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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 matrix metalloproteinases. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 was shown 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 the breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8.

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.

International audience; Transthyretin (TTR) is a beta-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-beta, playing a protective role in Alzheimer's disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal.

The most commonly used antidepressant drugs are the serotonin transporter inhibitors. Their effects depend strongly on the selectivity for a single monoamine transporter compared to other amine transporters or receptors, and the selectivity is roughly influenced by the spatial protein structure. Here, we provide a computational study on three human monoamine transporters, i.e., DAT, NET, and SERT. Starting from the construction of hDAT and hNET models, whose three-dimensional structure is unknown, and the prediction of the binding pose for 19 known inhibitors, 3D-QSAR models of three human transporters were built. The training set variability, which was high in structure and activity profile, was validated using a set of in-house compounds. Results concern more than one aspect. First of all, hDAT and hNET three-dimensional structures were built, validated, and compared to the hSERT one; second, the computational study highlighted the differences in binding site arrangement statistically correlated to inhibitor selectivity; third, the profiling of new inhibitors pointed out a conservation of the inhibitory activity trend between rabbit and human SERT with a difference of about 1 order of magnitude; fourth, binding and functional studies confirmed 4-(benzyloxy)-4-phenylpiperidine 20a–d and 21a–d as potent SERT inhibitors. In particular, one of the compounds (compound 20b) revealed a higher affinity for SERT than paroxetine in human platelets.

Matrix metalloproteinase-12 (MMP-12) selective inhibitors could play a role in the treatment of lung inflammatory and cardiovascular diseases. In the present study, the previously reported 4-methoxybiphenylsulfonyl hydroxamate and carboxylate based inhibitors (1b and 2b) were modified to enhance their selectivity for MMP-12. In the newly synthesized thioaryl derivatives, the nature of the zinc binding group (ZBG) and the sulfur oxidation state were changed. Biological assays carried out in vitro on human MMPs with the resulting compounds led to identification of a sulfide, 4a, bearing an N-1-hydroxypiperidine-2,6-dione (HPD) group as new ZBG. Compound 4a is a promising hit compound since it displayed a nanomolar affinity for MMP-12 with a marked selectivity over MMP-9, MMP-1, and MMP-14. Solution complexation studies with Zn2+ were performed to characterize the chelating abilities of the new compounds and confirmed the bidentate binding mode of HPD derivatives. X-ray crystallography studies using MMP-12 a...

Protein homodimers play important roles in physiological and pathological processes, including cancer invasion and metastasis. Recently, MMP-9 natural homodimerization via the PEX domain has been correlated with high migration rates of aggressive cancer cells. Here we propose that bifunctional MMP-9 inhibitors designed to impair natural MMP-9 homodimerization promoted by PEX-PEX interactions might be an effective tool to fight cancer cell invasion. Elaborating a previously described dimeric hydroxamate inhibitor 1, new ligands were synthesized with different linker lengths and branch points. Evaluation of the modified bifunctional ligands by X-ray crystallography and biological assays showed that 7 and 8 could reduce invasion in three glioma cell lines expressing MMP-9 at different levels. To rationalize these results, we present a theoretical model of full-length MMP-9 in complex with 7. This pioneering study suggests that a new approach using MMP-9 selective bifunctional inhibitors might lead to an effective therapy to reduce cancer cell invasion.

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.

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.

Matrix metalloproteinases (MMPs) have been shown to be involved in tumor-induced angiogenesis. In particular, MMP-2, MMP-9, and MMP-14 have been reported to be crucial for tumor angiogenesis and the formation of metastasis, thus becoming attractive targets in cancer therapy. Here, we report our optimization effort to identify novel N-isopropoxy-arylsulfonamide hydroxamates with improved inhibitory activity toward MMP-2, MMP-9, and MMP-14 with respect to the previously discovered compound 1. A new series of hydroxamates was designed, synthesized, and tested for their antiangiogenic activity using in vitro assays with human umbilical vein endothelial cells (HUVECs). A nanomolar MMP-2, MMP-9, and MMP-14 inhibitor was identified, compound 3, able to potently inhibit angiogenesis in vitro and also in vivo in the matrigel sponge assay in mice. Finally, X-ray crystallographic and docking studies were conducted for compound 3 in order to investigate its binding mode to MMP-9 and MMP-14.

Despite recent progress in the fight against malaria, the emergence and spread of drug-resistant parasites remains a serious obstacle to the treatment of infections. We recently reported the development of a novel antimalarial drug that combines the 4-aminoquinoline pharmacophore of chloroquine with that of clotrimazole-based antimalarials. Here we describe the optimization of this class of hybrid drug through in-depth structure-activity relationship studies. Antiplasmodial properties and mode of action were characterized in vitro and in vivo, and interactions with the parasite's 'chloroquine resistance transporter' were investigated in a Xenopus laevis oocyte expression system. These tests indicated that piperazine derivatives 4b and 4d may be suitable for coadministration with chloroquine against chloroquine-resistant parasites. The potential for metabolism of the drugs by cytochrome P450 was determined in silico, and the lead compounds were tested for toxicity and mutagenicity. A preliminary pharmacokinetic analysis undertaken in mice indicated that compound 4b has an optimal half-life.

Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to μm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the μm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.3 and 8.0 μm as demonstrated by measurements of steady state ATPase activity as well as single cycle charge transfer. Characterization of sequential reactions comprising the ATPase catalytic and transport cycle then demonstrated that NF1058, and similarly CLT, interferes with the mechanism of Ca(2+) binding and Ca(2+)-dependent enzyme activation (E(2) to E(1)·Ca(2) transition) required for formation of phosphorylated intermediate by ATP utilization. On the other hand, Ca(2+) independent phosphoenzyme formation by utilization of P(i) (i.e. reverse of the hydrolytic reaction in the absence of Ca(2+)) was not inhibited by NF1058 or CLT. Comparative experiments showed that the high affinity inhibitor thapsigargin interferes not only with Ca(2+) binding and phosphoenzyme formation with ATP but also with phosphoenzyme formation by utilization of P(i) even though this reaction does not require Ca(2+). It is concluded that NF1058 and CLT inhibit SERCA by stabilization of an E(2) state that, as opposed to that obtained with thapsigargin, retains the functional ability to form E(2)-P by reacting with P(i).