Specific alterations in riboproteomes composition of isonicotinic acid treated arabidopsis seedlings.

Plants have developed strategies to deal with the great variety of challenges they are exposed to. Among them, common targets are the regulation of transcription and translation to finely modulate protein levels during both biotic and abiotic stresses. Increasing evidence suggests that ribosomes are highly adaptable modular supramolecular structures which remodel to adapt to stresses. Each Arabidopsis thaliana ribosome consists of approximately 81 distinct ribosomal proteins (RPs), each of which is encoded by two to seven genes. To investigate the identity of ribosomal proteins of the small subunit (RPS) and of the large subunit (RPL) as well as ribosomes-associated proteins, we analysed by LC/MS/MS immunopurified ribosomes from A. thaliana leaves treated with isonicotinic acid (INA), an inducer of plant innate immunity. We quantified a total of 2084 proteins. 165 ribosome-associated proteins showed increased abundance while 52 were less abundant. Of the 52 identified RPS (from a possibility of 104 encoding genes), 15 were deregulated. Similarly, from the 148 possible RPL, 80 were detected and 9 were deregulated. Our results revealed potential candidates involved in innate immunity that could be interesting targets for functional genomic studies.

Isonicotinic acid N-oxide, from isoniazid biotransformation by Aspergillus niger, as an InhA inhibitor antituberculous agent against multiple and extensively resistant strains supported by in silico docking and ADME prediction.

Biotransformation of isoniazid produced isonicotinic acid (1), isonicotinic acid N-oxide (2), and isonicotinamide (3) which were isolated by column chromatography using silica gel and Sephadex LH 20 and elucidated using various spectroscopies. This is the first report for isolation of 2. Antituberculosis activity was evaluated against Mycobacterium tuberculosis strains: drug sensitive (DS), multiple drug resistant (MDR) and extensively drug resistant (XDR). 1-3 and isoniazid showed MICs of 63.49, 0.22, 15.98 and 0.88 µM, respectively, against the DS strain. For the MDR strain, 2 and 3 exhibited MICs of 28.06 and > 1000 µM, respectively, while 1 was inactive. Moreover, 2 had an MIC of 56.19 µM against XDR strain, while 1 and 3 were inactive. Docking simulation using enoyl ACP reductase (InhA) revealed favorable protein-ligand interactions. In silico study of pharmacokinetics and hepatotoxicity predicted 1-3 to have good oral bioavailability and 2 to have a lower hepatoxicity probability than isoniazid.

Structural insight into the molecular mechanism of cilofexor binding to the farnesoid X receptor.

Farnesoid X receptor (FXR) is a bile acid-related nuclear receptor and is considered a promising target to treat several liver disorders. Cilofexor is a selective FXR agonist and has already entered phase III trials in primary sclerosing cholangitis (PSC) patients. Pruritis caused by cilofexor treatment is dose dependent. The binding characteristics of cilofexor with FXR and its pruritogenic mechanism remain unclear. In our research, the affinity of cilofexor bound to FXR was detected using an isothermal titration calorimetry (ITC) assay. The binding mechanism between cilofexor and FXR-LBD is explained by the cocrystal structure of the FXR/cilofexor complex. Structural models indicate the possibility that cilofexor activates Mas-related G protein-coupled receptor X4 (MRGPRX4) or G protein-coupled bile acid receptor 1 (GPBAR1), leading to pruritus. In summary, our analyses provide a molecular mechanism of cilofexor binding to FXR and provide a possible explanation for the dose-dependent pruritis of cilofexor.

Isonicotinylation is a histone mark induced by the anti-tuberculosis first-line drug isoniazid.

Isoniazid (INH) is a first-line anti-tuberculosis drug used for nearly 70 years. However, the mechanism underlying the side effects of INH has remained elusive. Here, we report that INH and its metabolites induce a post-translational modification (PTM) of histones, lysine isonicotinylation (Kinic), also called 4-picolinylation, in cells and mice. INH promotes the biosynthesis of isonicotinyl-CoA (Inic-CoA), a co-factor of intracellular isonicotinylation. Mass spectrometry reveals 26 Kinic sites in histones in HepG2 cells. Acetyltransferases CREB-binding protein (CBP) and P300 catalyse histone Kinic, while histone deacetylase HDAC3 functions as a deisonicotinylase. Notably, MNase sensitivity assay and RNA-seq analysis show that histone Kinic relaxes chromatin structure and promotes gene transcription. INH-mediated histone Kinic upregulates PIK3R1 gene expression and activates the PI3K/Akt/mTOR signalling pathway in liver cancer cells, linking INH to tumourigenicity in the liver. We demonstrate that Kinic is a histone acylation mark with a pyridine ring, which may have broad biological effects. Therefore, INH-induced isonicotinylation potentially accounts for the side effects in patients taking INH long-term for anti-tuberculosis therapy, and this modification may increase the risk of cancer in humans.

Priming of seeds with INA and its transgenerational effect in common bean (Phaseolus vulgaris L.) plants.

Priming is a mechanism of defense that prepares the plant's immune system for a faster and/or stronger activation of cellular defenses against future exposure to different types of stress. This enhanced resistance can be achieved by using inorganic and organic compounds which imitate the biological induction of systemic acquired resistance. INA (2,6 dichloro-isonicotinic acid) was the first synthetic compound created as a resistance inducer for plant-pathogen interactions. However, the use of INA to activate primed resistance in common bean, at the seed stage and during germination, remains experimentally unexplored. Here, we test the hypothesis that INA-seed treatment would induce resistance in common bean plants to Pseudomonas syringae pv. phaseolicola, and that the increased resistance is not accompanied by a tradeoff between plant defense and growth. Additionally, it was hypothesized that treating seeds with INA has a transgenerational priming effect. We provide evidence that seed treatment activates a primed state for disease resistance, in which low nucleosome enrichment and reduced histone activation marks during the priming phase, are associated with a defense-resistant phenotype, characterized by symptom appearance, pathogen accumulation, yield, and changes in gene expression. In addition, the priming status for induced resistance can be inherited to its offspring.

Exploration of Structure-Activity Relationship of Aromatic Aldehydes Bearing Pyridinylmethoxy-Methyl Esters as Novel Antisickling Agents.

Aromatic aldehydes elicit their antisickling effects primarily by increasing the affinity of hemoglobin (Hb) for oxygen (O2). However, challenges related to weak potency and poor pharmacokinetic properties have hampered their development to treat sickle cell disease (SCD). Herein, we report our efforts to enhance the pharmacological profile of our previously reported compounds. These compounds showed enhanced effects on Hb modification, Hb-O2 affinity, and sickling inhibition, with sustained pharmacological effects in vitro. Importantly, some compounds exhibited unusually high antisickling activity despite moderate effects on the Hb-O2 affinity, which we attribute to an O2-independent antisickling activity, in addition to the O2-dependent activity. Structural studies are consistent with our hypothesis, which revealed the compounds interacting strongly with the polymer-stabilizing αF-helix could potentially weaken the polymer. In vivo studies with wild-type mice demonstrated significant pharmacologic effects. Our structure-based efforts have identified promising leads to be developed as novel therapeutic agents for SCD.

Copper amine oxidase 8 regulates arginine-dependent nitric oxide production in Arabidopsis thaliana.

Nitric oxide (NO) is a key signaling molecule in plants, regulating a wide range of physiological processes. However, its origin in plants remains unclear. It can be generated from nitrite through a reductive pathway, notably via the action of the nitrate reductase (NR), and evidence suggests an additional oxidative pathway, involving arginine. From an initial screen of potential Arabidopsis thaliana mutants impaired in NO production, we identified copper amine oxidase 8 (CuAO8). Two cuao8 mutant lines displayed a decreased NO production in seedlings after elicitor treatment and salt stress. The NR-dependent pathway was not responsible for the impaired NO production as no change in NR activity was found in the mutants. However, total arginase activity was strongly increased in cuao8 knockout mutants after salt stress. Moreover, NO production could be restored in the mutants by arginase inhibition or arginine addition. Furthermore, arginine supplementation reversed the root growth phenotype observed in the mutants. These results demonstrate that CuAO8 participates in NO production by influencing arginine availability through the modulation of arginase activity. The influence of CuAO8 on arginine-dependent NO synthesis suggests a new regulatory pathway for NO production in plants.

Why Can Unnatural Electron Acceptors Protect Photosynthesizing Organisms but Kill the Others?

The polychlorinated compounds captafol (CPL) and 2,6-dichloroisonicotinic acid (INA) are able to protect plants acting as a fungicide or an inductor of plant resistance, respectively. At the same time, CPL and INA are dangerous for the respiratory organisms, i.e. mammalians, bacteria, and fungi. The high electron-withdrawing ability of these compounds enables them to serve as unnatural electron acceptors in the cellular ambient near to electron transport pathways located in the thylakoid membrane of chloroplasts or in the mitochondrial respiratory chain. Low-energy electron attachment to CPL and INA in vacuo leads to formation of many fragment species mainly at thermal electron energy as it is shown using dissociative electron attachment spectroscopy. On the basis of the experimental findings, assigned with the support of density functional theory calculations it is suggested that the different bioactivity of CPL and INA in respiratory and photosynthetic organisms is due to the interplay between the dissociative electron attachment process and the energies of electrons leaked from the electron transport pathways.

Anti-inflammatory and Anti-nociceptive Activity of Ruthenium Complexes with Isonicotinic and Nicotinic Acids (Niacin) as Ligands.

This work evaluated the analgesic and anti-inflammatory activity of ruthenium(II) complexes trans-[Ru(NO(+))(NH3)4(L)](BF4)3 and [Ru(NH3)5(L)](BF4)3 containing the nonsteroidal anti-inflammatory drugs nicotinic acid (Hnic) and its isomer isonicotinic acid (ina) as ligands (L). The anti-nociceptive potential of these complexes and the free ligands (noncoordinated to ruthenium) was tested in different models with doses ranging from 1 to 100 µmol/kg. The ligands themselves were inactive; however, the ruthenium complexes containing Hnic and ina inhibited mechanical hyperalgesia induced by prostaglandin E2, carrageenan-induced hyperalgesia, and antigen-induced arthritis. Moreover, the ruthenium complexes inhibited overt nociception induced by formalin, acetic acid, capsaicin, and cinnamaldehyde. The mechanism involved in the anti-nociceptive effects of the ruthenium complexes suggested that ATP-sensitive K(+) channel pathways were not involved because glibenclamide did not affect their anti-nociceptive activities. However, the anti-nociceptive effect appears to be a consequence of the reduction in neutrophil migration and inhibition of the protein kinase C pathway.

Esters of the marine-derived triterpene sipholenol A reverse P-GP-mediated drug resistance.

Our previous studies showed that several sipholane triterpenes, sipholenol A, sipholenone E, sipholenol L and siphonellinol D, have potent reversal effect for multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp/ABCB1). Through comparison of cytotoxicity towards sensitive and multi-drug resistant cell lines, we identified that the semisynthetic esters sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate potently reversed P-gp-mediated MDR but had no effect on MRP1/ABCC1 and BCRP/ABCG2-mediated MDR. The results from [3H]-paclitaxel accumulation and efflux studies suggested that these two triterpenoids were able to increase the intracellular accumulation of paclitaxel by inhibiting its active efflux. In addition, western blot analysis revealed that these two compounds did not alter the expression levels of P-gp when treated up to 72 h. These sipholenol derivatives also stimulated the ATPase activity of P-gp membranes, which suggested that they might be substrates of P-gp. Moreover, in silico molecular docking studies revealed the virtual binding modes of these two compounds into human homology model of P-gp. In conclusion, sipholenol A-4-O-acetate and sipholenol A-4-O-isonicotinate efficiently inhibit the P-gp and may represent potential reversal agents for the treatment of multidrug resistant cancers.

Transport of the areca nut alkaloid arecaidine by the human proton-coupled amino acid transporter 1 (hPAT1).

OBJECTIVES: The pyridine alkaloid arecaidine is an ingredient of areca nut preparations. It is responsible for many physiological effects observed during areca nut chewing. However, the mechanism underlying its oral bioavailability has not yet been studied. We investigated whether the H⁺-coupled amino acid transporter 1 (PAT1, SLC36A1), which is expressed in the intestinal epithelium, accepts arecaidine, arecoline, isoguvacine and other derivatives as substrates. METHODS: Inhibition of L-[³H]proline uptake by arecaidine and derivatives was determined in Caco-2 cells expressing hPAT1 constitutively and in HeLa cells transiently transfected with hPAT1-cDNA. Transmembrane transport of arecaidine and derivatives was measured electrophysiologically in Xenopus laevis oocytes. KEY FINDINGS: Arecaidine, guvacine and isoguvacine but not arecoline strongly inhibited the uptake of L-[³H]proline into Caco-2 cells. Kinetic analyses revealed the competitive manner of L-proline uptake inhibition by arecaidine. In HeLa cells transfected with hPAT1-cDNA an affinity constant of 3.8 mm was obtained for arecaidine. Electrophysiological measurements at hPAT1-expressing X. laevis oocytes demonstrated that arecaidine, guvacine and isoguvacine are transported by hPAT1 in an electrogenic manner. CONCLUSION: We conclude that hPAT1 transports arecaidine, guvacine and isoguvacine across the apical membrane of enterocytes and that hPAT1 might be responsible for the intestinal absorption of these drug candidates.

The proteome response of potato leaves to priming agents and S-nitrosoglutathione.

The primed mobilization for more potent defense responses to subsequent stress has been shown for many plant species, but there is a growing need to identify reliable molecular markers for this unique phenomenon. In the present study a proteomic approach was used to screen similarities in protein abundance in leaves of primed potato (Solanum tuberosum L.) treated with four well-known inducers of plant resistance, i.e. ß-aminobutyric acid (BABA), γ-aminobutyric acid (GABA), Laminarin and 2,6-dichloroisonicotinic acid (INA), respectively. Moreover, to gain insight into the importance of nitric oxide (NO) in primed protein accumulation the potato leaves were supplied by S-nitrosoglutathione (GSNO), as an NO donor. The comparative analysis, using two-dimensional electrophoresis and mass spectrometry, revealed that among 25 proteins accumulated specifically after BABA, GABA, INA and Laminarin treatments, 13 proteins were accumulated also in response to GSNO. Additionally, overlapping proteomic changes between BABA-primed and GSNO-treated leaves showed 5 protein spots absent in the proteome maps obtained in response to the other priming agents. The identified 18 proteins belonged, in most cases, to functional categories of primary metabolism. The selected proteins including three redox-regulated enzymes, i.e. glyceraldehyde 3-phosphate dehydrogenase, carbonic anhydrase, and fructose-bisphosphate aldolase, were discussed in relation to the plant defence responses. Taken together, the overlapping effects in the protein profiles obtained between priming agents, GSNO and cPTIO treatments provide insight indicating that the primed potato exhibits unique changes in the primary metabolism, associated with selective protein modification via NO.

[Transformation of 2- and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria].

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt 1 strain possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 strain containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5-4 times lower compared to 4-cyanopyridine and 1.6-2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt 1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on china clay was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carbonic acids.

Modulation of anticonvulsant effects of cannabinoid compounds by GABA-A receptor agonist in acute pentylenetetrazole model of seizure in rat.

Cannabinoid system plays an important role in controlling neuronal excitability and brain function. On the other hand, modulation of gamma-aminobutyric acid (GABA) transmission is one of the initial strategies for the treatment of seizure. The aim of the present study was to evaluate possible interaction between cannabinoidergic and GABAergic systems in pentylenetetrazole (PTZ)-induced acute seizure in rat. Drugs were administered by intracerebroventricular (i.c.v.) administration 20 min before a single intraperitoneal (i.p.) injection of PTZ and the latency to the first generalized tonic-clonic seizure was measured. Both the cannabinoid receptor agonist WIN55212-2 (10, 30, 50 and 100 µg/rat) and the GABA-A receptor agonist isoguvacine (IGN; 10, 30 and 50 µg/rat) significantly increased the latency of seizure occurrence. Moreover, the fatty acid amide hydrolase inhibitor URB597 showed no anticonvulsive effect while the monoacyl glycerol lipase (MAGL) inhibitor URB602 (10, 50 and 100 µg/rat) protected rats against PTZ-induced seizure. Moreover, co-administration of IGN and cannabinoid compounds attenuated the anticonvulsant action of both WIN55212-2 and IGN in this model of seizure. Our data suggests that exogenous cannabinoid WIN55212-2 and MAGL inhibitor URB602 imply their antiseizure action in part through common brain receptorial system. Moreover, the antagonistic interaction of cannabinoids and IGN in protection against PTZ-induced seizure could suggest the involvement of GABAergic system in their anticonvulsant action.

Aminopyridinecarboxamide-based inhaled IKK-2 inhibitors for asthma and COPD: Structure-activity relationship.

Installation of sites for metabolism in the lead compound PHA-767408 was the key focus of the IKK-2 inhaled program. This paper reports our efforts to identify a novel series of aminopyridinecarboxamide-based IKK-2 inhibitors, which display low nanomolar potency against IKK-2 with long duration of action (DOA), and metabolically labile to phase I and/or phase II metabolizing enzymes with potential capability for multiple routes of clearance. Several compounds have demonstrated their potential usefulness in the treatment of asthma and chronic obstructive pulmonary disease (COPD).

Inhibition of chorismate-utilising enzymes by 2-amino-4-carboxypyridine and 4-carboxypyridone and 5-carboxypyridone analogues.

Several 2-amino-4-carboxypyridine, 4- and 5-carboxypyridone-based compounds were prepared and tested against three members of the chorismate-utilising enzyme family, anthranilate synthase, isochorismate synthase and salicylate synthase. Most compounds exhibited low micromolar inhibition of these three enzymes. The most potent inhibitor was a 4-carboxypyridone analogue bearing a lactate side chain on the pyridyl nitrogen which exhibited inhibition constants of 5, 91 and 54 muM against anthranilate synthase, isochorismate synthase and salicylate synthase respectively.

Expression, purification and functional characterization of IkappaB kinase-2 (IKK-2) mutants.

NF-kappaB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-kappaB regulation, IKK-2 is a viable target for drug discovery. In order to enable structure-based design of IKK-2 inhibitors, we carried out a rational generation of IKK-2 mutants based on induced-fit docking of a selective IKK-2 inhibitor, PHA-408, into the homology model of IKK-2. One mutant we have characterized is a catalytically inactive form of IKK-2, D145A IKK-2, wherein the catalytic aspartic acid, D145 was replaced with alanine. Unlike the WT enzyme, D145A IKK-2 is devoid of kinase activity despite its ability to bind ATP with high affinity and is not phosphorylated at the T loop. In addition, this mutant binds a diverse collection of inhibitors with comparable binding affinities to WT IKK-2. Another interesting mutant we have characterized is F26A IKK-2 (F26 is an aromatic residue located at the very tip of the Gly-rich loop). Pre-incubation of F26A IKK-2 with PHA-408 revealed the role of F26 in the time-dependent binding of this inhibitor. Thus, functional characterization of these mutants provides the first evidence showing the role of a Gly-rich loop residue of a kinase in binding kinetics. These two mutants along with others that we have identified could be used to validate homology models and probe the interactions of IKK-2 with a variety of inhibitors.

Continuous hydrolysis of 4-cyanopyridine by nitrilases from Fusarium solani O1 and Aspergillus niger K10.

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 degrees C with a deactivation constant (k (d)) and enzyme half-life of 0.014 h(-1) and 50 h, respectively, but the latter exhibited an even higher stability characterized by k (d) = 0.008 h(-1) and half-life of 87 h at 40 degrees C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.

Exploring the PI3K alpha and gamma binding sites with 2,6-disubstituted isonicotinic derivatives.

A homology model of the p110alpha catalytic subunit of PI3Kalpha was generated from the p110gamma crystal structure. Using this model, an isonicotinic scaffold was designed for chemically exploring the PI3Kalpha and gamma binding sites. A focused library of derivatives was synthesized and tested. The morpholine acids 5a and 5b proved to be the most potent analogs.

Infrared and Raman study of some isonicotinic acid metal(II) halide and tetracyanonickelate complexes.

In this study the M(IN)(2)Ni(CN)(4) [where M: Co, Ni, and Cd, and IN: isonicotinic acid, abbreviated to M-Ni-IN] tetracyanonickelate and some metal halide complexes with the following stoichiometries: M(IN)(6)X(2) (M: Co; X: Cl and Br, and M: Ni; X: Cl, Br and I) and Hg(IN)X(2) (X: Cl, Br, and I) were synthesized for the first time. Certain chemical formulas were determined using elemental analysis results. The FT-IR and Raman spectra of the metal halide complexes were reported in the 4000-0 cm(-1) region. The FT-IR spectra of tetracyanonickelate complexes were also reported in the 4000-400 cm(-1) region. Vibrational assignments were given for all the observed bands. For a given series of isomorphous complexes, the sum of the difference between the values of the vibrational modes of the free isonicotinic acid and coordinated ligand was found to increase in the order of the second ionization potentials of metals. The frequency shifts were also found to be depending on the halogen. The proposed structure of tetracyanonickelate complexes consists of polymeric layers of /M-Ni(CN)(4)/(infinity) with the isonicotinic acid molecules bound directly to the metal atom.