The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for ...antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC
50
, 32-368 µM). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.
To verify the effect of supplementation of different doses of Whey Proteins for 12 weeks on the gene expression of MTOR, MURF-1, MAFBX and on the alteration of the cellular diameter of the ...gastrocnemius muscle. Sample: 38 Wistar rats and supplemented for 12 weeks. The white portion of the right gastrocnemius muscle was embedded in Rna Latter solution and stored at -80 ° C and subjected to real-time polymerase chain reaction analysis. The left gastrocnemius muscle was intended for histological analysis. Gene expression of MTOR mRNA had lower statistical significance in the groups of Whey 2, Whey 4, Whey 6 than in the control group, in the order of 100%, 97%, 96%, respectively. Likewise, the gene expression of MURF-1 mRNA had lower statistical significance in the groups of Whey 2, Whey 4, Whey 6 than the control group, in the order of 65%, 75%, 84%, respectively. In line, the gene expression MAFBX mRNA had lower statistical significance in the groups Whey 2, Whey 4, Whey 6 in relation to the control group, in the order of 99%, 97%, 99%, respectively. There was no statistical difference in the cellular area of the gastrocnemius muscle between the groups. Therefore, supplementation of Whey Proteins at doses of 2, 4, 6 g / kg / day for 12 weeks in sedentary animals was not able to increase the gene expression of MTOR mRNA, however, it was able to reduce the gene expression of MURF -1 mRNA, MAFBX mRNA.
Verificar o efeito da suplementação de diferentes doses de Whey Proteins por 12 semanas na expressão gênica de MTOR, MURF-1, MAFBX e na alteração do diâmetro celular do músculo gastrocnêmio. Amostra: 38 ratos Wistar e suplementados por 12 semanas. A porção branca do músculo gastrocnêmio direito foi embebida em solução de Rna Latter e armazenada a -80°C e submetida à análise de reação em cadeia da polimerase em tempo real. O músculo gastrocnêmio esquerdo foi destinado à análise histológica. A expressão gênica do mRNA MTOR teve menor significância estatística nos grupos Whey 2, Whey 4, Whey 6 do que no grupo controle, na ordem de 100%, 97%, 96%, respectivamente. Da mesma forma, a expressão gênica do mRNA de MURF-1 teve menor significância estatística nos grupos Whey 2, Whey 4, Whey 6 do que no grupo controle, na ordem de 65%, 75%, 84%, respectivamente. Em linha, a expressão gênica MAFBX mRNA teve menor significância estatística nos grupos Whey 2, Whey 4, Whey 6 em relação ao grupo controle, na ordem de 99%, 97%, 99%, respectivamente. Não houve diferença estatística na área celular do músculo gastrocnêmio entre os grupos. Portanto, a suplementação de Whey Proteins nas doses de 2, 4, 6 g/kg/dia por 12 semanas em animais sedentários não foi capaz de aumentar a expressão gênica de MTOR mRNA, porém, foi capaz de reduzir a expressão gênica de MURF - 1 mRNA, mRNA MAFBX.
•The crystal structures of MurF in complex with ATP are reported.•The ATP-binding mode including a carbamoylated lysine and two Mg2+ ions is revealed.•Interactions of MurF with UDP, a part of ...substrate, are described.
MurF adds d-Ala-d-Ala dipeptide to UDP-N-acetylmuramyl-l-Ala-γ-d-Glu-m-DAP (or l-Lys) in an ATP-dependent manner, which is the last step in the biosynthesis of monomeric precursor of peptidoglycan. Here we report crystal structures of two MurF–ATP complexes: the MurF–ATP complex and the MurF–ATP–UDP complex. The ATP-binding mode revealed by the crystal structure of the MurF–ATP complex confirms the previous biochemical demonstration that a carbamoylated lysine and two Mg2+ ions are required for enzyme activity of MurF. The UDP–MurF interactions observed in the crystal structure of the MurF–ATP–UDP complex depict the characteristic substrate-binding mode of MurF. The emergence and dissemination of multidrug-resistant Acinetobacter baumannii strains are great threats to public health. Therefore, the structural information on A. baumannii MurF as a validated target for drug discovery will provide a framework to develop antibacterial agents against multidrug-resistant A. baumannii infections as well as to understand the reaction mechanism of MurF.
زمینه و هدف: آتروفی عضلانی یک فرآیند فعال کنترل شده بوسیله مسیرهای پیام‌‌رسانی و برنامـه‌‌های رونویسی ویژه است. آتروژین-1/ MAFbx و MuRF1 در مدل‌‌های مختلف آتروفی عضلانی، به طور مثبت تنظیم می‌‌شوند و ...مسئول افزایش تجزیه پروتئین از طریق سیستم پروتئازوم یوبیکوئیتین هستند. هدف از این مطالعه بررسی تاثیر یک دوره تمرین مقاومتی با شد‌‌ت‌‌های بالا و متوسط بر بیان ژن‌‌های ژن‌‌های miRNA-23a، MuRF و آتروژین- 1 در عضلات تند و کند انقباض رت‌‌های نر سالمند نژاد ویستار بود. روشتحقیق: در این تحقیق 30 سر موش صحرایی سالمند (23 ماهه) نژاد ویستار به شکل تصادفی به سه گروه (10=n) شامل تمرین مقاومتی با شدت متوسط (MIRT)، تمرین مقاومتی با شدت بالا (HIRT) و کنترل تقسیم شدند. برنامه تمرین مقاومتی شامل هشت هفته تمرین بالا رفتن از نردبان با شدت زیاد و متوسط (به ترتیب با 80 و 60 درصد حداکثر ظرفیت حمل ارادی) و با تکرار پنج روز در هفته بود. جداسازی عضله نعلی و عضله خم کننده دراز شست پا بلافاصله بعد از خونگیری در شرایط استریل انجام شد. بیان ژن MuRF، آتروژین -1 و miR-23a با تکنیک RT-PCR اندازهگیری شد. تجزیه تحلیل آماری با استفاده از آزمون تحلیل واریانس یک طرفه و دو طرفه در سطح معنیداری 05/0>p صورت گرفت. یافتهها: بیان ژن‌‌های MuRF و آتروژین-1 پس از هشت هفته در دو گروه مقاومتی نسبت به گروه کنترل کاهش معنیدار و بیان miR-23a افزایش معنیداری پیدا کرد (05/0>p)؛ اما اختلاف تأثیر دو مداخله صورت گرفته بر miR-23a، MuRF و آتروژین-1 در هر دو عضله معنیدار نبود (05/0<p). نتیجهگیری: به نظر میرسد تمرین مقاومتی با شد‌‌ت‌‌های متوسط و بالا، میتواند باعث کاهش سطوح استراحتی عوامل مرتبط با آتروفی عضلانی و ژن‌‌های هدف آن در رت‌‌های سالمند شود و پیشرفت سارکوپنیا در دوره سالمندی را مهار نماید.
Current therapeutic strategies for several diseases, including Mycobacterium tuberculosis infection, have evolved from an initial single-target treatment to a multitarget one. A multitarget ...antitubercular drugs targeting different mycobacterial proteins are more effective at suppressing bacterial growth. In this study, a high throughput virtual screening was performed to identify hits to the potential antitubercular multitarget: murA, murB, murC, murD, murE, murF, murG and murI from M. tuberculosis that is involved in peptidoglycan biosynthesis. In the virtual screening, we were docked 56,400 compounds of the ChEMBL antimycobacterial library and re-scored and identified the top 10 ranked compounds as antitubercular drug candidates. Further, the best common docked complex CHEMBL446262 was subjected to molecular dynamics simulation to understand the molecule's stability in the presence of an active site environment. After that, we have calculated binding free energy the top-ranked docked complexes using the MM/PBSA method. These ligands exhibited the highest binding affinity; find out novel drug-likeness might show the M. tuberculosis effect's inhibitor by interacting with multitarget Mur enzymes. New antitubercular therapies that include multitarget drugs may have higher efficacy than single-target medicines and provide a more straightforward antitubercular therapy regimen.
Communicated by Ramaswamy H. Sarma
Summary
Advancing age is associated with a progressive loss of skeletal muscle (SkM) mass and function. Given the worldwide aging demographics, this is a major contributor to morbidity, escalating ...socio‐economic costs and ultimately mortality. Previously, it has been established that a decrease in regenerative capacity in addition to SkM loss with age coincides with suppression of insulin/insulin‐like growth factor signalling pathways. However, genetic or pharmacological modulations of these highly conserved pathways have been observed to significantly enhance life and healthspan in various species, including mammals. This therefore provides a controversial paradigm in which reduced regenerative capacity of skeletal muscle tissue with age potentially promotes longevity of the organism. This paradox will be assessed and considered in the light of the following: (i) the genetic knockout, overexpression and pharmacological models that induce lifespan extension (e.g. IRS‐1/s6K KO, mTOR inhibition) versus the important role of these signalling pathways in SkM growth and adaptation; (ii) the role of the sirtuins (SIRTs) in longevity versus their emerging role in SkM regeneration and survival under catabolic stress; (iii) the role of dietary restriction and its impact on longevity versus skeletal muscle mass regulation; (iv) the crosstalk between cellular energy metabolism (AMPK/TSC2/SIRT1) and survival (FOXO) versus growth and repair of SkM (e.g. AMPK vs. mTOR); and (v) the impact of protein feeding in combination with dietary restriction will be discussed as a potential intervention to maintain SkM mass while increasing longevity and enabling healthy aging.
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Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of ...the bacterial Mur ligase family—MurC, MurD, MurE and MurF—are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park’s nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC–MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC–MurF enzymes in biochemical inhibition assays and molecules 10–14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC–MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents.
•Lymphatic filariasis is a vector borne parasitic disease that infects human lymphatic system by nematode Brugia malayi.•Peptidoglycan biosynthesis pathway from the Wolbachia endosymbiont of Brugia ...malayi represents an attractive therapeutic target for the development of novel anti-filarial agents.•Molecular modeling combined with molecular dynamics simulations and structure-based virtual screening were performed to identify potent lead molecules against UDP-N-acetylmuramoyl-tripeptide-d-alanyl-d-alanine ligase (MurF).•Based on the Glide score, predicted ADMET properties, binding affinity and mode of interactions with the MurF, five potent lead molecules were selected from different databases.•Promising lead molecules against MurF were identified.
Lymphatic filariasis is a parasitic disease caused by the worms Wuchereria bancrofti, Brugia malayi and Brugia timori. Three anti-filarial drugs namely Diethylcarbamazine, Ivermectin and Albendazole and their combinations are used as the control strategies for filariasis. The disease has received much attention in drug discovery due to the unavailability of vaccines and the toxic pharmaceutical properties of the existing drugs. In Wolbachia endosymbiont Brugia malayi, the UDP-N-acetylmuramoyl-tripeptide-d-alanyl-d-alanine ligase (MurF) plays a key role in peptidoglycan biosynthesis pathway and therefore can be considered as effective drug target against filariasis disease. Therefore, in the present study, MurF was selected as the therapeutic target to identify specific inhibitors against filariasis. Homology modeling was performed to predict the three-dimensional structure of MurF due to the absence of the experimental structure. Further molecular dynamics simulation and structure-based high throughput virtual screening with three different chemical databases (Zinc, Maybridge and Specs) were carried out to identify potent inhibitors and also to check their conformations inside the binding site of MurF, respectively. Top three compounds with high docking score and high relative binding affinity against MurF were selected. Further, validation studies, including predicted ADME (Absorption, Distribution, Metabolism, Excretion) assessment, binding free energy using MM-GBSA (Molecular Mechanics Generalized Born Surface Area) and DFT (Density Functional Theory) calculations were performed for the top three compounds. From the results, it was observed that all the three compounds were predicted to show high reactivity, acceptable range of pharmacokinetic properties and high binding affinity with the drug target MurF. Overall, the results could provide more understanding on the inhibition of MurF enzyme and the screened compounds could lead to the development of new specific anti-filarial drugs.
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•Ethyl pyridine substituted 3-cyanothiophene is identified as best docked compound.•Protein residues, Thr42 and Asp43 are involved in stabilizing the complex.•Molecular dynamic ...simulation revealed stable nature of the complex.•RDF and novel AFD analysis further supported the overall stability of the complex.•MM(PB/GB)SA energy calculation revealed dominancy of van der Waals energies.
MurF ligase catalyzes the final cytoplasmic step of bacterial peptidoglycan biosynthesis and, as such, is a validated target for therapeutic intervention. Herein, we performed molecular docking to identify putative inhibitors of Acinetobacter baumannii MurF (AbMurF). Based on comparative docking analysis, compound 114 (ethyl pyridine substituted 3-cyanothiophene) was predicted to potentially be the most active ligand. Computational pharmacokinetic characterization of drug-likeness of the compound showed it to fulfil all the parameters of Muegge and the MDDR rule. A molecular dynamic simulation of 114 indicated the complex to be stable on the basis of an average root mean square deviation (RMSD) value of 2.09Å for the ligand. The stability of the complex was further supported by root mean square fluctuation (RMSF), beta factor and radius of gyration values. Analyzing the complex using radial distribution function (RDF) and a novel analytical tool termed the axial frequency distribution (AFD) illustrated that after simulation the ligand is positioned in close vicinity of the protein active site where Thr42 and Asp43 participate in hydrogen bonding and stabilization of the complex. Binding free energy calculations based on the Poisson-Boltzmann or Generalized–Born Surface Area Continuum Solvation (MM(PB/GB)SA) method indicated the van der Waals contribution to the overall binding energy of the complex to be dominant along with electrostatic contributions involving the hot spot amino acids from the protein active site. The present results indicate that the screened compound 114 may act as a parent structure for designing potent derivatives against AbMurF in specific and MurF of other bacterial pathogens in general.
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