scholarly journals Mycobacterial MMAR_2193 catalyzes O-methylation of diverse polyketide cores

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262241
Author(s):  
Gorkha Raj Giri ◽  
Priti Saxena
Keyword(s):  
Wide Spectrum ◽  
Docking Studies ◽  
Mycobacterium Marinum ◽  
Bioactive Molecules ◽  
Wild Type ◽  
Type Iii ◽  
Methyltransferase Gene ◽  
Functional Analyses ◽  
Genomic Cluster

O-methylation of small molecules is a common modification widely present in most organisms. Type III polyketides undergo O-methylation at hydroxyl end to play a wide spectrum of roles in bacteria, plants, algae, and fungi. Mycobacterium marinum harbours a distinctive genomic cluster with a type III pks gene and genes for several polyketide modifiers including a methyltransferase gene, mmar_2193. This study reports functional analyses of MMAR_2193 and reveals multi-methylating potential of the protein. Comparative sequence analyses revealed conservation of catalytically important motifs in MMAR_2193 protein. Homology-based structure-function and molecular docking studies suggested type III polyketide cores as possible substrates for MMAR_2193 catalysis. In vitro enzymatic characterization revealed the capability of MMAR_2193 protein to utilize diverse polyphenolic substrates to methylate several hydroxyl positions on a single substrate molecule. High-resolution mass spectrometric analyses identified multi-methylations of type III polyketides in cell-free reconstitution assays. Notably, our metabolomics analyses identified some of these methylated molecules in biofilms of wild type Mycobacterium marinum. This study characterizes a novel mycobacterial O-methyltransferase protein with multi-methylating enzymatic ability that could be exploited to generate a palette of structurally distinct bioactive molecules.

Novel Spiro/non-Spiro Pyranopyrazoles: Eco-Friendly Synthesis, In-vitro Anticancer Activity, DNA Binding, and In-silico Docking Studies

2019 ◽  
Vol 15 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Paritosh Shukla ◽  
Ashok Sharma ◽  
Leena Fageria ◽  
Rajdeep Chowdhury
Keyword(s):  
Anticancer Activity ◽  
Anticancer Agents ◽  
In Silico ◽  
Antimicrobial Agents ◽  
Docking Studies ◽  
Bioactive Molecules ◽  
Microwave Assisted Synthesis ◽  
Binding Affinities ◽  
In Silico Docking

Background: Cancer being a deadly disease, many reports of new chemical entities are available. Pyranopyrazole (PPZ) compounds have also been disclosed as bioactive molecules but mainly as antimicrobial agents. Based on one previous report and our interest in anticancer drug design, we decided to explore PPZs as anticancer agents. To the best of our knowledge, we found that a comprehensive study, involving synthesis, in-vitro biological activity determination, exploration of the mechanism of inhibition and finally in-silico docking studies, was missing in earlier reports. This is what the present study intends to accomplish. Methods: Ten spiro and eleven non-spiro PPZ molecules were synthesized by environment-friendly multicomponent reaction (MCR) strategy. After subjecting each of the newly synthesized molecules to Hep3b hepatocellular carcinoma cell lines assay, we selectively measured the Optical Density (OD) of the most active ones. Then, the compound exhibiting the best activity was docked against human CHK- 1 protein to get an insight into the binding affinities and a quick structure activity relationship (SAR) of the PPZs. Results: The two series of spiro and non-spiro PPZs were easily synthesized in high yields using microwave assisted synthesis and other methods. Among the synthesized compounds, most compounds showed moderate to good anticancer activity against the MTT assay. After performing the absorbance studies we found that the non-spiro molecules showed better apoptosis results and appeared to bind to DNA causing disruption in their structures. Finally, the docking results of compound 5h (having N,Ndimethylamino substituted moiety) clearly showed good binding affinities as predicted by our experimental findings. Conclusion: The paper describes a comprehensive synthesis, in-vitro and docking studies done on new PPZs. The newly synthesized series of spiro and non-spiro PPZs were found to possess antineoplasmic activity as evinced by the studies on hep3b cells. Also, the UV visible absorbance study gave clues to the possible binding of these molecules to the DNA. Docking studies corroborated well with the experimental results. Thus, these new molecules appear to be potential anticancer agents, but further studies are required to substantiate and elaborate on these findings.

scholarly journals Regulation of the Ysa Type III Secretion System of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR

2004 ◽  
Vol 186 (13) ◽  
pp. 4056-4066 ◽  
Author(s):  
Kimberly A. Walker ◽  
Virginia L. Miller
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii ◽  
Regulatory Cascade ◽  
Two Component

ABSTRACT Yersinia enterocolitica biovar 1B contains two type III secretion systems (TTSSs), the plasmid-encoded Ysc-Yop system and the chromosomally encoded Ysa-Ysp system. Proteins secreted from the Ysa TTSS (Ysps) have only been detected in vitro when cells are cultured at 26°C in a high-NaCl medium. However, the exact role of the Ysa TTSS is unclear. Thus, investigations into the regulation of this system may help elucidate the role of the Ysps during the life cycle of Y. enterocolitica. Here we present evidence that the AraC-like regulator YsaE acts together with the chaperone SycB to regulate transcription of the sycByspBCDA operon, a phenomenon similar to that seen in the closely related Salmonella SPI-1 and Shigella flexneri Mxi-Spa-Ipa TTSSs. Deletion of either sycB or ysaE results in a twofold reduction in the activity of a sycB-lacZ fusion compared to the wild type. In a reconstituted Escherichia coli system, transcription of sycB was activated sixfold only when both YsaE and SycB were present, demonstrating that they are necessary for activation. ysrR and ysrS are located near the ysa genes and encode a putative two-component regulatory system. Mutations in either gene indicated that both YsrR and YsrS were required for secretion of Ysps. In addition, transcription from sycB-lacZ and ysaE-lacZ fusions was decreased 6.5- and 25-fold, respectively, in the ysrS mutant compared to the wild type. Furthermore, in the absence of NaCl, the activity of ysaE-lacZ was reduced 25-fold in the wild-type and ΔysrS strains, indicating that YsrS is probably required for the salt-dependent expression of the ysa locus. These results suggest that the putative two-component system YsrRS may be a key element in the regulatory cascade for the Ysa TTSS.

scholarly journals β-Defensins Coordinate In Vivo to Inhibit Bacterial Infections of the Trachea

Vaccines ◽  
2018 ◽  
Vol 6 (3) ◽  
pp. 57 ◽  
Author(s):  
Lisa Ryan ◽  
Jichuan Wu ◽  
Kyell Schwartz ◽  
Sunghan Yim ◽  
Gill Diamond
Keyword(s):  
Epithelial Cells ◽  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Airway Epithelial Cells ◽  
Mrna Levels ◽  
Wild Type ◽  
Type Iii ◽  
Primary Mouse

β-defensins are predicted to play an important role in innate immunity against bacterial infections in the airway. We previously observed that a type III-secretion product of Bordetella bronchiseptica inhibits the NF-κB-mediated induction of a β-defensin in airway epithelial cells in vitro. To confirm this in vivo and to examine the relative roles of other β-defensins in the airway, we infected wild-type C57BL/6 mice and mice with a deletion of the mBD-1 gene with B. bronchiseptica wild-type strain, RB50 and its mutant strain lacking the type III-secretion system, WD3. The bacteria were quantified in the trachea and the nasal tissue and mRNA levels of mouse β-defensin-3 (mBD-3) were assessed after 24 h. Infection with the wild-type bacterial strain resulted in lower mBD-3 mRNA levels in the trachea than in mice infected with the type III-deficient strain. Furthermore, we observed an increase in bacterial numbers of RB50 only in the tracheas of mBD-1-deficient mice. Neutrophils were also more abundant on the trachea in RB50 infected WT mice but not in the bronchiolar lavage fluid (BAL), compared with WD3 infected WT and mBD-1−/− mice, indicating that the coordination of β-defensin chemotactic effects may be confined to tracheal epithelial cells (TEC). RB50 decreased the ability of mice to mount an early specific antibody response, seven days after infection in both WT and mBD-1−/− mice but there were no differences in titers between RB50-infected WT and mBD-1−/− mice or between WD3-infected WT and mBD-1−/− mice, indicating mBD-1 was not involved in induction of the humoral immune response to the B. bronchiseptica. Challenge of primary mouse TEC in vitro with RB50 and WD3, along with IL-1β, further corroborated the in vivo studies. The results demonstrate that at least two β-defensins can coordinate early in an infection to limit the growth of bacteria in the trachea.

Inhibition of Cyclooxygenase Enzyme by Bioflavonoids in Horsegram Seeds Alleviates Pain and Inflammation

2020 ◽  
Vol 23 (9) ◽  
pp. 931-938 ◽  
Author(s):  
Malarvizhi Ramalingam ◽  
Veeresh K. Sali ◽  
Meenakshi Bhardwaj ◽  
Sugumar Mani ◽  
Hannah R. Vasanthi
Keyword(s):  
Animal Experiments ◽  
Docking Studies ◽  
Bioactive Molecules ◽  
Cyclooxygenase Inhibitors ◽  
Analgesic Agent ◽  
In Silico Docking ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Dose Dependent

Background: Inflammation and pain, mainly induced by the prostaglandins synthesized by the cyclooxygenase enzymes, may cause distress. To overcome this unpleasant stress in a safer manner, numerous natural molecules are proven for modulating the COX enzymes. Epicatechin and daidzein are two bioactive natural compounds present in horsegram, a legume known for its medicinal properties. Objective: The present study aims at evaluating the potential of horsegram, and some of its bioactive molecules, to be used as an anti-inflammatory and analgesic agent mediated by the inhibition of COX enzymes, which can be recommended as a substitute for chemically synthesized NSAIDs. Methods: The present work involved the quantification of epicatechin and daidzein present in horsegram seeds. The COX enzyme inhibitory nature of epicatechin and daidzein was tested using in silico docking analysis with Autodock software and was further confirmed by in vitro COX inhibitory biochemical assays. Furthermore, the anti-inflammatory and analgesic activities of the horsegram seeds were evaluated in animal experiments. Results: Horsegram seeds contain 158.1 microgram/g and 6.51 microgram/g of epicatechin and daidzein respectively. The docking studies reveal that both the bioactive molecules exhibit better binding efficiency with COX-2 protein as compared to COX-1. Hence, in vitro COX-2 inhibitory assay was performed for epicatechin, daidzein and compared with known analgesic agent diclofenac which revealed a pronounced dose dependent inhibitory activity. Furthermore, the analgesic and anti-inflammatory activity of horsegram in experimental animals exhibited a dose dependent effect which might be due to the presence of the bioactive compounds such as epicatechin and daidzein. Conclusion: The results suggest that epicatechin and daidzein present in horsegram are potent cyclooxygenase inhibitors and thus would be helpful in the management of inflammation and pain.

scholarly journals Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4510-4519 ◽  
Author(s):  
E Sporeno ◽  
R Savino ◽  
L Ciapponi ◽  
G Paonessa ◽  
A Cabibbo ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Interleukin 6 ◽  
Wide Spectrum ◽  
Myeloma Cell ◽  
Wild Type ◽  
Full Spectrum ◽  
Myeloma Cells ◽  
Interleukin 6 Receptor

Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super- antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.

The potent inhibition of human cytosolic sulfotransferase 1A1 by 17α-ethinylestradiol is due to interactions with isoleucine 89 on loop 1

2014 ◽  
Vol 20 (3) ◽  
Author(s):  
Katie Jo Rohn-Glowacki ◽  
Charles N. Falany
Keyword(s):  
Docking Studies ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Binding Assays ◽  
Wild Type Enzyme ◽  
Sulfotransferase 1A1 ◽  
Mechanism Of Inhibition ◽  
Potent Inhibition

AbstractDrug-drug interactions (DDI) with oral contraceptives containing 17α-ethinylestradiol (EE2) have been well characterized with regard to interactions with phase I drug metaolizing enzymes; however, DDI with EE2 and phase II enzymes have not been as thoroughly addressed. Our laboratory recently reported that in vitro EE2 potently inhibits human cytosolic sulfotransferase (SULT) 1A1 while EE2 was not sulfated until micromolar concentrations. Molecular docking studies demonstrated that Tyr169 and isoleucine 89 (Ile89) may play a role in the inhibitory and/or catalytic positioning of EE2 within the active site of SULT1A1. Therefore, the current study focused on determining the role of Ile89 in the inhibition of SULT1A1 utilizing site-directed mutagenesis. Ile89 was mutated to an alanine and the effect of the mutation was characterized using kinetic and binding assays. SULT1A1-Ile89Ala was found to have a Km for EE2 that was 11-fold greater than wild-type enzyme. A decreased affinity (Kd) of EE2 for SULT1A1-Ile89Ala was apparently responsible for the increase in Km, and also resulted in the loss of the potent inhibition. Molecular modeling was used in an attempt to determine the atomic level changes in binding of EE2 to SULT1A1-Ile89Ala. However, analysis of the effect of the single Ile89 mutation on both the open and closed homology models was not consistent with the docking and kinetic results. Overall, the mechanism of inhibition of EE2 for SULT1A1 is apparently the result of interactions of Ile89 with EE2 holding it in a potent inhibitory conformation, and mutation of the Ile89 significantly decreases the inhibition.

scholarly journals Type III Secretion-Dependent Modulation of Innate Immunity as One of Multiple Factors Regulated by Pseudomonas aeruginosa RetS

2006 ◽  
Vol 74 (7) ◽  
pp. 3880-3889 ◽  
Author(s):  
Irandokht Zolfaghar ◽  
David J. Evans ◽  
Reza Ronaghi ◽  
Suzanne M. J. Fleiszig
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Innate Immunity ◽  
Type Iii Secretion ◽  
Bacterial Colonization ◽  
Published Data ◽  
Wild Type ◽  
Type Iii ◽  
Severity Scores ◽  
Peripheral Ring

ABSTRACT Mutation of retS (rtsM) of Pseudomonas aeruginosa strain PA103 reduces its virulence in both ocular and respiratory murine models of infection. In vitro, retS mutants exhibit loss of the ExsA-regulated type III secretion system (TTSS), reduced twitching motility, and a decrease in association with, invasion of, and survival within corneal epithelial cells. In addition, transcription of multiple other virulence genes is positively and negatively affected by retS mutation. Since our published data show that ExoU and ExoT, the two TTSS effectors encoded by strain PA103, each confer virulence in this corneal model, we hypothesized that loss of virulence of retS mutants follows loss of type III secretion. Corneal pathology, bacterial colonization, and phagocyte infiltration were compared for wild-type PA103, retS mutants, and various TTSS mutants after infection with ∼106 CFU bacteria. Results showed that either a retS or an exsA (TTSS) mutation delayed disease progression, as illustrated by reduced severity scores and colonization levels during the first 48 h postinfection. Surprisingly, retS mutant infections then became more severe than those involving exsA mutants. By day 7, colonization levels of retS mutants even surpassed those of wild-type bacteria (more than twofold, P = 0.028). Although retS mutants caused more severe opacification of central corneas than both the wild type and the exsA mutants, neither mutant caused the peripheral ring opacity commonly associated with wild-type infection, suggesting that the TTSS was involved. Histological experiments with retS and various TTSS mutants showed that ring opacification required ExoU but not ExoT and that it consisted of dense polymorphonuclear phagocyte infiltration at the corneal periphery and the absence of any cell type in the central cornea. These data suggest that these P. aeruginosa TTSS effectors have different effects on innate immunity and that RetS influences virulence beyond its effects on the TTSS.

Molecular dynamics assisted mechanistic insight of Val430-Ala mutation of Rv1592c protein in isoniazid resistant Mycobacterium tuberculosis

2020 ◽  
Vol 16 ◽  
Author(s):  
Arbind Kumar ◽  
Pradeep Kumar Anand ◽  
Saahil Chandel ◽  
Anju Shrivatava ◽  
Jagdeep Kaur
Keyword(s):  
Molecular Dynamics ◽  
Resistant Strain ◽  
Shuttle Vector ◽  
Agar Plate ◽  
Docking Studies ◽  
Lipolytic Enzyme ◽  
Drug Resistant ◽  
Wild Type ◽  
The Impact

Background:: Multi drug-resistant tuberculosis is a major health threat to humans. Whole genome sequencing of several isoniazid (INH) resistant strains of M. tuberculosis revealed mutations in several genes. Rv1592c was demonstrated as lipolytic enzyme and its expression was up-regulated during isoniazid (INH) treatment. The valine at position 430 of Rv1592c was mutated to alanine frequently in the INH resistant strain of M. tuberculosis. Methods: In this report, an array of computational approaches was used to understand the role of Val430-Ala mutation in Rv1592c in INH resistance. The impact of mutations on structural stability and degree of INH modification was demonstrated using the molecular dynamics method. The mutation in the Rv1592c gene at V430 position was created by the PCR primer walking method. Mutant and wild type gene was cloned into E. coli-mycobacteria shuttle vector (pVV-16) and expressed in Mycobacterium smegmatis system. The isoniazid susceptibility assay was performed by agar plate culture spot and CFUs count assay. Results: This study demonstrated that the Val430 in Rv1592c makes the part of flap covering the substrate binding cavity. Mutation at Val430-Ala in Rv1592c caused the displacement of the flap region, resulting in uncovering a cavity, which allows accessibility of substrate to the active site cleft. The Val430-Ala mutation in Rv1592c created its structure energetically more stable. RMSD, RMSF and Rg simulation of mutant maintained overall stability throughout the simulation period while the native protein displayed comparatively more fluctuations. Moreover, docking studies showed that INH was bound into the active pocket of the mutant with considerable binding energy (−6.3 kcal/mol). In order to observe constant binding for INH, complexes were simulated for 50 ns. It was observed that after simulation, INH remained bound in the pocket with an increased molecular bonding network with the neighbor amino acid residues. In vitro studies clearly suggested that M. smegmatis expressing mutant has a better survival rate in isoniazid treatment as compared to wild type. Conclusion: Overall, this study at the outset suggested that the mutation observed in drug resistant strain provides stability to the Rv1592c protein and increased affinity towards the INH due to flap displacement, leading to the possibility for its modification. In vitro results supported our in silico findings.

scholarly journals Discovery of Bis-sydnone styryl ketone as a selective cyclooxygenase-2 inhibitor

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Abdualrahman Mohammed Abdualkader ◽  
Muhammad Taher ◽  
Nik Idris Nik Yusoff ◽  
Mohamed Alaama
Keyword(s):  
Wide Spectrum ◽  
Binding Pocket ◽  
Antimicrobial Activities ◽  
Bioactive Molecules ◽  
Unsaturated Ketone ◽  
Inhibitory Effects ◽  
Cox 2 ◽  
Anti Inflammatory ◽  
Cox 1

Abstract Background Various literature sources have documented a wide spectrum of therapeutic properties of sydnones including anti-inflammatory, anticancer, antimicrobial activities. Phenyl styryl ketones and their derivatives as members of the chalcone family have also been reported as significant bioactive molecules. The current study was initiated to evaluate the anti-inflammatory activity of sydnone-based compounds including some novel bis-sydnone styryl ketone hybrids. Results Twenty-five sydnone-containing compounds were successfully synthesized. Compounds 46-48 and 56-58 were reported as new sydnone derivatives. Whereas, compounds 61-63 were synthesized as novel molecules containing two sydnone rings linked via α,β-unsaturated ketone. The structures of the synthesized compounds were confirmed by FTIR, 1H NMR, 13C NMR and ToF-MS analyses. The in vitro COX inhibition assay showed varied activity. Compounds 47, 51, 58 and 63 showed the most potent COX inhibitory effects at a concentration of 200 μM. The selectivity index revealed that 63 was the best selective COX-2 inhibitor. Acetylation of the sydnone ring at C-4 was fruitful for the COX inhibitory effects. Docking analysis showed that COX-2 selectivity was due to a favourable positive charged interaction occurring between the sydnone ring of 63 and Arg513 of COX-2. Compound 51 was hydrogen bonded to Arg513. On the other hand, the low inhibitory effect of 63 against COX-1 was due to an unfavourable polar interaction with His513 in the binding pocket of COX-1. Conclusions The compounds were successfully synthesized and characterized. Compound 63 had a common architecture and pharmacophoric features with known selective COX-2 inhibitors (the coxib family) which make it a suitable candidate for the designing of selective and safe NSAID.

scholarly journals The Type III Secretion System of Biocontrol Pseudomonas fluorescens KD Targets the Phytopathogenic Chromista Pythium ultimum and Promotes Cucumber Protection

2005 ◽  
Vol 18 (9) ◽  
pp. 991-1001 ◽  
Author(s):  
Fabio Rezzonico ◽  
Christian Binder ◽  
Geneviève Défago ◽  
Yvan Moënne-Loccoz
Keyword(s):  
Pseudomonas Fluorescens ◽  
Type Iii Secretion ◽  
Plant Protection ◽  
Type Iii Secretion System ◽  
Pythium Ultimum ◽  
Secretion System ◽  
Activity Level ◽  
Wild Type ◽  
Type Iii

The type III secretion system (TTSS) is used by Proteobacteria for pathogenic or symbiotic interaction with plant and animal hosts. Recently, TTSSgenes thought to originate from the phytopathogen Pseudomonas syringae were evidenced in Pseudomonas fluorescens KD, which protects cucumber from the oomycete Pythium ultimum (kingdom Chromista/Stramenopila). However, it is not known whether te TTSS contributes to plant protection by the bacterium and, if so, whether it targets the plant or the phytopathogen. Inactivation of TTSS gene hrcV following the insertion of an omega cassette strongly reduced the biocontrol activity of the pseudomonad against P. ultimum on cucumber when compared with the wild type, but had no effect on its root-colonization ability. Analysis of a plasmid-based transcriptional hrpJ′-inaZ reporter fusion revealed that expression in strain KD of the operon containing hrcV was strongly stimulated in vitro and in situ by the oomycete and not by the plant. In vitro, both strain KD and its hrcV mutant reduced the activity level of the pectinase polygalacturonase (a key pathogenicity factor) from P. ultimum, but the reduction was much stronger with the wild type. Together, these results show that the target range of bacterial TTSS is not restricted to plants and animals but also can include members of Chromista/Stramenopila, and suggest that virulence genes acquired horizontally from phytopathogenic bacteria were functionally recycled in biocontrol saprophytic Pseudomonas spp., resulting in enhanced plant protection by the latter.

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