H274Y's Effect on Oseltamivir Resistance: What Happens Before the Drug Enters the Binding Site.

Increased reports of oseltamivir (OTV)-resistant strains of the influenza virus, such as the H274Y mutation on its neuraminidase (NA), have created some cause for concern. Many studies have been conducted in the attempt to uncover the mechanism of OTV resistance in H274Y NA. However, most of the reported studies on H274Y focused only on the drug-bound system, so the direct effects of the mutation on NA itself prior to drug binding still remain unclear. Therefore, molecular dynamics simulations of NA in apo form, followed by principal component analysis and interaction energy calculations, were performed to investigate the structural changes of the NA binding site as a result of the H274Y mutation. It was observed that the disruption of the NA binding site due to the H274Y mutation was initiated by the repulsive effect of Y274 on the 250-loop, which in turn altered the hydrogen-bonding network around residue 274. The rotated W295 side chain caused the upward movement of the 340-loop. Consequently, sliding box docking results suggested that the binding pathway of OTV was compromised because of the disruption of this binding site. This study also highlighted the importance of the functional group at C6 of the sialic acid mimicry. It is hoped that these results will improve the understanding of OTV resistance and shed some light on the design of a novel anti-influenza drug.

Branched isomeric 1,2,3-triazolium-based ionic liquids: new insight into structure-property relationships.

A series of four isomeric 1,2,3-triazolium-based ionic liquids (ILs) with vary degree of branching were synthesized and characterized to investigate the effect of ion branching on thermal and physical properties of the resulting IL. It was found that increased branching led to a higher ionicity and higher viscosity. The thermal properties were also altered significantly and spectral changes in the near edge X-ray absorption fine structure (NEXAFS) spectra show that branching affects intermolecular interaction. While the ionicity and viscosity varying linearly with branching, the MDSC and NEXAFS measurements show that the cation shape has a stronger influence on the melting temperature and absorptive properties than the number of branched alkyl substituents.

Experimental and DFT studies of porous carbon covalently functionalized by polyaniline as a corrosion inhibition barrier on nickel-based alloys in acidic media.

In acidic medium, nickel alloys severely suffer from long term corrosion problems as a result of the breakdown of their passivating oxide. The present study considers polyaniline functionalized fish-scale graphitic carbon as an anticorrosion coating on the nickel alloy surface. The fish-scale porous carbon materials are characterized by XRD, ATR-FITR, UV, Raman, TGA, SS NMR, FESEM, and TEM methods. The surface of the alloy is covalently bound with a polyaniline long chain protonated polymer so that the polyaniline functionalized honeycomb fish-scale carbon structure can exchange electrons with the metal surface. The corrosion inhibition efficiency has been investigated in different acid media like sulfuric acid and hydrochloric acid by electrochemical methods. Polyaniline functionalized porous carbon showed in 1 M H2SO4 inhibition efficiency around 64% and in 1 M HCl inhibition efficiency was around 74%. The inhibition efficiency was higher in HCl because chloride ions were not able to penetrate the graphitic sheet. The novelty of this coating is in the fact that the polyaniline functionalized porous carbon has high conductivity and is electrochemically stable in acidic medium. It is able to donate electrons to the polarized metal surface.

Potential New H1N1 Neuraminidase Inhibitors from Ferulic Acid and Vanillin: Molecular Modelling, Synthesis and in Vitro Assay.

We report the computational and experimental efforts in the design and synthesis of novel neuraminidase (NA) inhibitors from ferulic acid and vanillin. Two proposed ferulic acid analogues, MY7 and MY8 were predicted to inhibit H1N1 NA using molecular docking. From these two analogues, we designed, synthesised and evaluated the biological activities of a series of ferulic acid and vanillin derivatives. The enzymatic H1N1 NA inhibition assay showed MY21 (a vanillin derivative) has the lowest IC50 of 50 µM. In contrast, the virus inhibition assay showed MY15, a ferulic acid derivative has the best activity with the EC50 of ~0.95 µM. Modelling studies further suggest that these predicted activities might be due to the interactions with conserved and essential residues of NA with ΔGbind values comparable to those of oseltamivir and zanamivir, the two commercial NA inhibitors.

A general method to quantify quasi-equivalence in icosahedral viruses.

A quantitative, atom-based, method is described for comparing protein subunit interfaces in icosahedral virus capsids with quasi-equivalent surface lattices. An integrated, normalized value (between 0 and 1) based on equivalent residue contacts (Q-score) is computed for every pair of subunit interactions and scores that are significantly above zero readily identify interfaces that are quasi-equivalent to each other. The method was applied to all quasi-equivalent capsid structures (T=3, 4, 7 and 13) in the Protein Data Bank and the Q-scores were interpreted in terms of their structural underpinnings. The analysis allowed classification of T=3 structures into three groups with architectures that resemble different polyhedra with icosahedral symmetry. The preference of subunits to form dimers in the T=4 human Hepatitis B virus capsid (HBV) was clearly reflected in high Q-scores of quasi-equivalent dimers. Interesting differences between the classical T=7 capsid and polyoma-like capsids were also identified. Application of the method to the outer-shell of the T=13 Blue tongue virus core (BTVC) highlighted the modest distortion between the interfaces of the general trimers and the strict trimers of VP7 subunits. Furthermore, the method identified the quasi 2-fold symmetry in the inner capsids of the BTV and reovirus cores. The results show that the Q-scores of various quasi-symmetries represent a "fingerprint" for a particular virus capsid architecture allowing particle classification into groups based on their underlying structural and geometric features.

Binding preferences of hydroxamate inhibitors of the matrix metalloproteinase human fibroblast collagenase.

In this paper we report molecular dynamics (MD) and free energy perturbation (FEP) studies carried out on enzyme-inhibitor (two hydroxamates that only differ by a carbon-carbon double bond) complexes of human fibroblast collagenase to obtain insights into the structural and energetic preferences of these inhibitors. We have developed a bonded model for the catalytic and structural zinc centers (Hoops, S. C.; et al. J. Am. Chem. Soc. 1991, 113, 8262-8270) where the electrostatic representation for this model was derived using a novel quantum-mechanical/molecular-mechanical (QM/MM) minimization procedure followed by electrostatic potential fitting. The resulting bonded model for the zinc ions was then used to generate MD trajectories for structural analysis and FEP studies. This model has satisfactorily reproduced the structural features of the active site, and furthermore, the FEP simulations gave relative free energies of binding in good agreement with experimental results. MD simulations in conjunction with the FEP are able to provide a structural explanation regarding why one hydroxamate inhibitor is favored over the other, and we are also able to make predictions about changes in the inhibitor that would enhance protein-inhibitor interactions.

Mechanism(s) regulating inhibition of thymidylate synthase and growth by gamma-L-glutaminyl-4-hydroxy-3-iodobenzene, a novel melanin precursor, in melanogenic melanoma cells.

A proposed mechanism for the melanoma specific activity of phenolic amines is based upon the ability of the enzyme tyrosinase to oxidize these prodrugs to toxic intermediates. In this study, we synthesized an iodinated analog of gamma-L-glutaminyl-4-hydroxybenzene (GHB) with increased antimelanoma activity in both human and murine melanoma cell lines. GHB and gamma-L-glutaminyl-4-hydroxy-3-iodobenzene (I-GHB) were shown to be substrates for both mammalian and mushroom tyrosinase. Glutathione, a cellular antioxidant, inhibited tyrosinase mediated formation of gamma-L-glutaminyl-3,4-benzoquinone (GBQ) from GHB, inhibited melanin production, and blocked the inhibition of the enzyme thymidylate synthase by oxidized GHB. Buthionine sulfoximine (BSO) depletion of cellular glutathione enhanced the growth inhibitory activity and the inhibition of in situ thymidylate synthase by phenolic amines in melanoma cells. GHB and I-GHB were shown to be approximately 5- and 10-fold more cytotoxic, respectively, in highly metastatic B16-BL6 cells than in weakly metastatic B16-F1 cells with approximately equal tyrosinase activity. B16-BL6 cells had approximately 20-fold higher gamma-glutamyltranspeptidase (gamma-GTPase) activity than B16-F1 cells which suggested the possible involvement of this enzyme in the activation of the cytotoxicity of the phenolic amines. 4-Aminophenol, a product of gamma-GTPase reaction with GHB, was a substrate for tyrosinase and a potent inhibitor of in situ thymidylate synthase activity in melanogenic cells. In pigmented melanoma cells containing the enzyme tyrosinase, the quinone mediated mechanism of phenolic amine cytotoxicity may be uniquely important and the cellular antioxidant glutathione essential in the detoxification of these quinone-generated intermediates.

Radiotoxicity of 17 alpha-[125I]iodovinyl-11 beta-methoxyestradiol in MCF-7 human breast cancer cells.

Therapeutic strategies for human breast cancer using 125I-labeled steroid hormones are clinically attractive in light of the estrogen dependence of many human breast cancers and the favorable microdosimetry resulting from 125I decay. We determined the uptake specific estrogen receptor binding and radiotoxicity of 17 alpha-[125I]iodovinyl-11 beta-methoxyestradiol (125IVME2) in vitro using cultured MCF-7 human breast carcinoma cells. 125IVME2 rapidly enters MCF-7 cells and reaches a plateau in the presence of competing 10(-7) M 17 beta-estradiol. In the absence of competitor, uptake is substantially greater before reaching a plateau. Efflux of 125IVME2 from cells incubated in the absence of estradiol decreases to levels corresponding to specific binding. Under equilibrium conditions and in the absence of competitor, 125IVME2 binds to both specific and nonspecific sites but, in the presence of excess 17 beta-estradiol, the observed binding is nonspecific. 125IVME2 is cytotoxic to exponentially growing MCF-7 cells and produces a survival curve typical of those observed for [125I]iododeoxyuridine and 16 alpha-[125I]iodoestradiol.

Solid state NMR of a molecular self-assembly: multinuclear approach to the cyanuric acid-melamine system.

[see structure]. Trinuclear MAS NMR, involving naturally abundant (13C, 15N) and easily deuterated (2H) nuclei, is shown to offer newer opportunities to study molecular self-assembly in noncrystalline supramolecular systems.

Synthesis of a precursor for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide.

Starting from 11 beta-hydroxytestosterone, we achieved the synthesis of a strategic precursor, C-9 (11) unsaturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (9a), for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide. We optimized the reaction conditions for catalytic reduction employing hydrogen and subsequent base hydrolysis followed by purification on Amberlite XAD-2 resin to obtain the saturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.

Synthesis of metabolic intermediates of diethylstilbestrol.

This report details the synthesis of 1) 3,4,4'-trihydroxy-alpha, alpha'-diethyl-trans-stilbene; 2) 3,4-bis-(p-hydroxyphenyl)-trans-3-hexenol; 3) 3,4-bis-(p-hydroxyphenyl)-2,4-cis,cis-hexadienol; 4) 3,4-bis-(3'-methoxy-4'-hydrophenyl)-trans-3-hexene; 5) 3,4-bis-(3',4'-dimethoxyphenyl)-trans-3-hexene. These compounds are suspected metabolites of diethylstilbestrol.

A comparison of DMPC- and DLPE-based lipid bilayers.

A 250 ps molecular dynamics simulation of the dimyristoylphosphatidylcholine (DMPC)-based lipid bilayer, including explicit water molecules, is reported. The solvent environment of the head groups and other structural properties of the bilayer have been analyzed and compared with experimental results as well as our previous simulation of the dilauroylphosphatidylethanolamine (DLPE)-based bilayer. From this comparison we find that the solvent structure around the DMPC head group (clathrate shell) is significantly different than that around the DLPE head group (typical hydrogen bonding interactions). We have modeled the probable relationship between the different solvent environments around the R-N(CH3)3+ (DMPC) and R-NH3+ (DLPE) head groups and the different interlammelar distances in these systems by performing potential of mean force (PMF) simulations on two N(CH3)4+ and NH4+ ions in water. From the PMF simulations it appears that the differences in the hydration of the DMPC and DLPE head groups is not responsible for the differences in the hydration force observed for these systems. We also find that the orientational polarization of DLPE and DMPC is similar, which suggests that solvent polarization is not responsible for the differences in the hydration repulsion behavior observed in these systems. We also examined the order parameters for DMPC and found them to be in reasonable agreement with experiment. Given the different characteristics of the DLPE and DMPC head groups, we suggest an explanation of the differences in the interlammellar spacings of bilayers composed of these like-charged lipids. From our DLPE simulations we find that the R-NH3+ head groups can interact with the nonesterified oxygens of the phosphate group in an intraleaflet or an interleaflet manner. For the latter a "cross link" between two leaflets can be formed, which causes a stabilization of the interlamellar spacings at fairly short distances. Moreover, due to the strong intraleaflet interaction we find that the DLPE interface is relatively "flat" (as opposed to DMPC-based bilayers), which results in a surface that has regions of positive and negative charge that reside in the same plane along the bilayer normal. Based on this we propose that the DLPE bilayer interface can correlate itself with another DLPE interface by alignment of the regions of positive (or negative) charge on one leaflet with the opposite charges on the opposing leaflet.

Structure and dynamics of the dilauroylphosphatidylethanolamine lipid bilayer.

A 200-ps molecular dynamics (MD) simulation trajectory of a model dilauroylphosphatidylethanolamine (DLPE) bilayer in water at 315 K has been generated. Segmental order parameters, electron density profiles, and water pair distribution functions have been calculated. Comparison to experiment is made where possible. The dynamics of the system has been studied by analyzing the velocity autocorrelation functions (VAF) of both water and lipid atoms. Furthermore, the diffusive properties of water have been analyzed by computing the mean square displacement (MSD) and orientational correlation function (OCF) of water in two regions around the bilayer. The calculated order parameters show a behavior similar to the liquid crystalline phase of other bilayers, but the region around C1-C3 does not show the expected behavior. The electron density profile shows features that are characteristic of the liquid crystalline phase. The radial distribution functions suggest ordering of water near the charged head groups, which results in about 15 water molecules solvating each lipid molecule. We find from the VAF, MSD, and OCF calculation that the water molecules near the head groups of the lipid bilayer move more slowly than those further away. The VAF of the hydrocarbon chains have features of low-frequency motions that are probably cooperative nature in addition to the high-frequency motions associated with bond angle and torsional motions.

Interaction of small peptides with lipid bilayers.

Molecular dynamics simulations of the tripeptide Ala-Phe-Ala-O-tert-butyl interacting with dimyristoylphosphatidylcholine lipid bilayers have been carried out. The lipid and aqueous environments of the peptide, the alkyl chain order, and the lipid and peptide dynamics have been investigated with use of density profiles, radial distribution functions, alkyl chain order parameter profiles, and time correlation functions. It appears that the alkyl chain region accommodates the peptides in the bilayer with minimal perturbation to this region. The peptide dynamics in the bilayer bound form has been compared with that of the free peptide in water. The peptide structure does not vary on the simulation time scale (of the order of hundreds of picoseconds) compared with the solution structure in which a random structure is observed.

Gamma-glutamyltranspeptidase expression regulates the growth-inhibitory activity of the anti-tumor prodrug gamma-L-glutaminyl-4-hydroxy-3-iodobenzene.

gamma-L-glutaminyl-4-hydroxy-3-iodobenzene (I-GHB), a novel iodinated analog of gamma-L-glutaminyl-4-hydroxybenzene (GHB), demonstrates greater anti-tumor activity in human and in murine melanoma cell lines. These phenolic amides are substrates for gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a cell-membrane-associated ecto-enzyme which is elevated in a number of tumor systems. We now present data to show that the growth-inhibitory activity of I-GHB and GHB may be mediated via GGTP-catalyzed reactions. The growth-inhibitory activity of I-GHB and GHB in pigmented B16-BL6 melanoma cells was blocked significantly by rabbit anti-rat GGTP polyclonal antibodies. The combination of L-serine and sodium borate, a specific transition-state inhibitor of GGTP, as well as acivicin, a glutamine antagonist and irreversible GGTP inhibitor, inhibited the killing of BL6 cells by GHB and I-GHB. To further define the role of GGTP expression in the regulation of phenolic amide cytotoxicity, GGTP-negative Chinese hamster ovary cells (CHO-K1) were transfected with a functional rat renal cDNA representing the full-length GGTP transcript. I-GHB and GHB were significantly more cytotoxic in GGTP cDNA transfected Chinese hamster ovary (CHO-K1-GGTP) cells than in non-transfected CHO-K1 cells. The combination of L-serine and sodium borate blocked the cytotoxic activity of these pro-drugs and also inhibited GGTP-catalyzed formation of polymerized products from these phenolic amides in intact BL6 melanoma and CHO-K1-GGTP cells. Furthermore, melanin formation from GHB was not observed in non-transfected CHO-K1 cells lacking GGTP expression. The combined data strongly suggest that GGTP-catalyzed hydrolysis of the anti-tumor pro-drugs I-GHB and GHB to 4-aminophenols mediates the expression of antitumor activity.

Rapid, convenient radioimmunoassay of estrone sulfate.

We developed a specific, simple, and rapid RIA for the direct quantification of estrone sulfate (E1S) and established its performance characteristics. The assay has a dynamic range of 0.05-90 micrograms/L with a detection limit of 0.009 microgram/L. Intraassay CVs were 9.2%, 4.5%, and 4.6% at 0.35, 9.0, and 60 micrograms/L, respectively. Interassay CVs were 8.8%, 5.1%, and 5.5% at 0.076, 0.5, and 12 micrograms/L, respectively. Linearity of dilution studies showed values of 80-105% of expected, and recovery of E1S added to serum samples ranged from 82% to 102%. Cross-reactivities with structurally related estrogens were < 5%. When compared with a conventional assay (involving hydrolysis of E1S and indirect measurement of estrone), the present RIA showed excellent correlation (r = 0.99, slope = 1.54, Sy/x = 2.14, n = 71). Mean E1S concentrations measured with this RIA for normal men (n = 20) and women in follicular (n = 20) and luteal (n = 25) phases of their menstrual cycle were 0.96, 0.96, and 1.74 microgram/L, respectively. Mean E1S concentrations for oral contraceptive users (n = 20) and postmenopausal women without hormone replacement therapy (n = 21) or on hormone replacement therapy (n = 22) were 0.74, 0.13, and 2.56 micrograms/L, respectively. Serum concentrations of E1S in pregnant women in their first (n = 14), second (n = 17), and third (n = 15) trimesters were 20, 66, and 105 micrograms/L, respectively. Availability of this simple RIA should provide a useful tool for the assessment of estrogen status in women.