Human Neuraminidases: Structures and Stereoselective Inhibitors.

This Perspective describes the classification, structures, substrates, mechanisms of action, and implications of human neuraminidases (hNEUs) in various pathologies. Some inhibitors have been developed for each isoform, leading to more precise interactions with hNEUs. Although crystal structure data are available for NEU2, most of the findings are based on NEU1 inhibition, and limited information is available for other hNEUs. Therefore, the synthesis of new compounds would facilitate the enrichment of the arsenal of inhibitors to better understand the roles of hNEUs and their mechanisms of action. Nevertheless, due to the already known inhibitors of human neuraminidase enzymes, a structure-activity relationship is presented along with different approaches to inhibit these enzymes for the development of potent and selective inhibitors. Among the different emerging strategies, one is the inhibition of the dimerization of NEU1 or NEU3, and the second is the inhibition of certain receptors located close to hNEU.

Targeting Class IIa HDACs: Insights from Phenotypes and Inhibitors.

This review summarizes key literature defining the phenotypes of individual class IIa HDAC proteins and compounds that selectively target their enzymatic catalytic domain (CD). The focus is on the effects of class IIa HDACs in physiological and pathological conditions, both in vitro and in vivo, and on their mode of action in regulating genes, upstream proteins and signaling pathways. Phenotype studies further demonstrate either beneficial or detrimental effects of silencing selected class IIa HDACs or their enzymatic properties. We also summarize the knowledge gained from structure-activity relationships of CD inhibitors as well as molecular mechanisms underpinning isozyme selectivity where crystal structures or modelling studies are available. Given that the number of genes affected by silencing class IIa HDACs is much smaller than class I, the role of gene regulation of class IIa HDACs could be much more selective. Since class IIa HDACs have restricted tissue distributions and multiple functions independent of their CD, targeting the CD of class IIa HDACs could lead to more selective therapeutic agents with significantly fewer side-effects than other HDAC ligands.

Interest of new alkylsulfonylhydrazide-type compound in the treatment of alcohol use disorders.

RATIONALE: Recent preclinical research suggested that histone deacetylase inhibitors (HDACIs) and specifically class I HDAC selective inhibitors might be useful to treat alcohol use disorders (AUDs). OBJECTIVE: The objective of this study was to find a new inhibitor of the HDAC-1 isoenzyme and to test its efficacy in an animal model of AUDs. METHODS: In the present study, we prepared new derivatives bearing sulfonylhydrazide-type zinc-binding group (ZBG) and evaluated these compounds in vitro on HDAC-1 isoenzyme. The most promising compound was tested on ethanol operant self-administration and relapse in rats. RESULTS: We showed that the alkylsulfonylhydrazide-type compound (ASH) reduced by more than 55% the total amount of ethanol consumed after one intracerebroventricular microinjection, while no effect was observed on motivation of the animals to consume ethanol. In addition, one ASH injection in the central amygdala reduced relapse. CONCLUSIONS: Our study demonstrated that a new compound designed to target HDAC-1 is effective in reducing ethanol intake and relapse in rats and further confirm the interest of pursuing research to study the exact mechanism by which such inhibitor may be useful to treat AUDs.

Class I HDAC Inhibitors: Potential New Epigenetic Therapeutics for Alcohol Use Disorder (AUD).

Alcohol use disorder (AUD) represents a serious public health issue, and discovery of new therapies is a pressing necessity. Alcohol exposure has been widely demonstrated to modulate epigenetic mechanisms, such as histone acetylation/deacetylation balance, in part via histone deacetylase (HDAC) inhibition. Epigenetic factors have been suggested to play a key role in AUD. To date, 18 different mammalian HDAC isoforms have been identified, and these have been divided into four classes. Since recent studies have suggested that both epigenetic mechanisms underlying AUD and the efficacy of HDAC inhibitors (HDACIs) in different animal models of AUD may involve class I HDACs, we herein report the development of class I HDACIs, including information regarding their structure, potency, and selectivity. More effort is required to improve the selectivity, pharmacokinetics, and toxicity profiles of HDACIs to achieve a better understanding of their efficacy in reducing addictive behavior.

[Epigenetic mechanisms and alcohol use disorders: a potential therapeutic target]. / Mécanismes épigénétiques et troubles de l'usage d'alcool : une cible thérapeutique intéressante?

Alcohol use disorder is a devastating illness with a profound health impact, and its development is dependent on both genetic and environmental factors. This disease occurs over time and requires changes in brain gene expression. There is converging evidence suggesting that the epigenetic processes may play a role in the alcohol-induced gene regulations and behavior such as the intervention of DNA methylation and histone acetylation. Histone acetylation, like histone methylation, is a highly dynamic process regulated by two classes of enzymes: histone acetyltransferases and histone deacetylases (HDACs). To date, 18 human HDAC isoforms have been characterized, and based on their sequence homologies and cofactor dependencies, they have been phylogenetically categorized into 4 main classes: classes I, II (a and b), III, and IV. In the brain, expression of the different classes of HDACs varies between cell types and also in their subcellular localization (nucleus and/or cytosol). Furthermore, we recently showed that a single ethanol exposure inhibits HDAC activity and increases both H3 and H4 histone acetylation within the amygdala of rats. In the brain of alcoholic patients, ethanol has been shown to induce histone-related and DNA methylation epigenetic changes in several reward regions involved in reward processes such as hippocampus, prefrontal cortex, and amygdala. We recently demonstrated alteration of histone H3 acetylation levels in several brain regions from the reward circuit of rats made dependent to alcohol after chronic and intermittent exposure to ethanol vapor. In neuronal cell line culture, ethanol was shown to induce HDAC expression. In mouse and rat brain, numerous studies reported epigenetic alterations following ethanol exposure. We also demonstrated that both the expression of genes and the activity of enzymes involved in epigenetic mechanisms are changed after repeated administrations of ethanol in mice sensitized to the motor stimulant effect of ethanol (a model of drug-induced neuroplasticity). Numerous studies have shown that HDAC inhibitors are able to counter ethanol-induced behaviors and the ethanol-induced changes in the levels of HDAC and/or levels of acetylated HDAC. For example, trichostatin A (TSA) treatment caused the reversal of ethanol-induced tolerance, anxiety, and ethanol drinking by inhibiting HDAC activity, thereby increasing histone acetylation in the amygdala of rats. Another study demonstrated that TSA prevented the development of ethanol withdrawal induced anxiety in rats by rescuing deficits in histone acetylation induced by increased HDAC activity in the amygdala. We have demonstrated that treatment with the HDAC inhibitor sodium butyrate blocks both the development and the expression of ethanol-induced behavioral sensitization in mice. In this context, converging evidence indicates that HDAC inhibitors could be useful in counteracting ethanol-induced gene regulations via epigenetic mechanisms, that is, HDAC inhibitors could affect different acetylation sites and may also alter the expression of different genes that could in turn counteract the effect of ethanol. Recent work in rodents has shown that systemic administration of pan HDAC class I and II inhibitors, TSA and N-hydroxy-N-phenyl-octanediamide [SuberoylAnilide Hydroxamic Acid] (SAHA), and of the more selective inhibitor (mainly HDAC1 and HDAC9) MS-275, decrease binge-like alcohol drinking in mice. SAHA selectively reduced ethanol operant self-administration and seeking in rats. Our previous study revealed that MS-275 strongly decreased operant ethanol self-administration in alcohol-dependent rats when administered 30 minutes before the session at the second day of injection. We also demonstrated that intra-cerebro-ventricular infusion of MS-275 increases acetylation of Histone 4 within the nucleus accumbens and the dorsolateral striatum, associated to a decrease in ethanol self-administration by about 75%. MS-275 also diminished both the motivation to consume ethanol (25% decrease), relapse (by about 50%) and postponed reacquisition after abstinence. Both literature and several of our studies strongly support the potential therapeutic interest of targeting epigenetic mechanisms in excessive alcohol drinking and strengthen theinterest of focusing on specific isoforms of histone deacetylases.

Design, Synthesis, and Use of MMP-2 Inhibitor-Conjugated Quantum Dots in Functional Biochemical Assays.

The development of chemically designed matrix metalloprotease (MMP) inhibitors has advanced the understanding of the roles of MMPs in different diseases. Most MMP probes designed are fluorogenic substrates, often suffering from photo- and chemical instability and providing a fluorescence signal of moderate intensity, which is difficult to detect and analyze when dealing with crude biological samples. Here, an inhibitor that inhibits MMP-2 more selectively than Galardin has been synthesized and used for enzyme labeling and detection of the MMP-2 activity. A complete MMP-2 recognition complex consisting of a biotinylated MMP inhibitor tagged with the streptavidin-quantum dot (QD) conjugate has been prepared. This recognition complex, which is characterized by a narrow fluorescence emission spectrum, long fluorescence lifetime, and negligible photobleaching, has been demonstrated to specifically detect MMP-2 in in vitro sandwich-type biochemical assays with sensitivities orders of magnitude higher than those of the existing gold standards employing organic dyes. The approach developed can be used for specific in vitro visualization and testing of MMP-2 in cells and tissues with sensitivities significantly exceeding those of the best existing fluorogenic techniques.

Neutrophil elastase as a target in lung cancer.

Human neutrophil elastase (HNE), a main actor in the development of chronic obstructive pulmonary diseases, has been recently involved in non-small cell lung cancer progression. It can act at several levels (i) intracellularly, cleaving for instance the adaptor molecule insulin receptor substrate-1 (IRS-1) (ii) at the cell surface, hydrolyzing receptors as CD40 (iii) in the extracellular space, generating elastin fragments i.e. morphoelastokines which potently stimulate cancer cell invasiveness and angiogenesis. Since decades, researchers identified natural compounds and/or synthesized agents which antagonize HNE activity that will be described in this review article. Some of these compounds might be of value as therapeutic agents in lung cancer. However, it is now widely accepted that lung tumor invasion and metastasis involve proteolytic cascades. Accordingly, we will here mainly focus our attention to natural substances able to display a dual inhibitory capacity (i.e. lipids and derivatives, phenolics) towards HNE and matrix metalloproteinases (MMPs), particularly MMP-2. To that purpose, we recently synthesize substances named "LipoGalardin" (Moroy G. et al., Biochem. Pharmacol., 2011, 81(5), 626-635) exhibiting such inhibitory bifunctionality. At last, we will propose an original synthetic scheme for designing a potent biheaded HNE/MMP-2 inhibitor.

Inhibition of human leukocyte elastase, plasmin and matrix metalloproteinases by oleic acid and oleoyl-galardin derivative(s).

Molecular modeling was undertaken at aims to analyze the interactions between oleic acid and human leukocyte elastase (HLE), plasmin and matrix metalloproteinase-2 (MMP-2), involved in the inhibitory capacity of fatty acid towards those proteases. The carboxylic acid group of the fatty acid was found to form a salt bridge with Arg(217) of HLE while unsaturation interacted with Phe(192) and Val(216) at the S(3) subsite, and alkyl end group occupied S(1) subsite. In keeping with the main contribution of kringle 5 domain in plasmin-oleic acid interaction [Huet E et al. Biochem Pharmacol 2004;67(4):643-54], docking computations revealed that the long alkyl chain of fatty acid inserted within an hydrophobic groove of this domain with the carboxylate forming a salt bridge with Arg(512). Finally, blind docking revealed that oleic acid could occupy both S'(1) subsite and Fn(II)(3) domain of MMP-2. Several residues involved in Fn(II)(3)/oleic acid interaction were similarly implicated in binding of this domain to collagen. Oleic acid was covalently linked to galardin (at P'(2) position): OL-GAL (CONHOH) or to its carboxylic acid counterpart: OL-GAL (COOH), with the idea to obtain potent MMP inhibitors able to also interfere with elastase and plasmin activity. OL-GALs were found less potent MMP inhibitors as compared to galardin and no selectivity for MMP-2 or MMP-9 could be demonstrated. Docking computations indicated that contrary to oleic acid, OL-GAL binds only to MMP-2 active site and surprisingly, hydroxamic acid was unable to chelate Zn, but instead forms a salt bridge with the N-terminal Tyr(110). Interestingly, oleic acid and particularly OL-GALs proved to potently inhibit MMP-13. OL-GAL was found as potent as galardin (K(i) equal to 1.8nM for OL-GAL and 1.45nM for GAL) and selectivity for that MMP was attained (2-3 log orders of difference in inhibitory potency as compared to other MMPs). Molecular modeling studies indicated that oleic acid could be accommodated within S'(1) pocket of MMP-13 with carboxylic acid chelating Zn ion. OL-GAL also occupied such pocket but hydroxamic acid did not interact with Zn but instead was located at 2.8Å from Tyr(176). Since these derivatives retained, as their oleic acid original counterpart, the capacity to inhibit the amidolytic activity of HLE and plasmin as well as to decrease HLE- and plasmin-mediated pro MMP-3 activation, they might be of therapeutic value to control proteolytic cascades in chronic inflammatory disorders.

In silico study of MMP inhibition.

Lack of enzyme inhibition selectivity is frequently the major drawback preventing the development of enzyme inhibitors. Sulfonylhydrazides have recently been suggested to act as zinc ligands. Consequently, such derivatives potentially possess important industrial or therapeutic implications. DFT calculations (B3LYP/6-31G**+LANL2DZ theory level) of the binding modes and free energies of binding of a variety of N-acetyl-N'-sulfonylhydrazides in the presence of a Zn(2+) ion embedded in an MMP active site model show that protonated and deprotonated sulfonylhydrazides bind the Zn(2+) ion according to different modes. These results strongly suggest that sulfonylhydrazides can be developed as selective metalloprotease inhibitors, and the results of molecular docking computations fully support this hypothesis.

Control of melanoma invasiveness by anticollagenolytic agents: a reappraisal of an old concept.

Collagen, the major constituent of human dermis, represents the main barrier against progression of melanoma cells. Several matrix metalloproteinases (MMPs), i.e. collagenase-1 (MMP-1), gelatinase A (MMP-2) and membrane-type 1-MMP (MMP-14), favor melanoma cell invasion through their capacity of degrading collagen and thus, are considered as main targets. Potent inhibitors, as hydroxamate-derived pseudopeptides were first proposed as pharmacological agents to control melanoma invasiveness. These molecules have major drawbacks linked to i) toxicity and ii) absence of specificity, in keeping with the high Zn chelating property of hydroxamates, that might hinder the contribution of the occupancy of other subsites in enzyme inhibition. To date, research focuses on the design of compounds which display a lower affinity for Zn in enzyme active site. For instance, hydroxamate can be replaced by phosphinic acid or hydrazide which further allows synthesis of both right- and left- hand side inhibitors and therefore occupancy of non-primed enzyme subsites. Novel types of selective MMP inhibitors also include non-competitive and mechanism-based inhibitors. Finally, collagenolysis may be controlled by modulating enzyme-substrate interaction through the identification of substances that bind to MMP exosites. Such compounds could be of value by impeding collagenases to associate to plasma-membrane of invading cancer cells.

Introduction of the 4-(4-bromophenyl)benzenesulfonyl group to hydrazide analogs of Ilomastat leads to potent gelatinase B (MMP-9) inhibitors with improved selectivity.

Hydrazide derivatives of Ilomastat, carrying either aryl groups or distinct alkyl and arylsulfonyl moieties were synthesized and evaluated for their MMP inhibitory activity. Potent and selective MMP-9 inhibition (IC(50)=3 nM) was observed for compound 3m (arylsulfonyl group: 4-(4-Br-C6H4)-C6H4-SO(2)-). Interaction with the S2 enzyme subsite is mainly responsible for the inhibitory properties of this derivative as confirmed by molecular docking computation.

Synthesis and conformational studies of pseudopeptides containing an unsymmetrical triazine scaffold.

Solid-phase synthesis and conformational studies of two pseudopeptides constituted by a triazine scaffold bound to two peptidic arms are described. In this paper, a new scaffold based on unsymmetrical triamino 1,3,5-triazine bearing two alkyl chains has been designed, assisted by molecular modelling, as a mimic of the backbone of the i + 1 and i + 2 residues of a beta-turn. The results confirm the ability of the triazine scaffold to induce extended conformations of the peptidic strands and point out that this scaffold is a good candidate as a template to induce anti-parallel beta-sheet structure.

Simultaneous presence of unsaturation and long alkyl chain at P'1 of Ilomastat confers selectivity for gelatinase A (MMP-2) over gelatinase B (MMP-9) inhibition as shown by molecular modelling studies.

Structural analogues of Ilomastat (Galardin), containing unsaturation(s) and chain extension carrying bulky phenyl group or alkyl moieties at P'1 were synthesized and purified by centrifugal partition chromatography. They were analyzed for their inhibitory capacity towards MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14, main endopeptidases involved in tumour progression. Presence of unsaturation(s) decreased the inhibitory potency of compounds but, in turn increased their selectivity for gelatinases. 2b and 2d derivatives with a phenyl group inhibited preferentially MMP-9 with IC50 equal to 45 and 38 nM, respectively, but also display activity against MMP-2 (IC50 equal to 280 and 120 nM, respectively). Molecular docking computations confirmed affinity of these substances for both gelatinases. With aims to obtain a specific gelatinase A (MMP-2) inhibitor, P'1 of Ilomastat was modified to carry one unsaturation coupled to an alkyl chain with pentylidene group. Docking studies indicated that MMP-2, but not MMP-9, could accommodate such substitution; indeed 2a proved to inhibit MMP-2 (IC50=123 nM), while displaying no inhibitory capacity towards MMP-9.

Synthesis of furano-epothilone D.

The total synthesis of furano-epothilone D by a convergent route is reported. The key fragments are available on a large scale to provide sufficient material for biological evaluation. The approach involves a palladium-catalyzed coupling that generates a highly functionalized aldehyde which is connected in a stereoselective aldol reaction to yield the framework of furano-epothilone D. Finally, a macrolactonization provides furano-epothilone D.

Nonpeptide RGD antagonists: a novel class of mimetics, the 5,8-disubstituted 1-azabicyclo[5.2.0]nonan-2-one lactam.

The 1-azabicyclo[5.2.0]nonan-2-one lactam 1 adequately substituted on both cycles A and B as scaffolds mimics the conformationally constrained beta-turn of the tripeptide RGD signaling motif of fibronectin. Using an in vitro assay, we establish that trans diastereoisomer 1b dissociates a soluble fibronectin-integrin alpha(5)beta(1) complex at concentrations comparable to those of a linear RGDS peptide as a competitor.