Synthesis and Evaluation of Monoaryl Derivatives as Transthyretin Fibril Formation Inhibitors

Transthyretin (TTR) is a homo-tetrameric protein characterized by four identical β-sheet rich monomers assembled together to form a tetramer that is crossed, along the 2-fold symmetry axis, by two similar binding pockets named thyroxin binding sites. Under unknown conditions, TTR can misfold and aggregate triggering the amyloidosis onset. One therapeutic approach consists to stabilize the tetramer with synthetic small molecules that bind TTR binding site hindering the first step of fibril formation. Here, we report the synthesis of new 2-((benzyloxy)imino)acetic, -propanoic and -butanoic acid derivatives, results of their turbidimetric UV assay and the docking study of new monoaryl compounds. The obtained results suggest that, for this class of compounds, (i) the chlorine atom in ortho position on the aromatic ring is the best substituent; (ii) the linker inversion still allows the interaction with thyroxine binding sites; and (iii) the steric hindrance in R1 position is detrimental for the activity

Synthesis, experimental evaluation and molecular modelling of hydroxamate derivatives as zinc metalloproteinase inhibitors

Manuscript. Published version available in European Journal of Medicinal Chemistry, Vol. 108, 27 January 2016, pp 141–153Enzymes of the M4 family of zinc-metalloproteinases are virulence factors secreted from gram-positive or gram-negative bacteria, and putative drug targets in the treatment of bacterial infections. In order to have a therapeutic value such inhibitors should not interfere with endogenous zinc-metalloproteinases. In the present study we have synthesised a series of hydroxamate derivatives and validated the compounds as inhibitors of the M4 enzymes thermolysin and pseudolysin, and the endogenous metalloproteinases ADAM-17, MMP-2 and MMP-9 using experimental binding studies and molecular modelling. In general, the compounds are stronger inhibitors of the MMPs than of the M4 enzymes, however, an interesting exception is LM2. The compounds bound stronger to pseudolysin than to thermolysin, and the molecular modelling studies showed that occupation of the S2’ subpocket by an aromatic group is favourable for strong interactions with pseudolysin

Discovery of Dimeric Arylsulfonamides as Potent ADAM8 Inhibitors

The metalloproteinase ADAM8 is upregulated in several cancers but has a dispensable function under physiological conditions. In tumor cells, ADAM8 is involved in invasion, migration and angiogenesis. The use of bivalent inhibitors could impair migration and invasion, through the double binding to a homodimeric form of ADAM8 located on the cell surface of tumor cells. Herein we report the rational design and synthesis of the first dimeric ADAM8 inhibitors selective over ADAM10 and MMPs. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 showed to inhibit ADAM8 proteolytic activity in vitro and in cell-based assays, at nanomolar concentration. Moreover, it was more effective than the parent monomeric compound in blocking invasiveness in breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8

Discovery of a new selective inhibitor of A Disintegrin And Metalloprotease 10 (ADAM-10) able to reduce the shedding of NKG2D ligands in Hodgkin's lymphoma cell models

Hodgkin's lymphoma (HL) is the most common malignant lymphoma in young adults in the western world. This disease is characterized by an overexpression of ADAM-10 with increased release of NKG2D ligands, involved in an impaired immune response against tumor cells. We designed and synthesized two new ADAM-10 selective inhibitors, 2 and 3 based on previously published ADAM-17 selective inhibitor 1. The most promising compound was the thiazolidine derivative 3, with nanomolar activity for ADAM-10, high selectivity over ADAM-17 and MMPs and good efficacy in reducing the shedding of NKG2D ligands (MIC-B and ULBP3) in three different HL cell lines at non-toxic doses. Molecular modeling studies were used to drive the design and X-ray crystallography studies were carried out to explain the selectivity of 3 for ADAM-10 over MMPs

Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells

Shedding of ADAM10 substrates, like TNFa, MICA or CD30, is reported to affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. Soluble forms of these molecules and ADAM10 can be carried and spread in the microenvironment by exosomes released by tumor cells. We reported new ADAM10 inhibitors able to prevent MICA shedding in Hodgkin lymphoma (HL), leading to recognition of HL cells by cytotoxic lymphocytes. In this paper, we show that the mature bioactive form of ADAM10 is released in exosome-like vesicles (ExoV) by HL cells and lymph node mesenchymal stromal cells (MSC). We demonstrate that ADAM10 inhibitors are released in ExoV by MSC or HL cells, endocytosed by bystander cells and localized in the endolysosomal compartment in HL MSC. ExoV released by HL cells can enhance MICA shedding by MSC, while ExoV from MSC induce TNFa or CD30 shedding by HL cells. Of note, ADAM10 sheddase activity carried by ExoV is prevented with the ADAM10 inhibitors LT4 and CAM29, pretreating either the ExoVproducing or the ExoV-receiving cells. In particular, both inhibitors reduce CD30 shedding maintaining the anti-tumor effects of the ADC Brentuximab-Vedotin or the anti-CD30 Iratumumab on HL cells. Thus, spreading of ADAM10 activity due to ExoV can result in the release of cytokines, like TNFa,a lymphoma growth factor, or soluble molecules, like sMICA or sCD30, that potentially interfere with host immune surveillance or immunotherapy. ADAM10 blockers can interfere with this process, allowing the development of anti-lymphoma immune response and/or efficient ADC-based or human antibody-based immunotherapy

ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

Hodgkin lymphoma (HL) resistant to conventional therapies is increasing, making of interest the search for new schemes of treatment. Members of the “A Disintegrin And Metalloproteases” (ADAMs) family, mainly ADAM10 or ADAM17, have been proposed as therapeutic targets in solid tumors and some ADAMs inhibitors have been shown to exert antitumor effects. We have previously described an overexpression of ADAM10 in HL, together with increased release of NKG2D ligands (NKG2D-L) and reduced activation of effector T lymphocytes with anti-lymphoma capacity. Aim of the present work was to verify whether inhibition of ADAM10 in HL cells could restore the triggering of NKG2D-dependent anti-lymphoma T cell response. As no selective ADAM10 blockers have been reported so far, we synthesized the two hydroxamate compounds LT4 and MN8 with selectivity for ADAM10 over metalloproteases (MMPs), LT4 showing higher specificity for ADAM10 over ADAM17. We show that (i) HL lymph nodes (LN) and cultured HL cells express high levels of the mature active membrane form of ADAM10; (ii) ADAM10 is the major sheddase for the NKG2D-L in HL cells; (iii) the new LT4 and MN8 compounds strongly reduce the shedding of NKG2D-L by HL cell lines and enhance the binding of NKG2D receptor; (iv) of note, these new ADAM10 inhibitors increase the sensitivity of HL cell lines to NKG2D-dependent cell killing exerted by natural killer and γδ T cells. Overall, the biologic activity of LT4 and MN8 appears to be more potent than that of the commercial inhibitor GI254023X

Selective Arylsulfonamide Inhibitors of ADAM-17: Hit Optimization and Activity in Ovarian Cancer Cell Models

Activated Leukocyte Cell Adhesion Mol. (ALCAM) is expressed at the surface of epithelial ovarian cancer (EOC) cells and is released in a sol. form (sALCAM) by ADAM-17-mediated shedding. This process is relevant to EOC cell motility and invasiveness, which is reduced by inhibitors of ADAM-17. In addn., ADAM-17 plays a key role in EGFR signalling and thus may represent a useful target in anticancer therapy. Herein we report our hit optimization effort to identify potent and selective ADAM-17 inhibitors, starting with previous mol. 1. A new series of secondary sulfonamido-based hydroxamates was designed and synthesized. The biol. activity of the newly synthesized compds. was tested in vitro on isolated enzymes and human EOC cell lines. The optimization process led to compd. 21, which showed an IC50 of 1.9 nM on ADAM-17 with greatly increased selectivity. This compd. maintained good inhibitory properties on sALCAM shedding in several in vitro assays

ADAM metalloproteinases as potential drug targets

The ADAMs, together with ADAMTSs and snake venom metalloproteases (SVMPs), are members of the Adamalysin family. Differences in structural organization, functions and localization are known and their domains, catalytic or non-catalytic, show key roles in the substrate recognition and protease activity. Some ADAMs, as membrane-bound enzymes, show sheddase activity. Sheddases are key to modulation of functional proteins such as the tumor necrosis factor, growth factors, cytokines and their receptors, adhesion proteins, signaling molecules and stress molecules involved in immunity. These activities take part in the regulation of several physiological and pathological processes including inflammation, tumor growth, metastatic progression and infectious diseases. On these bases, some ADAMs are currently investigated as drug targets to develop new alternative therapies in many fields of medicine. This review will be focused on these aspects

Matrix metalloproteinase inhibitors prevent the release and proteolytic activity of monocyte/macrophage-derived microparticles

Background: The role of monocyte/macrophage-derived microparticles (MPs) in the pathophysiology of cancer and chronic inflammatory diseases has been reported; nevertheless, the mechanism underlying microparticles release is currently unclear. The aim of the current study was to investigate whether matrix metalloproteinase (MMP) inhibitors can prevent MP shedding from stimulated human monocyte/macrophage. Methods: Microparticles were obtained by isolated peripheral blood mononuclear cells after stimulation with the calcium ionophore, A23187. MP shedding, intracellular calcium concentration, analysis of RhoA expression, and proteolytic activities of isolated MPs were assessed in the absence or presence of MMP inhibitors. Results: We demonstrated that MMP inhibitors remarkably prevented MP shedding in a concentration-dependent manner with IC50 values in the nano- to micromolar range. Such an effect was related to their ability to reduce the intracellular Ca2+ levels induced by the calcium ionophore and the consequent translocation of RhoA from cytosol to membrane. Furthermore, MMP inhibitors could inhibit the proteolytic activity of cell-derived MPs. Conclusions: The current study provide evidence that MMP inhibitors can prevent MPs shedding from stimulated human monocyte/macrophage and the proteolytic activity of released MPs. Finally, the most active compound tested might represent the lead compound of a new class of molecules with therapeutic potential in cancer and chronic inflammatory diseases

Multifunctional Small Molecules as Potential Anti-Alzheimer's Disease Agents

Alzheimer's disease (AD) is a severe multifactorial neurodegenerative disorder characterized by a progressive loss of neurons in the brain. Despite research efforts, the pathogenesis and mechanism of AD progression are not yet completely understood. There are only a few symptomatic drugs approved for the treatment of AD. The multifactorial character of AD suggests that it is important to develop molecules able to target the numerous pathological mechanisms associated with the disease. Thus, in the context of the worldwide recognized interest of multifunctional ligand therapy, we report herein the synthesis, characterization, physicochemical and biological evaluation of a set of five (1a-e) new ferulic acid-based hybrid compounds, namely feroyl-benzyloxyamidic derivatives enclosing different substituent groups, as potential anti-Alzheimer's disease agents. These hybrids can keep both the radical scavenging activity and metal chelation capacity of the naturally occurring ferulic acid scaffold, presenting also good/mild capacity for inhibition of self-Aβ aggregation and fairly good inhibition of Cu-induced Aβ aggregation. The predicted pharmacokinetic properties point towards good absorption, comparable to known oral drugs