Imidazoquinoline Derivatives as Potential Inhibitors of InhA Enzyme and Mycobacterium tuberculosis.

Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.

Fecal supernatants from dogs with idiopathic epilepsy activate enteric neurons.

Introduction: Alterations in the composition and function of the gut microbiome have been reported in idiopathic epilepsy (IE), however, interactions of gut microbes with the enteric nervous system (ENS) in this context require further study. This pilot study examined how gastrointestinal microbiota (GIM), their metabolites, and nutrients contained in intestinal contents communicate with the ENS. Methods: Fecal supernatants (FS) from healthy dogs and dogs with IE, including drug-naïve, phenobarbital (PB) responsive, and PB non-responsive dogs, were applied to cultured myenteric neurons to test their activation using voltage-sensitive dye neuroimaging. Additionally, the concentrations of short-chain fatty acids (SCFAs) in the FS were quantified. Results: Our findings indicate that FS from all examined groups elicited neuronal activation. Notably, FS from PB non-responsive dogs with IE induced action potential discharge in a higher proportion of enteric neurons compared to healthy controls, which exhibited the lowest burst frequency overall. Furthermore, the highest burst frequency in enteric neurons was observed upon exposure to FS from drug-naïve dogs with IE. This frequency was significantly higher compared to that observed in PB non-responsive dogs with IE and showed a tendency to surpass that of healthy controls. Discussion: Although observed disparities in SCFA concentrations across the various FS samples might be associated with the induced neuronal activity, a direct correlation remains elusive at this point. The obtained results hint at an involvement of the ENS in canine IE and set the basis for future studies.

Localization of nitric oxide-producing hemocytes in Aedes and Culex mosquitoes infected with bacteria.

Mosquitoes are significant vectors of various pathogens. Unlike vertebrates, insects rely solely on innate immunity. Hemocytes play a crucial role in the cellular part of the innate immune system. The gaseous radical nitric oxide (NO) produced by hemocytes acts against pathogens and also functions as a versatile transmitter in both the immune and nervous systems, utilizing cyclic guanosine monophosphate (cGMP) as a second messenger. This study conducted a parallel comparison of NO synthase (NOS) expression and NO production in hemocytes during Escherichia coli K12 infection in four vector species: Aedes aegypti, Aedes albopictus, Culex pipiens molestus, and Culex pipiens quinquefasciatus. Increased NOS expression by NADPH diaphorase (NADPHd) staining and NO production by immunofluorescence against the by-product L-citrulline were observed in infected mosquito hemocytes distributed throughout the abdomens. NADPHd activity and citrulline labeling were particularly found in periostial hemocytes near the heart, but also on the ventral nerve chord (VNC). Pericardial cells of Ae. aegypti and Cx. p. molestus showed increased citrulline immunofluorescence, suggesting their involvement in the immune response. Oenocytes displayed strong NADPHd and citrulline labeling independent of infection status. This comparative study, consistent with findings in other species, suggests a widespread phenomenon of NO's role in hemocyte responses during E. coli infection. Found differences within and between genera highlight the importance of species-specific investigations.

Discovery of new diaryl ether inhibitors against Mycobacterium tuberculosis targeting the minor portal of InhA.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) affects 10 million people each year and the emergence of resistant TB augurs for a growing incidence. In the last 60 years, only three new drugs were approved for TB treatment, for which resistances are already emerging. Therefore, there is a crucial need for new chemotherapeutic agents capable of eradicating TB. Enzymes belonging to the type II fatty acid synthase system (FAS-II) are involved in the biosynthesis of mycolic acids, cell envelope components essential for mycobacterial survival. Among them, InhA is the primary target of isoniazid (INH), one of the most effective compounds to treat TB. INH acts as a prodrug requiring activation by the catalase-peroxidase KatG, whose mutations are the major cause for INH resistance. Herein, a new series of direct InhA inhibitors were designed based on a molecular hybridization approach. They exhibit potent inhibitory activities of InhA and, for some of them, good antitubercular activities. Moreover, they display a low toxicity on human cells. A study of the mechanism of action of the most effective molecules shows that they inhibit the biosynthesis of mycolic acids. The X-ray structures of two InhA/NAD+/inhibitor complexes have been obtained showing a binding mode of a part of the molecule in the minor portal, rarely seen in the InhA structures reported so far.

A mesoporous metal-organic framework used to sustainably release copper(ii) into reducing aqueous media to promote the CuAAC click reaction.

The mesoporous metal-organic framework Cr-MIL-101-NH2 (MOF1) has been used to encapsulate, by a simple impregnation method, large amounts of copper sulfate. The resulting loaded material, Cu@MOF1, was successfully employed to slowly release copper(ii) into an appropriate reaction medium in which the reducing agent sodium ascorbate reduces copper(ii) to copper(i), thus allowing the well-known copper(i)-catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction to proceed in the absence of potentially high local copper(i) concentrations. The use of a MOF-based controlled copper release system such as Cu@MOF1 may be relevant for copper(i)-catalyzed reactions having substrates that could be degraded by potentially high local concentrations of copper(i). The copper chelating ligand TBTA (tris(benzyltriazolylmethyl)amine), a very useful ligand for click chemistry, has been successfully attached to the pores of MOF1. The resulting TBTA-functionalized MOF (MOF3) was compared with its non-functionalized version (MOF1). At copper loadings of ca. 3 mmol g-1, the results revealed that the performances of the two materials are strikingly similar. Upon immersion in methanol/water (95/5) containing sodium ascorbate, both materials slowly released copper encapsulated in their pores and could be recovered and reused efficiently for up to five reaction cycles without reloading with metal ion, while allowing the CuAAC reaction to proceed with excellent conversion rates and yields.

Caco-2/HT29-MTX co-cultured cells as a model for studying physiological properties and toxin-induced effects on intestinal cells.

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.

Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine.

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells.

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.

Exploring the chemical space of 1,2,3-triazolyl triclosan analogs for discovery of new antileishmanial chemotherapeutic agents.

Triclosan and isoniazid are known antitubercular compounds that have proven to be also active against Leishmania parasites. On these grounds, a collection of 37 diverse 1,2,3-triazoles based on the antitubercular molecules triclosan and 5-octyl-2-phenoxyphenol (8PP) were designed in search of novel structures with leishmanicidal activity and prepared using different alkynes and azides. The 37 compounds were assayed against Leishmania donovani, the etiological agent of leishmaniasis, yielding some analogs with activity at micromolar concentrations and against M. tuberculosis H37Rv resulting in scarce active compounds with an MIC of 20 µM. To study the mechanism of action of these catechols, we analyzed the inhibition activity of the library on the M. tuberculosis enoyl-ACP reductase (ENR) InhA, obtaining poor inhibition of the enzyme. The cytotoxicity against Vero cells was also tested, resulting in none of the compounds being cytotoxic at concentrations of up to 20 µM. Derivative 5f could be considered a valuable starting point for future antileishmanial drug development. The validation of a putative leishmanial InhA orthologue as a therapeutic target needs to be further investigated.

Crystal structure of a two-dimensional coordination polymer of formula [Zn(NDC)(DEF)] (H2NDC is naphthalene-2,6-di-carb-oxy-lic acid and DEF is N,N-di-ethyl-formamide).

A zinc metal-organic framework, namely poly[bis-(N,N-di-ethyl-formamide)(µ4-naphthalene-2,6-di-carboxyl-ato)(µ2-naphthalene-2,6-di-carboxyl-ato)dizinc(II)], [Zn(C12H6O4)(C15H11NO)] n , built from windmill-type secondary building units and forming zigzag shaped two-dimensional stacked layers, has been solvothermally synthesized from naphthalene-2,6-di-carb-oxy-lic acid and zinc(II) acetate as the metal source in N,N-di-ethyl-formamide containing small amounts of formic acid.

New potent bisubstrate inhibitors of histone acetyltransferase p300: design, synthesis and biological evaluation.

Bisubstrate-type compound Lys-CoA has been shown to inhibit the p300 histone acetyl transferase activity efficiently and may constitute a lead compound for a novel class of anticancer therapeutics. Based on this strategy, we synthesized a series of CoA derivatives and evaluated these molecules for their activity as p300 histone acetyltransferases inhibitor. The best activity was obtained with compound 3 bearing a C-5 spacing linker that connects the CoA moiety to a tert-butyloxycarbonyl (Boc) group. Based on docking simulations, this inhibitor exhibits favorable interactions with two binding areas, namely pockets P1 and P2, within the active site.

Superoxide-induced bleaching of streptocyanine dyes: Application to assay the enzymatic activity of superoxide dismutases.

With the aim of developing a novel superoxide dismutase (SOD) activity assay, a series of polymethinium salts (streptocyanines) were prepared and studied for their ability to be reduced by superoxide radical anion generated either from the pyrogallol autoxidation or by the xanthine oxidase-catalyzed oxidation of xanthine. The nonacarbon chain streptocyanine 9Cl(NEt(2))(2) was found to be relatively stable in neutral buffered aqueous solutions, to be reduced at a significant rate by superoxide, and addition of iron-dependent superoxide dismutase (Fe-SOD) prevented its bleaching, thus constituting a good candidate as a possible superoxide indicator in a spectrophotometric SOD assay. The values found to be optimal for a SOD assay were defined as pH 7.4, wavelength 728nm, xanthine and xanthine oxidase as superoxide source, and a reaction time of 5min. Based on the color change caused by the superoxide-induced bleaching of the streptocyanine, a qualitative colorimetric method for the SOD activity detection is proposed, enabling visual detection within a short time without any instrument.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity.

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 A and T. cruzi at 1.9 A resolution, and that from P. falciparum at a higher resolution (2.0 A) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.

Irreversible inhibition of aldolase by a phosphorylated alpha-dicarbonyl compound.

The preparation of a phosphorylated alpha-dicarbonyl compound designed to specifically react with arginine residues of enzymes accepting phosphorylated compounds as effectors is reported, and shown to inhibit rabbit muscle aldolase in a time-dependent and irreversible manner. This irreversible inhibition occured in a buffer devoid of borate ions, suggesting that the presence of the phosphate moiety contributes in the stabilization of the adduct formed with arginine residues. Under the same conditions, the metalloenzyme iron superoxide dismutase, in which an arginine is known to be critical for the catalytic function, is not significantly inhibited.

Synthesis and evaluation of new phosphonolipid compounds for gene delivery.

The preparation of a series of novel water soluble cationic lipid derivatives possessing phosphonate ester groups linked to the para-position of N-methyl pyridinium moieties and bearing either identical or different alkyl chains is reported. The obtained phospholipids were tested for transfection efficiency into three different mammalian cell lines alone and in conjunction with diphytanoylphosphatidylethanolamine (DiPPE) or dioleylphosphatidylethanolamine (DOPE), using an assay adapted for 96-well microplates based on the detection of a colorimetric change caused by the production of a chromogen induced by expressed secreted human placental alkaline phosphatase. In our conditions, the highest transfection activities of cells HEK293 and hard-to-transfect cell lines B16 and CHO were achieved with a 4-phosphonobutylpyridinium compound used at 1:5, 1:10 or 3:6 DNA/lipid ratio bearing two myristyl chains in the presence of the fusogenic helper lipid DiPPE.

Esterase-activated chromane-sydnonimine prodrug hybrids.

The preparation and characterization of two vitamin E analogs-sydnonimine conjugates, delta-tocopheryloxycarbonyl-3-morpholinosydnonimine (2) and troloxoxycarbonyl-3-morpholinosydnonimine (3), in which the hydroxyl group of the tocopheryl moieties is linked via an enzymatically cleavable urethane group to the sydnone moiety is described. In the presence of porcine liver esterase, these tocopheryl-sydnonimine conjugates generated the expected antioxidant moieties, i.e., delta-tocopherol or Trolox, and were found to convert oxyhemoglobin to methemoglobin at 37 degrees C in 50 mM phosphate buffer at pH 7.4, thus providing evidence for nitric oxide release. Their potency as antioxidants was indirectly studied by associating the two products of the hydrolysis, SIN-1, and delta-tocopherol or Trolox. Our findings suggest that unlike the other members of the sydnonimine family these chromane-sydnonimine derivatives do not act as peroxynitrite donors, and require enzymatic bioactivation before nitric oxide or nitroxyl anion (NO(-)) can be released.

Screening of Plasmodium falciparum iron superoxide dismutase inhibitors and accuracy of the SOD-assays.

In vitro evaluation of a chemical library of synthetic compounds using two consecutive assays has led to the discovery of fifteen compounds which have the ability to inhibit recombinant Plasmodium falciparum iron superoxide dismutase (PfSOD), suggested as a highly selective target for design of antiparasitic drugs. A large number of compounds were in fact excluded, because they were found to significantly interfere with the components of the assays, thus outlining the drawbacks relative to the use of standard SOD-assays for the research of compounds targeting SODs. The best of the selected compounds showed significant antimalarial activities against two strains of P. falciparum, including a strain moderately resistant to chloroquine.

Selective inhibition of Fe- versus Cu/Zn-superoxide dismutases by 2,3-dihydroxybenzoic acid derivatives.

A series of catechol derivatives were synthesised and tested for their ability to inactivate the iron-containing superoxide dismutase (Fe-SOD) from Escherichia coli and the bovine erythrocytes Cu/Zn-SOD. Incubation of catechols with Fe- or Cu/Zn SODs resulted in a time-dependent loss of enzyme activity with highly selective inhibition for the iron-dependent enzyme. Catechol-induced inactivation of SODs was correlated with the auto-oxidation of the catechol compounds to their corresponding ortho-quinone derivatives, which was found to be non-dependent on the presence of enzymes. Mass electrospray experiments on catechol-incubated Fe-SOD provided evidence for the irreversible nature of the inhibition process, yielding to a complex mixture of modified proteins.