennington, C. J., The ADAM metalloproteinases,
contribution to neurodegenerative disorders such as Alzheimer, Down syndrome and cerebral ischemia. On the other side, positive effects such as antiangiogenic properties have been recognised to some ADAMTSs. [1]
1.7 ADAMTS-5
Fig. 13 Crystal structure of ADAMTS-5 in complex with an inhibitor 14
A Disintegrin and Metalloproteinase with Thrombospondin motifs-5 (see Figure 12), or aggrecanase-2, has nowadays been recognised as a major target in osteoarthritis diseases.
The role of ADAMTS-5, in fact, is studied in mice due to its cleavage of aggrecan (cleavage site: Glu373↓374Ala), but also type II collagen, and other joint tissue substrates responsible of cartilage degradation, osteophyte formation and subchondral bone sclerosis. Mutant mice that lacked ADAMTS-5 showed protection against cartilage aggrecan loss in osteoarthritis models.
14
[Fig.13] Mosyak, L., Georgiadis, K., Shane, T., Svenson, K., Hebert, T., McDonagh, T., Mackie, S., Olland, S., Lin, L., Zhong, X., Kriz, R., Reifenberg, E.L., Collins-Racie, L.A., Corcoran, C., Freeman, B., Zollner, R., Marvell, T., Vera, M., Sum, P.E., Lavallie, E.R., Stahl, M., Somers, W., Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4
These activities classify ADAMTS-5 as the major aggrecan degrader together with ADAMTS-4, whose role however is not as fundamental as ADAMTS-5 in mice models studied, and both enzymes as effective ECM degrader together with the closely related MMP family. [15]
1. 8 Metzincins inhibitors
Misregulation of Metzincins activity is a major feature in many serious pathologies. In particular, the overexpression of these enzymes can lead to massive tissue degradation, not only dangerous in itself, but also for the promotion of cellular events such as tumour invasiveness and inflammation spreading.
The strong involvement of Metzincins in these critical diseases has been repeatedly confirmed by gene-direct studies and animal models so that many efforts have been invested in the last 30 years by pharmaceutical researchers for the development of Metzincins inhibitors.
1.8.1 MMPs inhibitors
Three generations of MMPs inhibitors (MMPI) have been developed in the last 30 years, following the evolution in these proteinases knowledge.
The first one (which was created approximately from 1995 to 1999) is nowadays considered as almost obsolete. The members of this generation in fact, such as
Batimastat, Marimastat and Ilomastat (see Figure 14), were small molecules whose
structure was based on peptidomimesis of the endogenous substrates, united with a zinc-binding group (ZBG). This binding group was generally a hydroxamate. This moiety selection was initially due to the patent covering restrictions existing in those years for ACE inhibitors structures. However, hydroxamates in their anionic form actually represent the most potent ZBG for MMPs, since their substitution leads to potency loss of the studied inhibitors. The zinc chelation mediated by hydroxamates proceeds with a bidentate structure, in which each oxygen is located at an optimal distance to the catalytic zinc. Also, hydroxamates effectiveness is potentiated by the hydrongen bonds formed between its heteroatoms and some residues conserved for all MMPs (reason why this moiety suffers poor selectivity).
The principal drawbacks of first generation inhibitors were the total lack of selectivity amongst the whole family of the MMPs united to poor in vivo availability (peptidic moieties are in fact rapidly hydrolysed). The development pathway of these compounds was sharply interrupted in clinical trials due to the major side effects caused by the inhibition of some MMPs, which actually provided positive indispensible effects even in pathologies related to these enzymes. The most important syndrome derived from the use of broad spectrum MMPI is MSS that is MusculoSkeletal Syndrome and has nowadays been ascribed to MMP-1 and MMP-14 inhibition. [16]
Starting from the failure of broad-spectrum peptidomimetic inhibitors, the second generation of MMPI was developed (approximately from 1999 to 2003). Sulfonamidic moieties were successfully used to replace the liable amidic bonds, increasing at the same time the binding strength of the inhibitors to MMPs. One of the oxygens of the sulfonamidic moiety in fact, is capable of forming two hydrogen bonds with the amide nitrogen of a Leu and an Ala residues.
Also, the first steps to gain in selectivity were made in those years by the starting of crystallographic studies of the principal interaction pocket, which is S1’. The differences in depth of this cavity amongst the MMPs family were acknowledged for the first time. The milestone of this generation is represented by compound CGS-27023A (see
Figure 15), one of the first sulfonamidic inhibitors, which is a potent MMP-12 inhibitor
(moderately selective over some other MMPs) and is now in clinical trials phase study. Its action could be applied for COPD treatment.
A direct derivative of this compound is represented by the well-known Prinomastat (see Figure 15), another promising MMPI, which has shown profound inhibition of tumour growth in human glioma tumour models. Prinomastat presents a moderate selectivity for MMP-2, MMP-3, MMP-9, MMP-13 and MMP-14.
However, the improvements made in selectivity by second generation MMPI were not enough and important side effects such as MSS are still observed with these compounds. [17]
It was only with third generation of MMPI that a more mature knowledge of the different MMPs implied in each pathology permitted to recognise TARGET MMPs against ANTITARGET MMPs. For example, MMP-12 overexpression by alveolar macrophages is involved in various pulmonary pathologies, including COPD. The overexpression of MMP-3 and MMP-13 can lead to dermal ulcers, while MMP-1, MMP-2, MMP-9 and MMP-14 are required for correct wound healing. MMP-13 represents at the same time an important osteoarthritis (OA) target, for the uncontrolled cleavage of type II collagen by this MMP observed in OA disease. MMPs also represent one of the most promising targets for cancer treatment. MT1-MMP is recognised as the primary mediator of proteolytic events on the cancer cells surface. MMP-1, MMP-9 and MMP-13 are involved in progression and invasion of some types of tumour, and MMP-9 is also associated to the release of proangiogenic factors. [17]
Third generation MMPI were thus developed aiming to achieve ki values ratio of
inhibition between antitarget/target MMPs higher than a 103 factor on isolated enzymes. These MMPI show various inhibition strategies and can be classified as follows.
Arylsulfonamidic hydroxamate-based MMPI
Conveying all the efforts derived from previous generations of MMPI, a general effective scaffold was detected (see Figure 16), modifying which the inhibitors selectivity can be studied and improved even for single MMP-selectivity goals.
The structure presents the hydroxamate moiety as ZBG, the sulfonamide scaffold with an arylic substitution (P1’) on sulfonyl group to increase lipophilicity, a substituent (P2’) on amide group and the proper spacer (P1) between the sulphonamide and the ZBG. Modifications of P1’ have been principally carried out to devoid inhibition of MMP-1, correlated to the most relevant side effects.
R S N O O R1 R2CONHOH P1' ZBG basic skeleton P2' P1 N H N OH O SO2 O A H N O H O S N O O N O HO B
Fig. 16 Basic structure of arylsulfonamidic hydroxamate-based MMPI, and two
Structure activity relationships of the synthesised compounds united to crystallographic studies results have permitted to deeper exploit the specific interaction of the different substituents tested.
For example, the presence of a biphenyl substitution in P1’ (by Rossello et al., [17]) as for compound A in Figure 16 confers selectivity for MMP-2 if united to the isopropyloxy moiety in P2’. This compound (A, Figure 16) is a promising antiangiogenic agent. Electron withdrawing substituents in para position of the biphenyl ring give selectivity towards MMP-3 and MMP-13, because of improved aryl-aryl stacking interactions (with His201 and Tyr223), while a methylthio substituent in this position confers potent inhibitory activity against MMP-13. [17]
While the P1’ chain elongation, as explained above, favours MMP-13 deep channel targeting, it has been found that restricting the rotation of the phenyl group, for example by introducing a third condensed ring, permits to achieve selectivity for the less flexible MMP-12 S1’ pocket.
Compound B (Figure 16) synthesised by Whitlock et al. [17] uses a reverse sulfonamidic moiety, which increases the steric hindrance near the ZBG. Thus, the compound results selective for MMP-3 due to the characteristic large opening of this enzyme S1’ pocket.
Resuming the three example compounds features, specific inhibitors for MMP-13 have been designed with elongated P1’ chains to probe deep into this enzyme S1’ pocket, while rigid backbones improve MMP-12 targeting. Moreover, emphasis on the opening of S1’ pocket rather than on its interior, has led to MMP-3 selectivity development. Other arylsulfonamidic hydroxamate-based MMPI are still studied since these class of compounds has achieved good results so far. [18]
Non hydroxamate MMPI
Potent carboxylate-based compounds have been developed in parallel with hydroxamate MMPI.
As an example, Weyth researchers conducted a study in 2005 [17] among a series of carboxylate compounds, which aimed to achieve selectivity for MMP-13 over MMP-2, a difficult result since the two enzymes present high homologous S1’ pockets. However, crystallographic studies indicated that MMP-2 loop region is two aminoacids shorter than MMP-13 analogue region, so that the Tyr229 of MMP-2 could collide with specific substituents in P1’ portion. This small difference permitted to select compound C (see
Figure 17) amongst a variety of 3,4,5-trisubstituted benzofuran 2-carboxamides
Fig. 17 Wyeth carboxylate-based MMP-13 selective inhibitor
This inhibitor retained potency on MMP-13 (IC50 = 2.3 nM) while gaining more than
700-fold selectivity over MMP-2, due to the displacement of the benzofuran system from planarity, which directs the 4-methoxy substituent towards the Tyr229 of MMP-2 S1’ pocket causing steric hindrance for the interaction.
Also the MMPI synthesised in this Thesis possess a sulfonamidic carboxylate-based scaffold, as to testify the still spreading relevance of this class of compounds. [4]
Novel ZBGs
The high potency of hydroxamate ZBG of the MMPI is unfortunately accompanied by low metabolic stability, since this moiety is highly subject to hydrolysis, reduction and glucuronidation so that hydroxamate-based inhibitors are rapidly eliminated in vivo. Many efforts have been invested over the years in the study of alternative ZBGs and a multitude of these have been described by recent literature. The most representative ones are illustrated in Figure 18 with the so far purposed coordination mechanism to the zinc ion.
Fig. 18 Binding modes of some novel ZBGs to Zn2+
ion 15
Firstly, a series of isosteres, such as hydrazide and sulfonylhydrazide, have been investigated for the hydroxamate-similar bidentate chelation. Substitution of Ilomastat (Figure 14) ZBG with these isosteric analogues, for example, led to considerably less potent compounds. However, a more in depth study of these ZBGs advantages has indicated that they can be derivatised to probe both the primed and the unprimed side pockets of the active site. New promising compounds that carry these ZBGs have then been developed.
N-hydroxyurea ZBG has also been studied to improve oral bioavailability of MMPI, but
the formation of an intramolecular hydrogen bond significantly reduces its capability of coordinating the zinc ion (which is only coordinated by the oxydrylic oxygen). Unsuccessful substitutions on the aminic N have also been evaluated.
Squaric acid based inhibitors were developed with promising increases in the potency
of the inhibition. The binding mode of this ZBG, if further investigated, could provide a means to uniquely position inhibitors into the substrate-binding pockets, with possible fruitful results.
A new phosphorous-based ZBG is carbamoyl phosphonate, which coordinates zinc through one oxygen of the phosphonate and the oxygen of the carbonyl in α-position to the phosphonate, thus forming a 5 membered chelation ring. Extra hydrogen bonds formed by the amidic portion could improve the inhibition activity. The negative charge of these compounds also prevents cell penetration and restricts these MMPI to extracellular action. Several inhibitors carrying this ZBG are currently being evaluated both in vitro and in vivo, showing remarkable potency of inhibition.
15
[Fig.18] Jacobsen, J. A., Jourden, J. L. M, Miller, M. T., Cohen, S. M., To bind zinc or not to
bind zinc: An examination of innovative approaches to improved metalloproteinase inhibition,
Novel nitrogen-based ZBGs, ranging from carboxylated pyrimidines to small aza-crowns, have been firstly introduced by Jacobsen et al. in 2006. Successively, other similar types of ZBGs have been investigated such as oxazolines, which direct the substituents in position 2 towards the S1’ pocket. The most studied nitrogen-based ZBGs are those that present a pyrimidintrione core. These ZBGs form additional hydrogen bonds with the active site peptide backbone and the derivatisation at position 5 provides access to target S1’ and S2’ pockets. Many compounds carrying this ZBG have been recently optimised for the development of anti-OA drugs.
Finally, heterocyclic bidentate chelators have been introduced by Puerta et al. [18], who also provided for a bioinorganic model complex able to mimic the MMPs active site zinc binding mode. Two examples of these ZBGs (1,2-HOPO and 1,2-HOPTO) are reported in Figure 18. These ZBGs show a rigid structure that should provide tighter zinc-binding, and are potentially more stable in vivo.
A better understanding of each ZBG role in generating selectivity towards specific MMPs may provide an additional means to obtain new MMPI. [18]
MMPI without ZBG
These compounds have been discovered by High Throughput Screening (HTS), and are fundamentally allosteric non competitive inhibitors, which show increased inhibition activity if combined with MMPI that bear ZBGs (a possible strategy also explored). The absence of zinc coordination permits the tailoring of the compounds into the depth of S1’ pocket. As expected, selective MMP-13 and MMP-12 inhibitors are principally found, since these enzymes S1’ pocket is a long, hydrophobic, open channel.
However, the real mechanism of inhibition of these MMPI has been revealed by crystallographic structure of MMP-MMPI complexes. Not only the inhibitors bind the bottom of S1’ cavity of MMP-13 but they also succeed to protrude in an additional side pocket, which is gained by a change in the conformation of the enzyme. The MMP-13 conformation, in fact, is flexible. The loop in the S1’ pocket shows low energetic costs in the stabilisation of a specific conformation that is here given by the rotation of certain glycine residues. These residues are not conserved in the MMPs family. The specific moieties of these inhibitors are capable of forming hydrogen bonds, which finally confer rigidity to the inhibitor-binding conformation while impeding protein-substrate interaction. [16]
Figure 19 represents the structure of a compound with no ZBG developed by Johnson
et al. [18] with an IC50 value of 0.67 nM for MMP-13 over other MMPs (> of 100 µM,
in OA rat models (68% dose-dependent reduction of cartilage lesion area) with good oral bioavailability. [18]
Fig. 19 One amongst the best MMP-13 selective inhibitors
Dublanchet et al. [16] have reported a new class of MMP-12 micromolar inhibitors in 2005. Selective inhibitors have been discovered by HTS also for MMP-8 (in 2009, [16]), which showed a S1’ side pocket similar to that observed for MMP-13. Also MMP-3 presents flexibility in the loop region of S1’, even if it occurs in a different portion. Strictly structural selectivity given by highly specific interaction is required for this class of MMPI, while possible interaction in vivo with other proteins that show similar deep cavities cannot be excluded. [16], [4]
Mechanism-based MMPI
This recent class of compounds was started by Mobashery et al. results in 2000 [18]. The research group synthesised the first of these MMPI (see Figure 20), which is capable of covalently bonding MMPs, and shows selectivity for MMP-2 and MMP-9. The binding is not fully irreversible, but the catalytic turnover is strongly reduced due to the low dissociation constant of the slow binding kinetic of SB-3CT. The inhibitor thiirane moiety is activated by the zinc coordination, then a glutamic residue attacks the ring, which opens, and forms an ester with the inhibitor. Activity is lost if the thiirane moiety is replaced by an epoxide. [18]
Fig. 20 First mechanism–based MMPI by Mobashery et al.
In vivo tests of the described compound have given promising results against
metastases and stroke neuronal damages. However, the metabolism of this compound is so fast that it has been hypotesised that the action observed is actually produced by
its metabolites. This stopped the pathway that should have led SB-3CT to clinical trials while its potent metabolites and other derivatives are now being studied. [18]
Mechanism-based inhibitors are still at their early stages, but may provide the trigger for a new approach to selective MMPI drug discovery.
Despite the many achievements described above, today only two old drugs are actually sold as MMPI: the doxycycline, a tetracycline (developed by Pfizer Inc. in the early 1960s) used for periodontal diseases, and glucosamine sulphate (firstly synthesised in 1876 by Georg Ledderhose) for OA. This situation highlights the still limited features of existing MMPI, though should not discourage from continuing the promising efforts invested so far. As highlighted by some of the above examples, MMPI drug discovery progresses together with the growth of MMPs structures and properties knowledge. [4]
1.8.2 ADAMTS inhibitors
The design and synthesis of ADAMTS inhibitors, unlike MMPI, are still at their earliest stages. The implication of these enzymes, particularly ADAMTS-5 and ADAMTS-4, in osteoarthritis, only recently discovered, inspired drug discovery studies of small-molecules that would control their aggrecanolytic activity.
In 2001, a structure-based approach by Yao et al. [19] converted some lead hydroxamate compounds previously studied as MMP-8 inhibitors (another enzyme that shows aggrecanolytic activity) in order to obtain a hybrid inhibitor that retained activity against 8 and gained activity against aggrecanases while sparing 1, MMP-2 and MMP-9.
This result was achieved by the introduction of a Tyr residue in P1’ position of the peptidic hydroxamate starting scaffold and was improved by the shift of the pseudotyrosine hydroxyl group from para to meta position. Also, for P2’ position a rigid structure was exploited, which increased potency and selectivity over MMP-8. Minor alterations of P1 side chain had great impact in the selectivity too. Three example compounds from this study (54, 55 and 56) and their inhibition activity evaluations are reported in Figure 21.
Fig. 21 Compounds 54, 55 and 56 synthesised by Yao et al. in 2001 16
The first study for the development of non hydroxamate ADAMTS-5 inhibitors that spares ADAMTS-4 has been reported in 2007 by researchers from Wyeth [19]. The identified scaffolds derived from high throughput screening studies. Two example inhibitors (57 and 58) are reported in Figure 22. However, as already seen for MMPI, even if these compounds succeed in sparing other targets, a significant decrease (from nano to micromolar IC50) in their affinity for ADAMTS-5 is also observed.
The same Wyeth has recently advanced an ADAMTS-4 and ADAMTS-5 inhibitor (AGG-523, structure under patent protection) to Phase I clinical trials for the treatment of OA. [19]
16
[Fig.21] Georgiadis, D., Yiotakis, A., Specific targeting of metzincin family members with
small-molecule inhibitors: Progress toward a multifarious challenge, Bioorg. Med. Chem.,
Fig. 22 Compounds 57 and 58 synthesised by Wyeth in 2007 17
17
[Fig.22] Georgiadis, D., Yiotakis, A., Specific targeting of metzincin family members with
small-molecule inhibitors: Progress toward a multifarious challenge, Bioorg. Med. Chem.,
2. INTRODUCTION TO THE EXPERIMENTAL PART
2.1 Design and synthesis of MMP inhibitorsMatrix Metalloproteinases (MMPs) are a family of enzymes that are attracting growing interest as therapeutic targets. They are proteases whose fundamental role has been recognised in the degradation of the Extracellular Matrix (ECM) components. Their expression is finely regulated at many levels (transcription, activation, inhibition) while unregulated profiles have been found in many pathological conditions such as cancer, arthritis, atherosclerosis, and inflammatory diseases.
Since the use of endogenous inhibitors is not easy to accomplish, the design and synthesis of new small molecules that could allow the regulation of these proteins is a vanguard in nowadays research of new anticancer drugs, as well as for other therapies.
Protein homodimers play important roles in physiological and pathological processes, including cancer metastasis. Some mechanisms of homo- and/or hetero-dimerisation have been described for MMPs, [20]. In particular, the natural homodimerisation of MMP-9 has been recently correlated with high migration rate of aggressive cancer cells. This effect appears to be related to a non-covalent dimerisation of MMP-9 via the hemopexin domain (PEX) with subsequent heterodimerisation with CD44 in a MMP-9-CD44-EGFR signaling pathway, [21]. Therefore small molecules that promote an alternative MMP-9 dimerisation mode, could restrain PEX mediated natural homodimerisation and reduce invasiveness and migration of cancer cells. In particular, an unnatural chemically induced MMP dimerisation could impair the natural PEX domain dimerisation.
An artificial protein dimerisation has been observed by X-ray crystallographic studies performed on MMP inhibitors developed by Prof. Rossello’s group in complex with some MMPs (particularly MMP-9 and MMP-12) [22]. The unnatural homodimerisation was induced by bi-functional ligands obtained by linking two identical heads (MMP binding moieties) with a proper spacer.
The best biological results were obtained with a dimeric dicarboxylate inhibitor (1,
Figure 23), a nanomolar inhibitor of MMP-9 and MMP-12, which was able to arrest
Fig. 23 Dimeric dicarboxylate inhibitor 1
On the basis of these recent findings, in my Thesis project I was involved in the synthesis of a novel series of dimeric carboxylate MMP inhibitors and their monomeric analogues. In particular, I carried out the synthesis of compounds 2-4 (see Figure 24), analogues of 1 with different substituents in the sulfonamide moiety (P1’ group). The substitution of the biphenyl with a para-bromo, para-iodo and 4-(4’-chlorobenzyloxy)-biphenyl substituent in P1’ should potentially increase the activity of the dimers against the target MMPs (MMP-9 and MMP-12) or at least improve their selectivity profile over the other MMPs.
Fig. 24 Derivatives of 1 synthesised in this Thesis
Similarly, I also synthesised monomeric compounds 5 and 6 as analogues of 7 (see
Figure 25), the previously described monomer of 1, in order to prove the effect of the
new P1’ substituents in the mono-carboxylate inhibitor. Finally, a fluorescent-labelled analogue of 7, compound 8 (Figure 25), was prepared with the aim of facilitating further studies on the activity of 7 in cells.
Fig. 25 Compound 7 and its derivatives 5, 6 and 8
For the synthesis of the dimeric compounds 3 and 4, the route described in Scheme 1 was followed. Commercially available para-bromo or para-iodobenzenesulfonyl chloride were respectively converted in sulfonamides 9a,b by reaction with D-valine in water and dioxane in the presence of triethylamine. (R)-Sulfonamides 9a and 9b were then protected on the carboxylic moiety as tert-butyl esters (R)-10a,b by using N,N-dimethylformamide-di-tert-butyl acetal. A Mitsunobu condensation of sulfonamides
10a,b with N-Boc protected ethanolamine 11 (prepared by treatment of ethanolamine
with di-tert-butyl-dicarbonate at 0 °C) in the presence of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine gave the tert-butyl esters (R)-12a,b. A Pd-catalyzed Suzuki coupling of commercially available 4-(4’-chlorobenzyloxy)-phenylboronic acid with para-bromo ester 12a in the presence of K3PO4 afforded (R)-tert-butyl ester 13. Tert-butyl esters 14b and 14c were obtained by selective deprotection of the amine
group of compounds 12b and 13 with trifluoroacetic acid in controlled conditions. Then one equivalent of compound 15, a di-NHS (N-hydroxy succinimido) ester activated linker already present in Prof. Rossello’s laboratory, was added to a DMF solution of two equivalents of the monomeric trifluoroacetate salts 14b,c in the presence of N,N-diisopropylethylamine (DIPEA) to give the tert-butyl diesters 16b and 16c. The deprotection in acid conditions of these diesters led to final dicarboxylates 3 and 4.
Scheme 2 shows the synthesis of dimeric compound 2, which was obtained in a
different way. Compound 12a, obtained as described above, was deprotected in more drastic conditions both on the amine and on the carboxylic moieties, using trifluoroacetic acid at RT conditions. The direct dimerisation of the carboxylic acid 17 was attempted in conditions similar to those utilised for the corresponding esters as seen in Scheme 1. The dicarboxylic final compound 2 was obtained with a lower yield (22.2%) compared to its analogues 3 and 4.
Scheme 2. Synthesis of dimeric compound 2
The synthesis of monomers 5 and 6 (reported in Scheme 3) started from compounds
14b and 14c, synthesised as described above. The ammonic groups of 14b,c were
acylated by reaction with benzoyl chloride in basic conditions to give compounds
18b,c. Acid cleavage of esters 18b,c yielded the corresponding carboxylic acids 5 and 6.
Scheme 3. Synthesis of monomeric compounds 5 and 6
The fluorescent-labelled compound 8 was synthesised as reported in Scheme 4, in a similar way as already described. (R)-Sulfonamide 19, already present in Prof. Rossello’s laboratory, was protected by reaction with N,N-DMF-di-tert-butyl acetal and the obtained ester (20) underwent the Mitsunobu coupling of the amine group with Boc-ethanolamine (11).
To label the final compound with fluorescein-5-(6)-isothiocyanate, two options were considered: to selectively remove the amine protection of compound 21, then label the product with fluorescein and finally remove the ester protection (three steps pathway); or to directly attack the free acid (compound 22), obtained by total deprotection of compound 21 with more drastic conditions (two steps pathway). Even if the first option was more likely to succeed, the latter was attempted in the first place in order to gain in yield. Compound 8 was thus obtained with an acceptable yield (27.74%).
HN COOH SO2 19 HN COOtBu SO2 20 N COOtBu SO2 21 HN Boc 11 N COOH SO2 -OOCF 3C+H3N 22 N COOH SO2 H N 8 H N S O O O OH HO N COOH SO2 H N 8 H N S HOOC O OH O N O O O OH HO (6)-isothiocyanate Fluorescein-5-C S N,N-DMF-di-tert-butyl acetal dry toluene 105°C PPh3, DIAD dry THF RT TFA RT DIPEA dry DMSO 40°C Boc N H OH
Scheme 4. Synthesis of fuorescein-labelled compound 8
To conclude this first part of my Thesis regarding MMP inhibitors, I tried to synthesise compound 23 (see Figure 26), as analogue of 1 bearing a polioxy spacer between the two carboxylic heads. The synthesis of this derivative was planned in order to evaluate the effect of the introduction of a more flexible and hydrophilic spacer on the inhibitory activity. Unfortunately, the synthesis of this compound resulted quite tricky and I was unable to get the final product.
