Revealing the astragalin mode of anticandidal action

Due to limited arsenal of systemically available antifungal agents, infections caused by Candida albicans are difficult to treat and the emergence of drug-resistant strains present a major challenge to the clinicians worldwide. Hence further exploration of potential novel and effective antifungal drugs is required. In this study we have explored the potential of a flavonoid, astragalin, in controlling the growth of C. albicans, in both planktonic and biofilm forms by microdilution method; and in regulating the morphological switch between yeast and hyphal growth. Astragalin ability to interfere with membrane integrity, ergosterol synthesis and its role in the regulation of genes encoding for efflux pumps has been addressed. In our study, astragalin treatment produced good antimicrobial and significant antibiofilm activity. Anticandidal activity of astragalin was not related to ERG11 downregulation, neither to direct binding to CYP51 enzyme nor was linked to membrane ergosterol assembly. Instead, astragalin treatment resulted in reduced expression of CDR1 and also affected cell membrane integrity without causing cytotoxic effect on human gingival fibroblast cells. Considering that astragalin-mediated decreased expression of efflux pumps increases the concentration of antifungal drug inside the fungal cells, a combinatorial treatment with this agent could be explored as a novel therapeutic option for candidiasis

Dynophore-Based Approach in Virtual Screening: A Case of Human DNA Topoisomerase IIα

In this study, we utilized human DNA topoisomerase IIα as a model target to outline a dynophore-based approach to catalytic inhibitor design. Based on MD simulations of a known catalytic inhibitor and the native ATP ligand analog, AMP-PNP, we derived a joint dynophore model that supplements the static structure-based-pharmacophore information with a dynamic component. Subsequently, derived pharmacophore models were employed in a virtual screening campaign of a library of natural compounds. Experimental evaluation identified flavonoid compounds with promising topoisomerase IIα catalytic inhibition and binding studies confirmed interaction with the ATPase domain. We constructed a binding model through docking and extensively investigated it with molecular dynamics MD simulations, essential dynamics, and MM-GBSA free energy calculations, thus reconnecting the new results to the initial dynophore-based screening model. We not only demonstrate a new design strategy that incorporates a dynamic component of molecular recognition, but also highlight new derivates in the established flavonoid class of topoisomerase II inhibitors

Amlodipine Benzenesulfonate: A Mechanistic Investigation of Its Industrial Preparation via Detritylation of N-tritylamlodipine and Related NMR Studies

Kinetics and product analysis of detritylation of N-tritylamlodipine by benzenesulfonic acid in methanol, methanol-chloroform (volume ratio 9:1), ethanol, 2-propanol, and methanol/2-propanol (mole ratio 1:1) have been investigated by HPLC; amongst these reaction conditions are ones closely similar to those of one method of manufacturing amlodipine benzenesulfonate. Kinetics of detritylation of Ntritylamlodipine have also been investigated in methanol-d4 by 1H NMR spectroscopy and the agreement with the results by HPLC is good. The rate of detritylation increases with increasing concentrations of benzenesulfonic acid, and p-methoxy-substituents in the trityl group have been shown to lead to faster reactions. In methanol, the rate is hardly affected by 10 % (vol. fraction) chloroform. These studies relate to mechanistic investigations of acid-catalysed deaminations of methoxy-substituted tritylalkylamines, and Arrhenius activation parameters (Ea and A) are similar indicating a common generic mechanism. Acid-catalysed trans-esterification has been shown by HPLC to accompany detritylation in methanol, and attendant protium-deuterium exchange in the methyl at C6 by reversible acid-catalysed iminium ion formation in the 4-aryl-1,4-dihydropyridine moiety of both N-tritylamlodipine and amlodipine has been investigated in deuteriated methanol by 1H, 13C, and 15N NMR spectroscopy

Discovery of (phenylureido)piperidinyl benzamides as prospective inhibitors of bacterial autolysin E from Staphylococcus aureus

Autolysin E (AtlE) is a cell wall degrading enzyme that catalyzes the hydrolysis of the β-1,4-glycosidic bond between the N-acetylglucosamine and N-acetylmuramic acid units of the bacterial peptidoglycan. Using our recently determined crystal structure of AtlE from Staphylococcus aureus and a combination of pharmacophore modeling, similarity search, and molecular docking, a series of (Phenylureido)piperidinyl benzamides were identified as potential binders and surface plasmon resonance (SPR) and saturation-transfer difference (STD) NMR experiments revealed that discovered compounds bind to AtlE in a lower micromolar range. (phenylureido)piperidinyl benzamides are the first reported non-substrate-like compounds that interact with this enzyme and enable further study of the interaction of small molecules with bacterial AtlE as potential inhibitors of this target

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

International audienceBacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents

Characterization of Asparagine Deamidation in Immunodominant Myelin Oligodendrocyte Glycoprotein Peptide Potential Immunotherapy for the Treatment of Multiple Sclerosis

Mannan (polysaccharide) conjugated with a myelin oligodendrocyte glycoprotein (MOG) peptide, namely (KG)5MOG35–55, represents a potent and promising new approach for the immunotherapy of Multiple Sclerosis (MS). The MOG35–55 epitope conjugated with the oxidized form of mannan (poly-mannose) via a (KG)5 linker was found to inhibit the symptoms of MOG35–55-induced experimental autoimmune encephalomyelitis (EAE) in mice using prophylactic and therapeutic vaccinated protocols. Deamidation is a common modification in peptide and protein sequences, especially for Gln and Asn residues. In this study, the structural solution motif of deaminated peptides and their functional effects in an animal model for MS were explored. Several peptides based on the MOG35–55 epitope have been synthesized in which the Asn53 was replaced with Ala, Asp, or isoAsp. Our results demonstrate that the synthesized MOG peptides were formed to the deaminated products in basic conditions, and the Asn53 was mainly modified to Asp. Moreover, both peptides (wild type and deaminated derivative) conjugated with mannan (from Saccharomyces cerevisiae) independently inhibited the development of neurological symptoms and inflammatory demyelinating spinal cord lesions in MOG35–55-induced EAE. To conclude, mannan conjugated with a deamidated product did not affect the efficacy of the parent peptide

Evaluation of the published kinase inhibitor set to identify multiple inhibitors of bacterial ATP-dependent mur ligases

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 (IC50, 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

The Binding Mode of Second-Generation Sulfonamide Inhibitors of MurD: Clues for Rational Design of Potent MurD Inhibitors

<div><p>A series of optimized sulfonamide derivatives was recently reported as novel inhibitors of UDP-<em>N</em>-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). These are based on naphthalene-<em>N</em>-sulfonyl-D-glutamic acid and have the D-glutamic acid replaced with rigidified mimetics. Here we have defined the binding site of these novel ligands to MurD using ¹H/¹³C heteronuclear single quantum correlation. The MurD protein was selectively ¹³C-labeled on the methyl groups of Ile (δ1 only), Leu and Val, and was isolated and purified. Crucial Ile, Leu and Val methyl groups in the vicinity of the ligand binding site were identified by comparison of chemical shift perturbation patterns among the ligands with various structural elements and known binding modes. The conformational and dynamic properties of the bound ligands and their binding interactions were examined using the transferred nuclear Overhauser effect and saturation transfer difference. In addition, the binding mode of these novel inhibitors was thoroughly examined using unrestrained molecular dynamics simulations. Our results reveal the complex dynamic behavior of ligand–MurD complexes and its influence on ligand–enzyme contacts. We further present important findings for the rational design of potent Mur ligase inhibitors.</p> </div

Nontrivial NOE connectivities and corresponding distances (Å) calculated from the transferred NOESY spectra for alkyloxy derivatives.

<p>For the sake of clarity, the atom labels do not strictly follow IUPAC rules for all compounds.</p>a<p>Distances from X-ray structure, PDB code 2JFF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052817#pone.0052817-Kotnik1" target="_blank">[7]</a>. ^b Observed in 1D trace from 2D spectrum.^c Medium NOE cross-peak that can belong either to H1” or H5” due to signal overlap.</p