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.

Pharmacokinetics, pharmacodynamics, safety and tolerability of cilofexor, a novel nonsteroidal Farnesoid X receptor agonist, in healthy volunteers.

Cilofexor is a nonsteroidal farnesoid X receptor (FXR) agonist being evaluated for treatment of nonalcoholic steatohepatitis (NASH) and primary sclerosing cholangitis (PSC). This work characterized the pharmacokinetics, pharmacodynamic, safety, and tolerability of cilofexor in healthy participants. Cilofexor single and multiple once-daily doses (10 to 300 mg fasting or fed and twice-daily doses [15 and 50 mg; fed]; tablet formulation) were evaluated. In each cohort, participants were randomized to active drug or placebo in a 4:1 ratio (planned n = 15/cohort). Multiple dosing was for 14 days. Pharmacokinetic and pharmacodynamic samples were collected and safety and tolerability were assessed. Overall, 120 participants were enrolled in the study and 118 participants received at least one dose of study drug. Cilofexor pharmacokinetics followed bi-exponential disposition and its exposure increased in a less-than-dose-proportional manner over the 10 to 300 mg dose range, with no significant accumulation with repeated dosing. Moderate-fat meal reduced cilofexor area under the plasma concentration versus time curve (AUC) by 21% to 45%. Cilofexor increased plasma levels of fibroblast growth factor19 (FGF19) and reduced the serum bile acid intermediate 7α-hydroxy-4-cholesten-3-one (C4) and bile acids in an exposure-dependent manner. Cilofexor doses >30 mg appeared to achieve the plateau of intestinal FXR activation. Cilofexor was generally well tolerated; all treatment-emergent adverse events (TEAEs) were mild or moderate in severity, with headache being the most frequently observed TEAE. The pharmacokinetics pharmacodynamic safety, and tolerability results from this study supported further evaluations, and informed dose selection, of cilofexor in phase II studies in patients with NASH and PSC.

The hydrogen bond continuum in solid isonicotinic acid.

The understanding and correct description of intermolecular hydrogen bonds are crucial in the field of multicomponent pharmaceutical solids, such as salts and cocrystals. Solid isonicotinic acid can serve as a suitable model for the development of methods that can accurately characterize these hydrogen bonds. Experimental solid-state NMR has revealed a remarkable temperature dependence and deuterium-isotope-induced changes of the chemical shifts of the atoms involved in the intermolecular hydrogen bond; these NMR data are related to changes of the average position of the hydrogen atom. These changes of NMR parameters were interpreted using periodic DFT path-integral molecular dynamics (PIMD) simulations. The small size of the unit cell of isonicotinic acid allowed for PIMD simulations with the computationally demanding hybrid DFT functional. Calculations of NMR parameters based on the hybrid-functional PIMD simulations are in excellent agreement with experiment. It is thus demonstrated that an accurate characterization of intermolecular hydrogen bonds can be achieved by a combination of NMR experiments and advanced computations.

Safety and efficacy of combination therapy with semaglutide, cilofexor and firsocostat in patients with non-alcoholic steatohepatitis: A randomised, open-label phase II trial.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is associated with increased risk of liver-related and cardiovascular morbidity and mortality. Given the complex pathophysiology of NASH, combining therapies with complementary mechanisms may be beneficial. This trial evaluated the safety and efficacy of semaglutide, a glucagon-like peptide-1 receptor agonist, alone and in combination with the farnesoid X receptor agonist cilofexor and/or the acetyl-coenzyme A carboxylase inhibitor firsocostat in patients with NASH. METHODS: This was a phase II, open-label, proof-of-concept trial in which patients with NASH (F2-F3 on biopsy, or MRI-proton density fat fraction [MRI-PDFF] ≥10% and liver stiffness by transient elastography ≥7 kPa) were randomised to 24 weeks' treatment with semaglutide 2.4 mg once weekly as monotherapy or combined with once-daily cilofexor (30 or 100 mg) and/or once-daily firsocostat 20 mg. The primary endpoint was safety. All efficacy endpoints were exploratory. RESULTS: A total of 108 patients were randomised to semaglutide (n = 21), semaglutide plus cilofexor 30 mg (n = 22), semaglutide plus cilofexor 100 mg (n = 22), semaglutide plus firsocostat (n = 22) or semaglutide, cilofexor 30 mg and firsocostat (n = 21). Treatments were well tolerated - the incidence of adverse events was similar across groups (73-90%) and most events were gastrointestinal in nature. Despite similar weight loss (7-10%), compared with semaglutide monotherapy, combinations resulted in greater improvements in liver steatosis measured by MRI-PDFF (least-squares mean of absolute changes: -9.8 to -11.0% vs. -8.0%), liver biochemistry, and non-invasive tests of fibrosis. CONCLUSIONS: In patients with mild-to-moderate fibrosis due to NASH, semaglutide with firsocostat and/or cilofexor was generally well tolerated. In exploratory efficacy analyses, treatment resulted in additional improvements in liver steatosis and biochemistry vs. semaglutide alone. Given this was a small-scale open-label trial, double-blind placebo-controlled trials with adequate patient numbers are warranted to assess the efficacy and safety of these combinations in NASH. CLINICAL TRIAL REGISTRATION NUMBER: NCT03987074. LAY SUMMARY: Non-alcoholic fatty liver disease and its more severe form, non-alcoholic steatohepatitis (NASH), are serious liver conditions that worsen over time if untreated. The reasons people develop NASH are complex and combining therapies that target different aspects of the disease may be more helpful than using single treatments. This trial showed that the use of 3 different types of drugs, namely semaglutide, cilofexor and firsocostat, in combination was safe and may offer additional benefits over treatment with semaglutide alone.

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.

Novel candidates in the clinical development pipeline for TB drug development and their synthetic approaches.

Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis (Mtb) and one of the deadliest infectious diseases in the world. Mtb has the ability to become dormant within the host and to develop resistance. Hence, new antitubercular agents are required to overcome problems in the treatment of multi-drug-resistant Tb (MDR-Tb) and extensively drug-resistant Tb (XDR-Tb) along with shortening the treatment time. Several efforts are being made to develop very effective new drugs for Tb, within the pharmaceutical industry, the academia and through public-private partnerships. This review will address the antitubercular activities, biological target, mode of action, synthetic approaches and thoughtful concept for the development of several new drugs currently in the clinical trial pipeline (up to October 2019) for tuberculosis. The aim of this review may be very useful in scheming new chemical entities (NCEs) for Mtb.

Antitumor efficacy of CHMFL-KIT-110 solid dispersion in mouse xenograft models of human gastrointestinal stromal tumors.

PURPOSE: CHMFL-KIT-110, a selective c-KIT kinase inhibitor for gastrointestinal stromal tumors (GISTs), possesses a poorly water-soluble, limiting the further development of the drug. This study was to investigate the antitumor efficacy of CHMFL-KIT-110 and CHMFL-KIT-110 solid dispersion (laboratory code: HYGT-110 SD) in GIST tumor xenograft models and to explore the PK/PD relationship of HYGT-110 SD. METHODS: Plasma concentrations of HYGT-110 and HYGT-110 SD were determined by LC-MS/MS in KM mice. Antitumor activity was evaluated by measuring tumor volume and weight in c-KIT-dependent GIST xenograft models. PK/PD relationship was assessed by LC-MS/MS and Western Blot in the GIST-T1 xenografted mice. RESULTS: HYGT-110 exhibited a low oral bioavailability (10.91%) in KM mice. Compared with HYGT-110 treatment, the Cmax and AUC0-t of HYGT-110 SD in mice plasma were substantially increased by 18.81 and 6.76-fold, respectively. HYGT-110 SD (10, 30, and 100 mg/kg/day) also could dose-dependently decrease the tumor volume and weight in the GIST-882 cell-inoculated xenograft mouse models and show 86.35% tumor growth inhibition (TGI) at 28 days at a 25 mg/kg bid dosage in the GIST-T1 cell-inoculated xenograft mouse model. The free concentration of HYGT-110 in plasma was closely correlated with the inhibition of c-KIT phosphorylation levels in tumor tissues. CONCLUSIONS: In comparison with the HPMC formulation, both improved PK and PD characteristics of the solid dispersion formulation of CHMFL-KIT-110 were observed in in vivo animal experiments.

The identification of farnesoid X receptor modulators as treatment options for nonalcoholic fatty liver disease.

INTRODUCTION: The farnesoid-x-receptor (FXR) is a ubiquitously expressed nuclear receptor selectively activated by primary bile acids. AREA COVERED: FXR is a validated pharmacological target. Herein, the authors review preclinical and clinical data supporting the development of FXR agonists in the treatment of nonalcoholic fatty liver disease. EXPERT OPINION: Development of systemic FXR agonists to treat the metabolic liver disease has been proven challenging because the side effects associated with these agents including increased levels of cholesterol and LDL-c and reduced HDL-c raising concerns over their long-term cardiovascular safety. Additionally, pruritus has emerged as a common, although poorly explained, dose-related side effect with all FXR ligands, but is especially common with OCA. FXR agonists that are currently undergoing phase 2/3 trials are cilofexor, tropifexor, nidufexor and MET409. Some of these agents are currently being developed as combination therapies with other agents including cenicriviroc, a CCR2/CCR5 inhibitor, or firsocostat an acetyl CoA carboxylase inhibitor. Additional investigations are needed to evaluate the beneficial effects of combination of these agents with statins. It is expected that in the coming years, FXR agonists will be developed as a combination therapy to minimize side effects and increase likelihood of success by targeting different metabolic pathways.

The novel pathogen-responsive glycosyltransferase UGT73C7 mediates the redirection of phenylpropanoid metabolism and promotes SNC1-dependent Arabidopsis immunity.

Recent studies have shown that global metabolic reprogramming is a common event in plant innate immunity; however, the relevant molecular mechanisms remain largely unknown. Here, we identified a pathogen-induced glycosyltransferase, UGT73C7, that plays a critical role in Arabidopsis disease resistance through mediating redirection of the phenylpropanoid pathway. Loss of UGT73C7 function resulted in significantly decreased resistance to Pseudomonas syringae pv. tomato DC3000, whereas constitutive overexpression of UGT73C7 led to an enhanced defense response. UGT73C7-activated immunity was demonstrated to be dependent on the upregulated expression of SNC1, a Toll/interleukin 1 receptor-type NLR gene. Furthermore, in vitro and in vivo assays indicated that UGT73C7 could glycosylate p-coumaric acid and ferulic acid, the upstream metabolites in the phenylpropanoid pathway. Mutations that lead to the loss of UGT73C7 enzyme activities resulted in the failure to induce SNC1 expression. Moreover, glycosylation activity of UGT73C7 resulted in the redirection of phenylpropanoid metabolic flux to biosynthesis of hydroxycinnamic acids and coumarins. The disruption of the phenylpropanoid pathway suppressed UGT73C7-promoted SNC1 expression and the immune response. This study not only identified UGT73C7 as an important regulator that adjusts phenylpropanoid metabolism upon pathogen challenge, but also provided a link between phenylpropanoid metabolism and an NLR gene.

Selective inhibition of JAK3 signaling is sufficient to reverse alopecia areata.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) are key intracellular mediators in the signal transduction of many cytokines and growth factors. Common γ chain cytokines and interferon-γ that use the JAK/STAT pathway to induce biological responses have been implicated in the pathogenesis of alopecia areata (AA), a T cell-mediated autoimmune disease of the hair follicle. We previously showed that therapeutic targeting of JAK/STAT pathways using the first-generation JAK1/2 inhibitor, ruxolitinib, and the pan-JAK inhibitor, tofacitinib, was highly effective in the treatment of human AA, as well as prevention and reversal of AA in the C3H/HeJ mouse model. To better define the role of individual JAKs in the pathogenesis of AA, in this study, we tested and compared the efficacy of several next-generation JAK-selective inhibitors in the C3H/HeJ mouse model of AA, using both systemic and topical delivery. We found that JAK1-selective inhibitors as well as JAK3-selective inhibitors robustly induced hair regrowth and decreased AA-associated inflammation, whereas several JAK2-selective inhibitors failed to restore hair growth in treated C3H/HeJ mice with AA. Unlike JAK1, which is broadly expressed in many tissues, JAK3 expression is largely restricted to hematopoietic cells. Our study demonstrates inhibiting JAK3 signaling is sufficient to prevent and reverse disease in the preclinical model of AA.

Crystal structures of two inhibitors in complex with histone lysine demethylase 4D (KDM4D) provide new insights for rational drug design.

Histone lysine demethylase 4D (KDM4D), also known as JMJD2D, plays an important role in cell proliferation and survival and has been associated with several tumor types. KDM4D has emerged as a potential target for the treatment of human cancer. Here, we reported crystal complex structures for two KDM4D inhibitors, OWS [2-(1H-pyrazol-3-yl)isonicotinic acid] and 10r (5-hydroxy-2-methylpyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile), which were both determined to 2.0 Å. OWS is a newly discovered KDM4D inhibitor (IC50 = 4.28 µM) and the critical pharmacophores of this compound are confirmed by the complex structure. Compound 10r is a KDM4D inhibitor reported by us previously. To clarify the binding mode in more detail, the crystal structure was determined and the comparison analysis revealed unique interactions that had never been observed before. Overall, our data provide new structural insights for rational design and offer an opportunity for optimization of KDM4D inhibitors.

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.

Synthesis of Highly Potent Anti-Inflammatory Compounds (ROS Inhibitors) from Isonicotinic Acid.

In search of anti-inflammatory compounds, novel scaffolds containing isonicotinoyl motif were synthesized via an efficient strategy. The compounds were screened for their in vitro anti-inflammatory activity. Remarkably high activities were observed for isonicotinates 5-6 and 8a-8b. The compound 5 exhibits an exceptional IC50 value (1.42 ± 0.1 µg/mL) with 95.9% inhibition at 25 µg/mL, which is eight folds better than the standard drug ibuprofen (11.2 ± 1.9 µg/mL). To gain an insight into the mode of action of anti-inflammatory compounds, molecular docking studies were also performed. Decisively, further development and fine tuning of these isonicotinates based scaffolds for the treatment of various aberrations is still a wide-open field of research.

Intestine-specific FXR agonists as potential therapeutic agents for colorectal cancer.

Colorectal cancer (CRC) is one of the most malignant cancers in the world. A major cause of death in CRC patients is the limited therapeutic options in its advanced stages. The Farnesoid X receptor (FXR) is a member of the nuclear superfamily, which is effective in slowing the progression of colorectal cancer in addition to its extraordinary role in regulating metabolic disorders. Due to the systemic side-effects caused by non-selective agonists, the intestine-restricted FXR agonists can induce a whole-body benefit without activating the hepatic FXR, suggesting intestinal FXR activation as a potentially safer therapy in the treatment of CRC. This review highlights the effects of FXR on the disturbed bile acid circulation and the carcinogenesis of CRC and with a specific emphasis on listing the functions of several intestinal-restricted FXR agonists.

Design, synthesis and evaluation of carbazole derivatives as potential antimicrobial agents.

Five series of novel carbazole derivatives containing an aminoguanidine, dihydrotriazine, thiosemicarbazide, semicarbazide or isonicotinic moiety were designed, synthesised and evaluated for their antimicrobial activities. Most of the compounds exhibited potent inhibitory activities towards different bacterial strains (including one multidrug-resistant clinical isolate) and one fungal strain with minimum inhibitory concentrations (MICs) between 0.5 and 16 µg/ml. Compounds 8f and 9d showed the most potent inhibitory activities (MICs of 0.5-2 µg/ml). Furthermore, compounds 8b, 8d, 8f, 8k, 9b and 9e with antimicrobial activities were not cytotoxic to human gastric cancer cell lines (SGC-7901 and AGS) or a normal human liver cell line (L-02). Structure-activity relationship analyses and docking studies implicated the dihydrotriazine group in increasing the antimicrobial potency and reducing the toxicity of the carbazole compounds. In vitro enzyme activity assays suggested that compound 8f binding to dihydrofolate reductase might account for the antimicrobial effect.

The pontine Kölliker-Fuse nucleus gates facial, hypoglossal, and vagal upper airway related motor activity.

The pontine Kölliker-Fuse nucleus (KFn) is a core nucleus of respiratory network that mediates the inspiratory-expiratory phase transition and gates eupneic motor discharges in the vagal and hypoglossal nerves. In the present study, we investigated whether the same KFn circuit may also gate motor activities that control the resistance of the nasal airway, which is of particular importance in rodents. To do so, we simultaneously recorded phrenic, facial, vagal and hypoglossal cranial nerve activity in an in situ perfused brainstem preparation before and after bilateral injection of the GABA-receptor agonist isoguvacine (50-70 nl, 10 mM) into the KFn (n = 11). Our results show that bilateral inhibition of the KFn triggers apneusis (prolonged inspiration) and abolished pre-inspiratory discharge of facial, vagal and hypoglossal nerves as well as post-inspiratory discharge in the vagus. We conclude that the KFn plays a critical role for the eupneic regulation of naso-pharyngeal airway patency and the potential functions of the KFn in regulating airway patency and orofacial behavior is discussed.