Characterization of a new class of inhibitors of the recombinant human liver UDP-glucuronosyltransferase, UGT1*6.

The inhibitory effect of a series of novel structurally related compounds on the human UDP-glucuronosyltransferase UGT1*6 stably expressed in a V79 cell line was investigated. The inhibitors contain a lipophilic N-acyl phenylaminoalcohol residue and a uridine moiety connected by a spacer varying for each compound. The effects of these compounds on the glucuronidation reaction measured with 4-methylumbelliferone as substrate were determined. The best inhibitor of the series, D-DPMSU, had an IC50 of 39 microM in the assay conditions. Low Ki values were found toward both UDP-glucuronic acid and 4-methylumbelliferone (17 and 21 microM, respectively). The inhibition was competitive toward both substrates. A similar strong and competitive inhibitory effect was observed with two other inhibitors, DHPASU and DHPASiU. Another compound, D-DPASiU, showed a pure competitive inhibition towards UDP-glucuronic acid, but a non-competitive inhibition towards the acceptor substrate. These data and the optimization of the structures of the inhibitors by molecular modeling suggest that D-DPMSU and DHPASiU compounds may be transition state analog inhibitors of the recombinant UGT1*6 enzyme.

Density functional theory and empirical derived force fields for the delocalized polaron form of polyaniline. Application to properties determination of an isolated oligomer using molecular dynamics simulations.

Normal mode analyses obtained from quantum chemical calculations at the DFT level of theory have been performed for the repetitive unit of the delocalized bipolaron form of polyaniline (PANI). Empirical molecular mechanics force field parameters were consecutively refined using the SPASIBA software and applied to the molecular dynamics properties of an isolated dodeca oligomer model of the leucoemeraldine form of PANI. It is shown that effects of protonation of the emeraldine base spread over the four rings constituting the repeating unit. Molecular dynamics simulations reveal alternative bendings of the whole chain with a time period of 18-20 ps for the model of PANI under study.

Theoretical and vibrational spectroscopic analysis of the CO stretching mode of cholesteryl alkanoates: the particular case of the cholesteryl acetate.

Structural and vibrational properties of the CO stretching bond of cholesteryl acetate and related steroids are investigated theoretically and by Micro-Raman spectroscopy. In this work, an analysis of the CO stretching mode for the cholesteryl acetate is presented. Experimental results in crystalline, isotropic liquid and liquid crystal phases are compared with quantum chemical calculations using semi empirical hamiltonians (AM1 and PM3) and the density functional theory. The calculations were performed on isolated molecules with different conformations as found on previous investigations giving strong evidence of their existence. Calculated frequencies are found to be very close to experiments and suggest the possible existence of the predicted conformers.

Specific histamine release capacity of peptides selected from the modelized Der p I protein, a major allergen of Dermatophagoides pteronyssinus.

Dust mite allergens are considered as a major cause of allergic disease and as a risk factor for asthma. Der p I, a 222 amino-acid residue globular glycoprotein, is one of the major allergens from Dermatophagoides pteronyssinus (Dpt) mites. In this study, we have used predictive conventional algorithms (i.e. hydrophilicity, mobility, accessibility) and a three-dimensional model of Der p I derived from comparison to actinidin and papain to select continuous amino acid sequences as potential B cell epitopes. Four peptides, 52-71, 117-133, 176-187, 188-199 were synthesized. Their antigenic reactivity was investigated, mainly by measuring their capacity to induce in vitro histamine release. Results indicated that only Dpt-sensitive patients react specifically to Der p I-derived peptides and more frequently to 52-71 and 117-133. For each peptide, the intensity of response was dependent on the patient tested and on the peptide concn. The capacity of peptides to induce histamine release was demonstrated to be correlated with the serum level of anti-Der p I IgE (r = 0.86; p less than 10(-2)). Taken together these data emphasize, in Dpt-sensitive patients, the heterogeneity of the specific response to synthetic Der p I-derived peptides and underline the possible variety of epitopes belonging to the allergen Der p I.

Amino acid residue: is it structural or functional?

A new approach is suggested for delineating the structural and functional amino acid residues in proteins with known three-dimensional structure, basing on the involvement of residues in intramolecular hydrophobic and hydrophilic interactions and additional information about the conservativity of the residues. The approach is applied to the families of homologous neurotoxins and cardiotoxins. The results obtained concerning the role of amino acid residues in both families of toxins accord well with the similarity of their fold, but different mechanisms of action. Current approach can be used for detailed characterization of protein spatial structures, as well as for rational protein engineering.

Molecular dynamics simulations of a monofucosylated biantennary glycan of the N-acetyllactosamine type: the human lactotransferrin glycan.

Molecular dynamics simulations were carried out to explore the conformational flexibility of the antennae of N-linked glycans. They were performed over 200 ps in vacuo on the complete disialylated monofucosylated biantennary glycan of the N-acetyllactosaminic type. Starting from a bird-conformation, the 3-D-structure evolved through 9 successive transitional states to a new, compact and energetically favorable conformation which had never been previously described. In this conformation, both antennae are organized in two coplanar loops rolled in a contrary direction and oriented perpendicularly to the plane of the di-N-acetyl chitobiose residue leading to a 'lobster conformation'. All the glycosidic linkages of the disialylated monofucosylated biantennary glycan, except the Fuc(alpha 1-6)GlcNAc(beta 1-), were modified by a phase transition. Particularly, the Man(beta 1-4) GlcNAc(beta 1-) linkage, which was previously described by NMR and X-ray diffraction as a rigid one, was involved in numerous conformational changes during 83 ps, even before the first transition phase. The freedom of mobility of the torsional angles of the Man(alpha 1-6)Man(beta 1-) linkage was limited, under these simulation conditions, to the angle psi which took three values: 30 degrees, 90 degrees and 180 degrees. Moreover, from 150 ps up to the end of the simulation, the value of the torsional angle omega of the NeuAc(alpha 2-6)Gal(beta 1-) linkage of the alpha-1,6-antenna continuously swung between 60 degrees and -60 degrees. Finally, we observed that the values of the torsional angles of the three linkages: NeuAc(alpha 2-6)Gal(beta 1-), Gal(beta 1-4)GlcNAc(beta 1-) and GlcNAc(beta 1-2)Man(beta 1-) of each of the two antennae were different, demonstrating their asymmetric conformation.

The SPASIBA force field as an essential tool for studying the structure and dynamics of saccharides.

The SPASIBA force field has been applied to the determination of the structure and dynamical properties of various disaccharides. It has been shown that the experimental properties (structure, dipole moment, conformational relative energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are confidently reproduced without specific terms for the alpha and beta anomers and the type of glycosidic linkages.

4-Mercaptoimidazoles derived from the naturally occurring antioxidant ovothiols 2. Computational and experimental approach of the radical scavenging mechanism.

The radical-scavenging mechanism of fourteen 4-mercaptoimidazoles, derived from the natural family of ovothiols, was studied via a QSAR approach, cyclic voltammetry, ESR and NMR spectroscopy. A significant correlation was found between the DPPH scavenging abilities of test compounds and thermodynamic parameters like overall ease of disulphide formation. The production of a disulphide compound via thiyl radical formation is proposed. Upon DPPH scavenging, hydrogen abstraction from thiols yields transient short-lived thiyl radicals, which were characterised by ESR and rapidly dimerise to form a disulphide compound. Cyclic voltammetry showed that the best DPPH scavengers exhibit low oxidation potentials for their oxidation to disulphides.

Stabilization of proteins by enhancement of inter-residue hydrophobic contacts: lessons of T4 lysozyme and barnase.

Although the hydrophobic interactions are considered as the main contributors to the protein stability, not much examples of protein stabilization by rational increasing of this type of interactions still can be found in literature. This is partly due to the lack of proper theoretical "measure" of hydrophobic interactions and their changes upon mutations. In the present paper the molecular hydrophobicity potential approach is used to assess how the changes in type and the strength of inter-residue contacts upon single amino acid mutations are correlated with the changes in thermodynamic stability of T4 lysozyme and barnase mutants, and which factors affect these correlations. Mutations changing unfavorable hydrophilic-to-hydrophobic contacts into favorable hydrophobic were found to enhance the thermodynamic stability in more than 81 % of cases, if these mutations do not create steric bumps and do not involve proline residues and hydrogen-bonded side-chains. Mutations increasing hydrophobic contributions (according to molecular hydrophobicity potential formalism) lead to increase of thermodynamic stability in more than 94% of cases for certain type of mutations (i.e., mutations not involving charged residues, Pro and residues with side-chain hydrogen bonds, when these mutations do not introduce steric bumps and do not involve strongly exposed residues and residues situated at helix N- and C-cap positions). For this type of mutations the correlation was found between the change in hydrophobic contributions of mutated residues deltaCphob and thermodynamic parameters deltaTm (change in melting temperature) and deltadeltaG (change in free energy of unfolding). Although the correlation coefficients were larger if the experimental structures of mutants were used for the calculations (correlation coefficients r(exp) deltaC,deltaT = .85 and r(exp) deltaC,deltadeltaG = 0.87) than if the modeled structures were used instead (r(mod) deltaC,deltaT = 0.74 and r(mod)deltaC,deltadeltaG = 0.76), the modelled structures of mutants in the vast majority of cases can be used for qualitative predictition of the protein stabilization. Basing on the analysis of mutations increasing hydrophobic contributions in T4 lysozyme the substitution matrix was derived, which can be used to decide which new residue should be put instead the old one to increase the stability of protein. The estimation shows that the number of potential mutation sites for enhancement of hydrophobic interactions in T4 lysozyme is quite large, and only approximately 10 per cent of them were studied thus far. Basing on the current analysis of T4 lysozyme and barnase mutations the algorithm for increasing of protein stability via increasing of hydrophobic interactions for the proteins with known spatial structure is proposed.

Atomic solvation parameters for proteins in a membrane environment. Application to transmembrane alpha-helices.

Several sets of atomic solvation parameters imitating: (i) nonpolar environment of hydrocarbon core of a membrane, (ii) aqueous solution, and (iii) weakly-polar solvents have been developed. The parameters have been incorporated into the ECEPP/2 and CHARMM force fields and employed in non-restrained Monte Carlo and molecular dynamics simulations of membrane-spanning alpha-helical peptides (segment A of bacteriorhodopsin, melittin). Through these simulations, the structure and energetics of the helices have been examined as a function of the solvation term in the potential energy function. For the peptides under study, the set (i) of atomic solvation parameters reveals good retention of the alpha-helical conformation. By contrast, the simulations in vacuum or with the parameters imitating a polar solvent (sets (ii) or (iii)) show fast helix destabilization and tight packing of the structure accompanied by significant decreasing of the surface area accessible to solvent. Increased helical propensity for amino acid residues, population of side-chain rotamers as well as hydrogen-bonding pattern in nonpolar membrane-like environment agree well with available experimental and computational data. The problems related to further applications of the membrane-mimicking sets of atomic solvation parameters to simulations of membrane proteins and peptides are addressed.

Molecular modeling of HIV-1 coreceptor CCR5 and exploring of conformational space of its extracellular domain in molecular dynamics simulation.

The chemokine receptor CCR5 functions as a major fusion coreceptor for macrophage-tropic human immunodeficiency virus entry into cell. Here we report a three-dimensional model of CCR5 built using molecular modeling approach. Because the virus binds to extracellular domain of the receptor, special attention was given to conformational flexibility, hydrogen bonding, and environmental polarity properties of this protein part. Such data were obtained in the result of molecular dynamics study of the extracellular domain. It was shown that during the simulation the extracellular segments form a compact globular domain with numerous long-range hydrogen bonds between them. First loop of the receptor stays quite rigid while N-terminal region and loops 2, 3 are rather flexible. A number of amino acid residues disposed in unfavourable environment and, therefore, potentially involved in binding of CCR5 to viral glycoproteins and chemokines, was delineated. Comparison of the results with available experimental data permits a proposal that such residues in loop-1 and N-terminal part of the receptor are important for HIV-1 entry, while those in loops 2 and 3 participate in ligand binding. Perspectives of rational alteration of virus-binding activity of CCR5 are discussed.

Detailed assessment of spatial hydrophobic and electrostatic properties of 2D NMR-derived models of neurotoxin II.

2D NMR-derived spatial structures of neurotoxin II (NtII) and several homologous toxins in solution were assessed by comparison with their own amino acid sequences using a three-dimensional (3D) profile method. 3D profiles of all the toxin models match the sequences well and, therefore, the method of 3D profile was demonstrated to work correctly for these well-resolved NMR structures in aqueous solution. At the same time, the profile window plots reveal low scores in the bottom tip of loop II (residues 22-34 in NtII) and in beta-strand of loop III (residues 49-52). Some residues in the first poor-scoring region are of functional importance being involved in binding with nicotinic acetylcholine receptor (AChR). Furthermore, the second segment participates in intermolecular hydrogen bonding upon dimerization of postsynaptic neurotoxins in solution resulting in increasing of the 3D-1D score for residues at the interface between monomers. Therefore, the 3D profile method can be useful for detection functionally-important regions in well-resolved protein structures.

A new method to characterize hydrophobic organization of proteins: application to rational protein engineering of barnase.

We present a new algorithm for characterization of protein spatial structure basing on the molecular hydrophobicity potential approach. The method is illustrated by the analysis of three-dimensional structure of barnase and barnase-barstar complex. Current approach enables identification of amino acid residues situated in unfavorable environment (these residues may be "active" for binding), and to map quantitatively hydrophobic, hydrophilic and unfavorable hydrophobic-hydrophilic intra- and inter-molecular contacts involving backbone and side-chain segments of amino acid residues. Calculation of individual contributions of amino acid residues to such contacts permits identification of structurally-important residues. The contact plots obtained with molecular hydrophobicity potential calculations, provide easy rules to choose sites for mutations, which can increase a strength of intra- or inter-molecular hydrophobic interactions. The unfavorable hydrophobic-hydrophilic contact can be mutated to favorable hydrophobic, and already existing weak hydrophobic contact can be strengthen by increasing hydrophobicity of residues in contact. Basing on the analysis of the contact plots, we suggest several mutations of barnase which are supposed to increase intramolecular hydrophobic interactions, and thus might lead to increased stability of the protein. Part of these mutations was studied previously experimentally, and indeed stabilized barnase. The other of predicted mutations were not studied experimentally yet. Several new mutations of barnase and barstar are also proposed to enhance the hydrophobic interactions on their binding interface.

[Not Available]. / Etude par spectroscopie Raman de quelques anorexigenes-I Anorexigenes N-substitues.

Raman spectra of a series of three anorexigenic drugs in the polycrystalline state have been recorded. Several spectral similarities have been obse.

Etude par spectroscopie raman du chlorhydrate de cocaine.

Raman spectra of cocaine hydrochloride in the polycrystalline state and in saturated aqueous solutions have been recorded at room temperature from 0 to 4000 cm(-1) and at 9 K from 0 to 200 cm(-1) (only for the polycrystalline state). They show that cocaine can be characterized by the following proposed assignments. For the tropane nucleus the frequencies 851 and 786 cm(-1) (piperidine) and 896, 870 cm(-1) (pyrrolidine); these frequencies are assigned to ring carbon stretching vibrations V(C-C) Bands for the ester functional groups can be observed at 1713 cm(-1) (v(CO)) and 1203 cm(-1) (v((C-O-O)). The benzene nucleus is also important in characterization of cocaine hydrochloride because of its bands at 616, 990, 1000, 1026 and 1596 cm(-1). Special reference is made first to the work in the low-frequency range at room temperature and 9 K and secondly to the polarization studies of saturated aqueous solutions in the range 700-1726 cm(-1). The results constitute a pool of analytical characteristics which can be used for toxicological investigations, and also show all the possibilities of Raman spectroscopy in this field.

[Not Available]. / Microspectrometrie raman multicanale: application a l'identification du trimethylacetate et du caproate de fluocortolone isoles d'une forme galenique.

This work is an application of the results obtained by classical Raman spectroscopy for fluocortolone and its trimethylacetate and caproate esters. These drugs are used together in a pharmaceutical preparation. To avoid fluorescence problems with excipients a solvent extraction was first applied, followed by an HPLC separation. The aim was to identify two corticosteroids through their molecular structure by applying Raman spectroscopy to HPLC eluates. Tests with the drugs in aqueous methanol solution proved unsatisfactory so recourse was made to use of the crystalline state and a Raman multichannel microprobe. This allowed the identification, with accuracy, of Delta(1,4)-3-one structure with sample quantities as small as 10(-12) g.

Etude par spectrometrie raman de corticosteroides derives de la fluocortolone: trimethylacetate et caproate.

Les spectres Raman de trois corticostéroïdes: fluocortolone base, triméthylacétate et caproate de fluocortolone (TMAF et CAF) ontétéenregistrésàl'état polycristallin entre 150 et 4000 cm(-1). Une attribution des raies observées est proposée. Elle a montréla complexitédu problème poséàsavoir la différenciation de trois composés de structure trés voisine. Cependant quelques intervalles de fréquences contenant des raies spécifiques sont susceptibles d'e^tre utilisés afin de permettre l'identification des composésétudiés. Il convient en premier lieu de distinguer tout particulièrement les raies situées entre 1500 et 1800 cm(-1). Dans l'intervalle 1580-1690 cm(-1) celles-ci sont intenses et spécifiques de la structure conjuguée Delta(1,4) 3-one des corticostéroi des ce qui permet de distinguer la classe de médicamentàlaquelle appartiennent les substancesétudiées. La zone de fréquence comprise entre 1690 et 1750 cm(-1) témoigne non seulement de la présence d'une fonction cétonique sur la chai^ne latérale mais aussi de l'existence d'une fonction ester suivant que l'on y trouve respectivement une ou deux raies (fluocortolone base 1701 cm(-1); TMAF 1728, 1745 cm(-1); CAF 1723, 1745 cm(-1). La fluocortolone base présenteà1635 cm(-1) une raie qui n'est pas retrouvée chez les dérivès estérifiés. En second lieu, nous retiendrons les zones spectrales représentatives des chai^nes latérales estrrifiées fixées sur le carbone C(21): chai^ne triméthylacétate 340-360, 570-610, 750-810 cm(-1) (dans cette dernière région commune au caproate et au triméthylacétate la différenciation des deux composés se fait grâceàla raieà796 cm(-1) absente dans le spectre du caproate); chai^ne caproate 1310-1350 cm(-1) (dans cet intervalle, le caproate présente un profil très particulier avec trois raies d'intensitévoisine). Cet ensemble de résultats doite^tre utilisépour identifier ces substances par spectroscopie Raman sur des quantitiés de l'ordre du picogramme provenant d'éluat d'HPLC.

Harmonic dynamics of beta-D-fructopyranose.

The vibrational spectra of beta-D-fructopyranose crystals have been recorded in the 4000-400 cm(-1) region using the infrared and in the 4000-20 cm(-1) region using the Raman. These spectra are used as an experimental basis in order to establish a force field for the beta-D-fructopyranose molecule in the crystalline state through a normal co-ordinates analysis. For this purpose, a modified Urey-Bradley-Shimanoushi force field was combined with an intermolecular potential energy function that includes the van-der-Walls interactions, the electrostatic terms, and an explicit hydrogen bond function. The force field parameters are derived from those of beta-D-glucose and are fitted so as to obtain a good agreement between the calculated and the observed frequencies. The results obtained demonstrate the reliability and the transferability of the set of parameters constituting the initial force field. The fitted force field reproduces the experimental spectra to a marked degree of accuracy.

Modified UBFF calculations of the alpha-L-fucopyranose molecule in the crystalline state.

Normal co-ordinate analysis has been realised in the crystalline state using a modified Urey-Bradley-Shimanouchi force field combined with an intermolecular potential energy function that includes van der Waals interactions, some electrostatic terms and an explicit hydrogen bond function. The vibrational spectra of the alpha-L-fucose molecule have been recorded in the crystalline state, in the 4000-500 cm(-1) spectral region for the mid-IR spectra, in the 500-100 cm(-1) spectral region for the far-IR spectra, and in the 4000-20 cm(-1) spectral range for Raman spectra. These spectra constitute the experimental support for the establishment of a force field for the molecule in the crystalline state through a normal co-ordinate analysis.

Harmonic dynamics of alpha- and beta-methyl-D-galactopyranoside in the crystalline state.

The study of the anomeric differences observed on the spectra of methyl-alpha- and methyl-beta-D-galactopyranoside is the essential goal of this investigation. Thus, after a careful examination of the IR and Raman spectra of these two compounds, several differences in the intensities and frequency shifts are observed. This is especially noted in the region 1000-700 cm(-1). In order to make some assignments with more precision, the normal modes analyses of the two compounds are performed in the crystalline state. For this purpose, a modified Urey-Bradley-Shimanouchi force field has been combined with an intermolecular potential energy function. The initial set of force constants comes from those of alpha- and beta-D-galactopyranosyl, then the force constants have been varied, so as to obtain a good agreement between the observed and the calculated vibrational frequencies. The obtained results have finally reproduced the experimental data and have confirmed the previous assignments made for the methyl-alpha- and methyl-beta-D-galactopyranoside. The calculations have demonstrated also the transferability of the set of parameters of the initial force field of D-galactose to methyl-D-galactopyranoside.