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Here, we evaluate the performance of our range of ensemble simulation based binding free energy calculation protocols, called ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) for use in fragment based drug design scenarios. ESMACS is designed to generate reproducible binding affinity predictions from the widely used molecular mechanics Poisson-Boltzmann surface area (MMPBSA) approach. We study ligands designed to target two binding pockets in the lactate dehydogenase A target protein, which vary in size, charge and binding mode. When comparing to experimental results, we obtain excellent statistical rankings across this highly diverse set of ligands. In addition, we investigate three approaches to account for entropic contributions not captured by standard MMPBSA calculations: (1) normal mode analysis, (2) weighted solvent accessible surface area (WSAS) and (3) variational entropy.

Drug-target residence time, the length of time for which a small molecule stays bound to its receptor target, has increasingly become a key property for optimization in drug discovery programs. However, its in silico prediction has proven difficult. Here we describe a method, using atomistic ensemble-based steered molecular dynamics (SMD), to observe the dissociation of ligands from their target G protein-coupled receptor in a time scale suitable for drug discovery. These dissociation simulations accurately, precisely, and reproducibly identify ligand-residue interactions and quantify the change in ligand energy values for both protein and water. The method has been applied to 17 ligands of the A2A adenosine receptor, all with published experimental kinetic binding data. The residues that interact with the ligand as it dissociates are known experimentally to have an effect on binding affinities and residence times. There is a good correlation ( R2 = 0.79) between the computationally calculated change in water-ligand interaction energy and experimentally determined residence time. Our results indicate that ensemble-based SMD is a rapid, novel, and accurate semi-empirical method for the determination of drug-target relative residence time.

2D-IR spectroscopy reveals a role for protein structural dynamics in antimicrobial-resistance., Antimicrobial resistance represents a growing global health problem. The emergence of novel resistance mechanisms necessitates the development of alternative approaches to investigate the molecular fundamentals of resistance, leading ultimately to new strategies for counteracting them. To gain deeper insight into antibiotic–target interactions, the binding of the frontline anti-tuberculosis drug isoniazid (INH) to a target enzyme, InhA, from Mycobacterium tuberculosis was studied using ultrafast two-dimensional infrared (2D-IR) spectroscopy and molecular simulations. Comparing wild-type InhA with a series of single point mutations, it was found that binding of the INH–NAD inhibitor to susceptible forms of the enzyme increased the vibrational coupling between residues located in the Rossmann fold co-factor binding site of InhA and suppressed dynamic fluctuations of the enzyme structure. The effect correlated with biochemical assay data, being reduced in the INH-resistant S94A mutant and absent in the biochemically-inactive P193A control. Molecular dynamics simulations and calculations of inter–residue couplings indicate that the changes in coupling and dynamics are not localised to the co-factor binding site, but permeate much of the protein. We thus propose that the resistant S94A mutation circumvents subtle changes in global structural dynamics caused by INH upon binding to the wild-type enzyme that may impact upon the formation of important protein–protein complexes in the fatty acid synthase pathway of M. tuberculosis.

Aim To evaluate the efficacy of ethanolic extract of Salvadora persica (S. persica) and BioPure MTAD (a mixture of a tetracycline isomer, an acid, and a detergent) in removing the intracanal smear layer of primary teeth. Materials and methods The root canal of 40 extracted human primary anterior teeth were cleaned, shaped and grouped into experimental (n = 30) and control (n = 10). The root canals of the positive (n = 5) and the negative control (n = 5) were irrigated for 3 minutes with 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) or saline respectively. The canals in the experimental groups were irrigated for 3 minutes with 5 ml of 1 mg/ml ethanolic extract of S. persica (n = 15) or BioPure MTAD (n = 15) and then flushed with 2 ml of saline. The presence or absence of smear layer at the coronal and middle portion of each canal were examined under a scanning electron microscope (SEM). Results A significant d ifference (p = 0 .004) i n s mear l ayer removal between S. persica and MTAD at the middle third of the canal was observed. MTAD solution was as effective as 17% EDTA in removing the smear layer. Conclusion MTAD was significantly more effective in smear layer removal than S. persica solution at the middle third of the canal wall. Clinical significance Both tested irrigant solutions have the ability to remove the intracanal smear from primary root dentin following cleaning and shaping of the root canal and could be an alternative to EDTA. How to cite this article Balto H, Salama F, Al-Mofareh S, Al-Yahya F. Evaluation of Different Irrigating Solutions on Smear Layer Removal of Primary Root Dentin. J Contemp Dent Pract 2015;16(3):187-191.

Aim The aim of this study was to assess the effect of repeated (twice) applications of two fluoride varnishes (Duraflor and CavityShield) on the surface micromorphology of a high-viscosity glass ionomer (Fuji IX GP), a compomer (F2000), and a flowable composite (Filtek™ Flow) using a profilometer and scanning electron microscope (SEM). Methods and Materials Fifteen specimens were prepared from each material, surface roughness (Ra) was measured with a profilometer, and an impression was made for epoxy replicas. The fluoride varnishes were applied to the experimental specimens of each material at repeated intervals of 48 hours. For all specimens, Ra was measured and SEM replicas were examined. Results The final Ra of glass ionomer was 3.49 ± 0.59 (mean ± SD) for CavityShield, 4.69 ± 1.33 for Duraflor, and 2.96 ± 1.53 for the controls. The final Ra of flowable composite was 0.53 ± 0.20 for CavityShield, 2.61 ± 3.08 for Duraflor, and 0.15 ± 0.09 for controls. For glass ionomer and flowable composite, Duraflor was associated with a significantly higher roughness at the final measurement compared to controls (P < 0.05). SEM micrographs showed differing surface topographies which in many specimens confirmed Ra analysis. Conclusion Fuji IX GP and Filtek™ Flow showed significantly higher roughness after two applications of Duraflor compared to controls. Citation Salama FS, Schulte KM, Iseman MF, Reinhardt JW. Effects of Repeated Fluoride Varnish Application on Different Restorative Surfaces. J Contemp Dent Pract 2006 November;(7)5:054-061.

The structural properties of uracil photohydrates at the monomer and dimer level in aqueous solution have been examined in detail by nmr spectroscopy. Based on such evidence, the absolute configurations of the two diastereomers have been assigned, and the conformational perturbations induced by photohydration have been evaluated. In all instances, photohydration shifts the /sup 2/E reversible /sup 3/E puckering equilibrium of the sugar ring of the uridylyl fragment towards /sup 2/E (from 12 to 18%). Significant base-base interactions were observed in both hydrated dimers despite the loss of the planar ..pi..-system in the uracil fragment. In addition, the rate of photohydration for a particular dimer pair is shown to be inversely dependent on the amount of base stacking in the parent dimer. This latter parameter has also been correlated with the ratio of the two possible diastereomers formed in the reaction and is associated with a preferential attack at one face of the pyrimidine base ring. The possible biological significance of the above-mentioned conformational aspects is discussed.

The intimate details of the conformational features and dynamics of the trinucleoside diphosphates CpCpA and ApCpC in aqueous solution have been arrived at by the complete analysis of their proton magnetic resonance spectra. In addition to the right-handed stacked species in which the phosphodiester torsions conform to the gauche-gauche domains, sugar puckers /sup 3/E, C4'-C5' approx. = 60/sup 0/, C5'-O5' approx. = 180 /sup 0/, C3'-O3' approx. = 205/sup 0/, and chi/sub CN/ approx. = 40/sup 0/, the trimers display a variety of spatial configurations, an important one being a bulged configuration in which the central nucleotide unit is bulged out, enabling stacking interactions between the end units. It is further shown that the 3' acceptor end of tRNA, CpCpA, displays considerable flexibility for the terminal adenine nucleotide unit. Theoretical NMR calculations demonstrate that the predominant solution conformation does not conform to the CCA terminus of tRNA as reported by four independent crystallographic studies of tRNA/sup Phe/. It is shown that the preferred intramolecular order of CCA in solution is such that chi/sub 1/ = chi/sub 2/ = chi/sub 3/ = 40/sup 0/, all the three sugars are in /sup 3/E, psi/sub 1/ = psi/sub 2/ = psi/sub 3/ =more » 60/sup 0/, phi/sub 2/ and phi/sub 3/ = 170 and 180/sup 0/, respectively, phi/sub 1/' = phi/sub 2/' = 205/sup 0/, and ..omega../sub 1//..omega../sub 1/' and 0/sub 2//..omega../sub 2/' = 240/205/sup 0/ and 295/265/sup 0/, respectively.« less

In order to obtain information about the conformational features in a polyribonucleotide at the nearest neighbor level, detailed nuclear magnetic resonance studies of the dinucleoside monophosphates ApA, ApG, GpA, UpU, CpC, UpC, and CpU were undertaken. Proton spectra were recorded at 100, 220, 270, or 300 MHz for D2O solutions, 0.01-0.03 M, pD 7.4 at 20+/-2 degrees C. Spectra of ApA, ApG, UpU, and UpC were also recorded in the temperature range of 70-90 degrees C. Unambiguous signal assignments of all proton resonances were made with the aid of selectively deuterated dimers. Complete, accurate sets of nuclear magnetic resonance (NMR) parameters were derived for each nucleotidyl unit by simulation-iteration methods. A complete set of chemical shift and coupling constant data was also obtained for all the constituent monomeric units at a concentration and ionization state comparable to that of the dimers. Conformational properties were evaluated quantitatively for most of the bonds in the dinucleoside monophosphates using procedures developed in earlier studies. All of the dimers have a flexible conformational framework in aqueous solution. While flexibility is allowed and alternate conformations are accessible, these molecules nevertheless attempt to achieve conformational identity by showing preferences--sometimes overwhelming preferences--for certain orientations. Thus the ribose rings exist as equilibrium mixtures of C2'-endo in equilibrium C3'-endo conformers with a bias for the C3'-endo pucker in most cases. The C4'-C5' bonds of both nucleotidyl units show significant preference (70-85%) for a gg conformation. Similarly, the dominant conformer (80-90%) about C5'-O5' is g'g'. Even though an unambiguous determination of the orientation about C3'-O3' cannot be made, there is suggestive evidence that the orientation of the 3' phosphate group is coupled to the ribose conformational equilibrium and it is likely that a 3Eg- in equilibrium 2Eg+ equilibrium exists with a bias for the 3Eg- coupled conformation in which the H3'-C3'-O3'-P dihedral angle is about 34-38 degrees. The individual nucleotidyl units in the dimers differ in several key ways from corresponding monomer conformations. Specifically, the ribose equilibrium C2'-endo in equilibrium C3'-endo shifts in favor of C3'-endo upon dimerization, the only exception being UpU. The C4'-C5' and C5'-O5' bonding network in the dimer forms a stable conformational unit and no correlation exists in the dimers between the conformational preference of this fragment and ribose conformer population. The temperature data for the dimers and dimerization data clearly indicate that the transition C2'-endo leads to C3'-endo is directly related to XCN changes brought about by dimerization and stacking...

Conformational properties of (2'-5') and (3'-5') CpC have been determined by proton magnetic resonance spectroscopy at 220 MHz. The ribose ring structures are predominantly 3E with the exception of the ring from the 2'-phosphate fragment of C(2'-5')pC which exhibits an 2E pucker. Bases are oriented anti with respect to the ribose and the conformations about C4'-C5', C5'-O5', C3'-O3' (C2'-O2') are gg, g'g', and g+ in equilibrium g-, respectively. The dimers exist as mixtures of stacked (g+g+ and g-g- about the P-O(C) bonds) and unstacked species at 20 degrees C. Stacking is estimated to be 35% in both dimers.

A detailed /sup 1/H 220-MHz NMR study has been made of 1-(..beta..-D-lyxofuranosyl)uracil cyclic 3',5'-monophosphate (1a, 3',5'-cLUMP), 1-(..beta..-D-lyxofuranosyl)-5,6-dihydrouracil cyclic 3',5'-monophosphate (1b, 3',5'-cLDHUMP), 1-(..beta..-D-lyxofuranosyl)uracil cyclic 2',5'-monophosphate (2a, 2',5'-cLUMP, 1-(..beta..-D-lyxofuranosyl)-5,6-dihydrouracil cyclic 2',5'-monophosphate (2b, 2',5'-cLDHUMP), and 9-(..cap alpha..-D-arabinofuranosyl)adenine cyclic 2',5'-monophosphate (3, ..cap alpha..-2',5'-cAAMP) in D/sub 2/O solution. Conformational analyses showed the cyclic 2',5'-phosphates (2a,b and 3) to exhibit sugar conformations in the range of /sup 2/E whereas the cyclic 3',5'-phosphates (1a,b) showed a preference for the /sup 3/E to /sub 4//sup 3/T conformation. The unusually large /sup 3/J/sub PH/ coupling of approx. 31 Hz which had previously been observed for J/sub 5'P/ in 9-(..beta..-D-arabinofuranosyl)adenine cyclic 2',5'monophosphate (2c, 2',5'-cAAMP) and for 1-(..beta..-D-arabinofuranosyl)cytosine cyclic 2',5'-monophosphate (2d, 2',5'-cACMP) was again apparent in 2a,b but not in 3. This difference is attributed to a steric interaction between the ..beta..-oriented base and the cyclic 2',5'-phosphate ring in 2a-d, which is not present in 3 where the base is oriented ..cap alpha..; this allows the phosphate ring to take up a less-strained conformation to that in 2a-d. The cyclic phosphate rings of the compounds described in this study fix the sugar rings into a particular conformation, precluding the /sup 2/E reversible /sup 3/E conformer equilibrium usually found in acyclic mononucleotides.more » In the cases of 1a and 2a, this permits an accurate evaluation of the solution properties of the parent lyxonucleoside and the conformations of lyxouridine and lyxoadenosine are shown to be different from a /sup 3/E//sub 4//sup 3/T reversible /sub 1//sup 2/T//sub 3//sup 2/T equilibrium predicted earlier for lyxouridine. Furthermore, a detailed NMR examination has been made of the 2a ..-->.. la isomerization in aqueous solution, and a mechanistic rationale is proposed.« less

A detailed 1 H 220-MHz n.m.r. study of 9-(β- d -xylofuranosyl)adenine 3′,5′-cyclic monophosphate (3′,5′-xylo-cAMP, 1 ) and 9-(∇- d -arabinofuranosyl)adenine 2′,5′-cyclic monophosphate (2′,5′-ara-cAMP, 2 ) in D 2 O solution is described. The sugar-ring conformations in 1 and 2 are shown to be 3 E and 2 E , respectively, and the phosphate rings are in a chair form. An unusual 4 J P,H coupling of 2.4 Hz is observed between H-4′ and phosphorus in 1 and a vicinal J P,H of 30.8 Hz between H-5′ and phosphorus in 2 . This latter coupling verifies a similar value found previously in the ara-cytidine analog of 2 . A comparison of the conformational properties of cyclic nucleotides having fused phosphate and sugar rings has been made, together with an assessment of the use of the Karplus constants in such ring-systems.

Isotherms are presented for the adsorption of phenol, nitrobenzene, and sodium benzene sulfonate from aqueous solution on several different carbons, including a channel black and adsorbent carbons. The content of chemisorbed oxygen on these carbons was modified by oxidation and reduction. The adsorption measurements indicate that chemisorbed oxygen substantially reduces the capacity of the carbons for the adsorbates studied, and this behavior appears to be reversible with respect to the introduction or removal of chemisorbed oxygen. Dilute phenol solutions display greater sensitivity to chemisorbed oxygen on the carbon than do more concentrated solutions of phenol. In the adsorption of sodium benzenesulfonate, the presence of chemisorbed oxygen does not appear to interfere with the action of aqueous calcium ions in enhancing the amount adsorbed.

High-resolution 31P-NMR and ESR spectroscopies are used to probe the role of manganese in oxygen metabolism, in vivo, by Staphylococcus aureus. The linewidth of the intracellular orthophosphate resonance in the 31P-NMR spectrum and the amplitude of the ESR sextet of signals due to Mn2+ hexaquo ions are found to be sensitive to the oxygenation state of the cells. These results are attributed to changes in the oxidation state of the manganese. It is concluded that manganous ions are oxidized to Mn3+ in oxygenated cells. Mn3+ is in turn reduced to Mn2+ under anaerobic conditions. The Mn2+ is also oxidized to Mn3+ by hydrogen peroxide probably as a result of the disproportionation of H2O2 to H2O and O2 by an active catalase in S. aureus. Addition of mercaptoethanol to a suspension of oxygenated cells results in the reduction of Mn3+ to Mn2+.

Isotherms are presented for the adsorption of phenol, nitrobenzene, and sodium benzene sulfonate from aqueous solution on several different carbons, including a channel black and adsorbent carbons. The content of chemisorbed oxygen on these carbons was modified by oxidation and reduction. The adsorption measurements indicate that chemisorbed oxygen substantially reduces the capacity of the carbons for the adsorbates studied, and this behavior appears to be reversible with respect to the introduction or removal of chemisorbed oxygen. Dilute phenol solutions display greater sensitivity to chemisorbed oxygen on the carbon than do more concentrated solutions of phenol. In the adsorption of sodium benzenesulfonate, the presence of chemisorbed oxygen does not appear to interfere with the action of aqueous calcium ions in enhancing the amount adsorbed.

Antimicrobial resistance represents a growing global health problem. The emergence of novel resistance mechanisms necessitates the development of alternative approaches to investigate the molecular fundamentals of resistance, leading ultimately to new strategies for counteracting them. To gain deeper insight into antibiotic-target interactions, the binding of the frontline anti-tuberculosis drug isoniazid (INH) to a target enzyme, InhA, from Mycobacterium tuberculosis was studied using ultrafast two-dimensional infrared (2D-IR) spectroscopy and molecular simulations. Comparing wild-type InhA with a series of single point mutations, it was found that binding of the INH-NAD inhibitor to susceptible forms of the enzyme increased the vibrational coupling between residues located in the Rossmann fold co-factor binding site of InhA and suppressed dynamic fluctuations of the enzyme structure. The effect correlated with biochemical assay data, being reduced in the INH-resistant S94A mutant and absent in the biochemically-inactive P193A control. Molecular dynamics simulations and calculations of inter-residue couplings indicate that the changes in coupling and dynamics are not localised to the co-factor binding site, but permeate much of the protein. We thus propose that the resistant S94A mutation circumvents subtle changes in global structural dynamics caused by INH upon binding to the wild-type enzyme that may impact upon the formation of important protein-protein complexes in the fatty acid synthase pathway of M. tuberculosis.

The ability to compute from first principles the infrared spectrum of a protein in solution phase representing a biological system would provide a useful connection to atomistic models of protein structure and dynamics. Indeed, such calculations are a vital complement to 2DIR experimental measurements, allowing the observed signals to be interpreted in terms of detailed structural and dynamical information. In this article, we have studied nine structurally and spectroscopically well‐characterized proteins, representing a range of structural types. We have simulated the equilibrium conformational dynamics in an explicit point charge water model. Using the resulting trajectories based on MD simulations, we have computed the one and two dimensional infrared spectra in the Amide I region, using an exciton approach, in which a local mode basis of carbonyl stretches is considered. The role of solvent in shifting the Amide I band (by 30 to 50 cm−1) is clearly evident. Similarly, the conformational dynamics contribute to the broadening of peaks in the spectrum. The inhomogeneous broadening in both the 1D and 2D spectra reflects the significant conformational diversity observed in the simulations. Through the computed 2D cross‐peak spectra, we show how different pulse schemes can provide additional information on the coupled vibrations. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc.