Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme

A series of 1,2,3-trisubstituted indolizines (2a–2f, 3a–3d, and 4a–4c) were screened for in vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b–2d, 3a–3d, and 4a–4c were active against the H37Rv-MTB strain with minimum inhibitory concentration (MIC) ranging from 4 to 32 µg/mL, whereas the indolizines 4a–4c, with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC = 16–64 µg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthranilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demonstrated no toxicity for these compounds. Thus, the indolizines warrant further development and may represent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and drug-resistant MTB strains.     

Molecular Mechanisms,Thermodynamics, and Dissociation Kinetics of Knob-Hole Interactions in Fibrin

Polymerization of fibrin, the primary structural protein of blood clots and thrombi, occurs through binding of knobs ‘A’ and ‘B’ in the central nodule of fibrin monomer to complementary holes ‘a’ and ‘b’ in the γ- and β-nodules, respectively, of another monomer. We characterized the A:a and B:b knob-hole interactions under varying solution conditions using molecular dynamics simulations of the structural models of fibrin(ogen) fragment D complexed with synthetic peptides GPRP (knob ‘A’ mimetic) and GHRP (knob ‘B’ mimetic). The strength of A:a and B:b knob-hole complexes was roughly equal, decreasing with pulling force; however, the dissociation kinetics were sensitive to variations in acidity (pH 5–7) and temperature (T = 25–37 °C). There were similar structural changes in holes ‘a’ and ‘b’ during forced dissociation of the knob-hole complexes: elongation of loop I, stretching of the interior region, and translocation of the moveable flap. The disruption of the knob-hole interactions was not an “all-or-none” transition as it occurred through distinct two-step or single step pathways with or without intermediate states. The knob-hole bonds were stronger, tighter, and more brittle at pH 7 than at pH 5. The B:b knob-hole bonds were weaker, looser, and more compliant than the A:a knob-hole bonds at pH 7 but stronger, tighter, and less compliant at pH 5. Surprisingly, the knob-hole bonds were stronger, not weaker, at elevated temperature (T = 37 °C) compared with T = 25 °C due to the helix-to-coil transition in loop I that helps stabilize the bonds. These results provide detailed qualitative and quantitative characteristics underlying the most significant non-covalent interactions involved in fibrin polymerization.     

Identification of a Pair of Phospholipid:Diacylglycerol Acyltransferases from Developing Flax (Linum usitatissimum L.) Seed Catalyzing the Selective Production of Trilinolenin

The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3^cisΔ9,12,15) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce ∼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.     

Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl–trimethyl–homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

A heterokont Bubblegum acyl-CoA synthetase (ACSBG), or lipidosin, is essential in Microchloropsis (Nannochloropsis) gaditana and thio-esterifies 16:1 and 18:3 fatty acids to Coenzyme A in vivo.     

A Lipidomic Approach to Understanding Free Fatty Acid Lipogenesis Derived from Dissolved Inorganic Carbon within Cnidarian-Dinoflagellate Symbiosis

The cnidarian-dinoflagellate symbiosis is arguably one of the most important within the marine environment in that it is integral to the formation of coral reefs. However, the regulatory processes that perpetuate this symbiosis remain unresolved. It is essential to understand these processes, if we are to elucidate the mechanisms that support growth and resource accumulation by coral host, and conversely, recently observed reduction and/or mortality of corals in response to rapid environmental change. This study specifically focused on one area of metabolic activity within the symbiosis, that of free fatty acid synthesis within both the dinoflagellate symbionts and cnidarian host. The main model system used was Aiptasia pulchella and Symbiodinium sp. in combination with aposymbiotic A. pulchella, the symbiotic coral Acropora millepora system and dinoflagellate culture. Fatty acids (FAs) were selected because of their multiple essential roles inclusive of energy storage (resource accumulation), membrane structure fluidity and cell signaling. The study addressed free FA lipogenesis by using a new method of enriched stable isotopic (¹³C) incorporation from dissolved inorganic carbon (DI¹³C) combined with HPLC-MS. FAs derived from DI¹³C aligned with a mixture of known lipogenesis pathways with the addition of some unusual FAs. After 120 hr, ¹³C-enriched FA synthesis rates were attributed to only a complex integration of both n–3 and n–6 lipogenesis pathways within the dinoflagellate symbionts. Furthermore, there was no detectible evidence of symbiont derived enriched isotope fatty acids, catabolized ¹³C derivatives or DI¹³C being directly utilized, in host late n–6 pathway long-chain FA lipogenesis. These findings do not align with a popular mutualistic translocation model with respect to the use of translocated symbiont photoassimilates in host long-chain FA lipogenesis, which has important connotations for linking nutrient sources with metabolite production and the dynamic regulation of this symbiosis.     

Discovery of 2,4-thiazolidinedione-tethered coumarins as novel selective inhibitors for carbonic anhydrase IX and XII isoforms

Different 2,4-thiazolidinedione-tethered coumarins 5a–b, 10a–n and 11a–d were synthesised and evaluated for their inhibitory action against the cancer-associated hCAs IX and XII, as well as the physiologically dominant hCAs I and II to explore their selectivity. Un-substituted phenyl-bearing coumarins 10a, 10 h, and 2-thienyl/furyl-bearing coumarins 11a–c exhibited the best hCA IX (K_Is between 0.48 and 0.93 µM) and hCA XII (K_Is between 0.44 and 1.1 µM) inhibitory actions. Interestingly, none of the coumarins had any inhibitory effect on the off-target hCA I and II isoforms. The sub-micromolar compounds from the biochemical assay, coumarins 10a, 10 h and 11a–c, were assessed in an in vitro antiproliferative assay, and then the most potent antiproliferative agent 11a was tested to explore its impact on the cell cycle phases and apoptosis in MCF-7 breast cancer cells to provide more insights into the anticancer activity of these compounds.     

Intestine-specific expression of acyl CoA:diacylglycerol acyltransferase 1 reverses resistance to diet-induced hepatic steatosis and obesity in Dgat1−/− mice

Mice deficient in acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme in triacylglycerol (TG) biosynthesis, are resistant to high-fat (HF) diet-induced hepatic steatosis and obesity. DGAT1-deficient (Dgat1^−/−) mice have no defect in quantitative absorption of dietary fat; however, they have abnormally high levels of TG stored in the cytoplasm of enterocytes, and they have a reduced postprandial triglyceridemic response. We generated mice expressing DGAT1 only in the intestine (Dgat1^IntONLY) to determine whether this phenotype contributes to resistance to HF diet-induced hepatic steatosis and obesity in Dgat1^−/− mice. Despite lacking DGAT1 in liver and adipose tissue, we found that Dgat1^IntONLY mice are not resistant to HF diet-induced hepatic steatosis or obesity. The results presented demonstrate that intestinal DGAT1 stimulates dietary fat secretion out of enterocytes and that altering this cellular function alters the fate of dietary fat in specific tissues.     

Discovery of a small-molecule inhibitor of the TRIP8b–HCN interaction with efficacy in neurons

Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide–gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b–HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b–HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b–HCN interaction.     

Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica

Diacylglycerol acyltransferases (DGAT) are involved in the acylation of sn-1,2-diacylglycerol. Palm kernel oil, extracted from Elaeis guineensis (oil palm) seeds, has a high content of medium-chain fatty acids mainly lauric acid (C12:0). A putative E. guineensis diacylglycerol acyltransferase gene (EgDGAT1-1) is expressed at the onset of lauric acid accumulation in the seed endosperm suggesting that it is a determinant of medium-chain triacylglycerol storage. To test this hypothesis, we thoroughly characterized EgDGAT1-1 activity through functional complementation of a Yarrowia lipolytica mutant strain devoid of neutral lipids. EgDGAT1-1 expression is sufficient to restore triacylglycerol accumulation in neosynthesized lipid droplets. A comparative functional study with Arabidopsis thaliana DGAT1 highlighted contrasting substrate specificities when the recombinant yeast was cultured in lauric acid supplemented medium. The EgDGAT1-1 expressing strain preferentially accumulated medium-chain triacylglycerols whereas AtDGAT1 expression induced long-chain triacylglycerol storage in Y. lipolytica. EgDGAT1-1 localized to the endoplasmic reticulum where TAG biosynthesis takes place. Reestablishing neutral lipid accumulation in the Y. lipolytica mutant strain did not induce major reorganization of the yeast microsomal proteome. Overall, our findings demonstrate that EgDGAT1-1 is an endoplasmic reticulum DGAT with preference for medium-chain fatty acid substrates, in line with its physiological role in palm kernel. The characterized EgDGAT1-1 could be used to promote medium-chain triacylglycerol accumulation in microbial-produced oil for industrial chemicals and cosmetics.     

Tobacco Mesophyll Protoplasts Synthesize 1,3-beta-Glucanase, Chitinases, and "Osmotins" during in Vitro Culture

Tobacco (Nicotiana tabacum) mesophyll protoplasts synthesize six basic proteins (a, a′, a₁, b, b′, and c) which are undetectable in the leaf and whose synthesis is reduced by auxin (Y Meyer, L Aspart, Y Chartier [1984] Plant Physiol 75: 1027-1033). Polypeptides a, a′, and a₁ were shown to have similar mobilities on two-dimensional electrophoresis as one 1,3-β-glucanase and two chitinases from tobacco mosaic virus-infected leaves. In immunoblotting experiments, polypeptide a was recognized by specific antibodies raised against the 1,3-β-glucanase and a′ and a₁ reacted with anti-chitinase antibodies. Similarly, b and b′ comigrated with osmotin and its neutral counterpart, two proteins characteristic of salt-adapted tobacco cells, and reacted with anti-osmotin antibodies. In addition it has been shown that 1,3-β-glucanase and chitinase activities increased at the same time as a, a′, and a₁ accumulated in cultivated protoplasts. Finally, polypeptide c was also detected in tobacco mosaic virus-infected leaves but could not be identified as any of the pathogenesis-related proteins characterized so far in tobacco. Thus, cultivated tobacco protoplasts synthesize and accumulate typical stress proteins.     

Potential of Piperazinylalkylester Prodrugs of 6-Methoxy-2-Naphthylacetic Acid (6-MNA) for Percutaneous Drug Delivery

Piperazinylalkyl ester prodrugs (4a–5d) of 6-methoxy-2-naphthylacetic acid (6-MNA) (1) were synthesized and evaluated in vitro for the purpose of percutaneous drug delivery. These ionizable prodrugs exhibited varying aqueous solubilities and lipophilicities depending on the pH of the medium. The prodrugs (4a–5c) showed higher aqueous solubility and similar lipophilicity at pH 5.0 and lower aqueous solubility and higher lipophilicity at pH 7.4 in comparison to 6-MNA. The chemical and enzymatic hydrolyses of the prodrugs was investigated in aqueous buffer solutions (pH 5.0 and 7.4) and in 80% human serum (pH 7.4) at 37°C. The prodrugs showed moderate chemical stability (t_1/2 = 6–60 h) but got readily hydrolyzed enzymatically to 6-MNA with half-life ranging from 10–60 min. In the in vitro permeation study using rat skin, the flux of 6-MNA and the prodrugs was determined in aqueous buffers of pH 5.0 and 7.4. The prodrug (5b) showed 7.9- and 11.2-fold enhancement in skin permeation compared to 6-MNA (1) at pH 5.0 and 7.4, respectively. It was concluded that the parent NSAIDs having favorable pharmacokinetic and pharmacodynamic properties coupled with increased skin permeability of their prodrugs could give better options for the treatment of rheumatic diseases.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0240-6) contains supplementary material, which is available to authorized users.KEY WORDS: 6-MNA, NSAID, piperazinylalkylester, prodrug, skin permeation     

Development of 3-methyl/3-(morpholinomethyl)benzofuran derivatives as novel antitumor agents towards non-small cell lung cancer cells

As one of the most lethal malignancies, lung cancer is considered to account for approximately one-fifth of all malignant tumours-related deaths worldwide. This study reports the synthesis and in vitro biological assessment of two sets of 3-methylbenzofurans (4a–d, 6a–c, 8a–c and 11) and 3-(morpholinomethyl)benzofurans (15a–c, 16a–b, 17a–b and 18) as potential anticancer agents towards non-small cell lung carcinoma A549 and NCI-H23 cell lines, with VEGFR-2 inhibitory activity. The target benzofuran-based derivatives efficiently inhibited the growth of both A549 and NCI-H23 cell lines with IC₅₀ spanning in ranges 1.48–47.02 and 0.49–68.9 µM, respectively. The three most active benzofurans (4b, 15a and 16a) were further investigated for their effects on the cell cycle progression and apoptosis in A549 (for 4b) and NCI-H23 (for 15a and 16a) cell lines. Furthermore, benzofurans 4b, 15a and 16a displayed good VEGFR-2 inhibitory activity with IC₅₀ equal 77.97, 132.5 and 45.4 nM, respectively.     

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication

Zinc pyrithione (1a), together with its analogues 1b–h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC₅₀=1.88 ± 0.49 µM) and PL^Pro (IC₅₀=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b–h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.     

Sharing primary percutaneous coronary intervention care: first experiences with South Limburg ST-elevation myocardial infarction network

BackgroundIn the region of South Limburg, the Netherlands, a shared ST-elevation myocardial infarction (STEMI) networking system (SLIM network) was implemented. During out-of-office hours, two percutaneous coronary intervention (PCI) centres—Maastricht University Medical Centre and Zuyderland Medical Centre—are supported by the same interventional cardiologist. The aim of this study was to analyse performance indicators within this network and to compare them with contemporary European Society of Cardiology guidelines.MethodsKey time indicators for an all-comer STEMI population were registered by the emergency medical service and the PCI centres. The time measurements showed a non-Gaussian distribution; they are presented as median with 25th and 75th percentiles.ResultsBetween 1 February 2018 and 31 March 2019, a total of 570 STEMI patients were admitted to the participating centres. The total system delay (from emergency call to needle time) was 65 min (53–77), with a prehospital system delay of 40 min (34–47) and a door-to-needle time of 22 min (15–34). Compared with in-office hours, out-of-office hours significantly lengthened system delays (55 (47–66) vs 70 min (62–81), p < 0.001), emergency medical service transport times (29 (24–34) vs 35 min (29–40), p < 0.001) and door-to-needle times (17 (14–26) vs 26 min (18–37), p < 0.001).ConclusionsWith its effective patient pathway management, the SLIM network was able to meet the quality criteria set by contemporary European revascularisation guidelines.     

SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis–induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis–stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre–mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis–induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection–induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis–induced inflammatory immune responses and mapped out a novel SGK1–TRAF2/3–c-Jun–NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.     

Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production

Key message

We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes.

Abstract

The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the “food versus fuel” issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and LEAFY COTYLEDON2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80 % increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.