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H D Höltje

Publications and source records attributed to H D Höltje.

At least 19 recordsLinked to original sources
PubMed2005

Development of models for cytochrome P450 2A5 as well as two of its mutants.

It is known that small changes in the amino acid sequence can change the catalytic activity of cytochromes towards substrates dramatically. With the aim to broaden our knowledge about the structural properties of cytochromes and their relation with substrate specificity a model of CYP2A5 was built by homology modelling based on the crystal structure of CYP2C5. Model stability was evaluated by subjection of the model to a free molecular dynamics simulation in a waterbox under almost physiological conditions using the GROMACS program. The protein folding remains stable over 1.5 ns under these conditions. The modelling procedure was repeated for two mutated forms of CYP2A5 with known differing substrate selectivities towards corticosterone and desoxycorticosterone. A detailed analysis of the models and their behaviour in long running molecular dynamics simulations allows an understanding of the requirements for enzyme activity as well as an explanation of respective experimental data on the molecular level.

Amino Acid Sequence↗
PubMed2001

Molecular dynamics simulations of stratum corneum lipid models: fatty acids and cholesterol.

We report the results of an investigation on stratum corneum lipids, which present the main barrier of the skin. Molecular dynamics simulations, thermal analysis and FTIR measurements were applied. The primary objective of this work was to study the effect of cholesterol on skin structure and dynamics. Two molecular models were constructed, a free fatty acid bilayer (stearic acid, palmitic acid) and a fatty acid/cholesterol mixture at a 1:1 molar ratio. Our simulations were performed at constant pressure and temperature on a nanosecond time scale. The resulting model structures were characterized by calculating surface areas per headgroup, conformational properties, atom densities and order parameters of the fatty acids. Analysis of the simulations indicates that the free fatty acid fraction of stratum corneum lipids stays in a highly ordered crystalline state at skin temperatures. The phase behavior is strongly influenced when cholesterol is added. Cholesterol smoothes the rigid phases of the fatty acids: the order of the hydrocarbon tails (mainly of the last eight bonds) is reduced, the area per molecule becomes larger, the fraction of trans dihedrals is lower and the hydrophobic thickness is reduced. The simulation results are in good agreement with our experimental data from FTIR analysis and NIR-FT Raman spectroscopy.

Body Temperature↗
PubMed2001

Mechanistic appraisal of the charge-transfer complexes of promethazine with chloranil: a modelling approach.

Various mechanisms are often used to explain the interaction between electron donors and acceptors. Commonly proposed mechanisms are those in which the acceptor interacts with the aromatic pi-systems in the donor molecule or the acceptor forms a weak interaction of the Lewis acid with Lewis base type. In this study, the above mechanisms were examined as well as other possible mechanisms. Promethazine was chosen as the model drug containing aromatic systems capable of pi-pi interaction as well as N-methyl group capable of forming a complex with the weak Lewis acid, p-chloranil. Our modelling studies revealed that the situation where the p-chloranil interacts with a protonated N-methyl group is the most significant mechanism of interaction, based on the calculated energies for the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), the Tripos force field energy terms and also the stability of the complexes during molecular dynamics simulations.

Chloranil↗
PubMed2001

Molecular modelling of 17 alpha-hydroxylase-17,20-lyase.

New methods in treatment of hormone-dependent diseases like prostate or breast cancer have become a major subject in medical and pharmaceutical research. Because of the direct correlation of cancer growth and hormone concentration inhibition of hormone biosynthesis reveals a promising strategy in cancer therapy. The key enzyme of androgen biosynthesis is the cytochrome P450 system 17 alpha-hydroxylase-17,20-lyase. To gain deeper insights into the structure and function of this enzyme, whose crystal structure is still unknown we present in this paper a theoretical 3D-model of the human 17 alpha-hydroxylase-17,20-lyase. The model was built by homology modelling using the crystal structure of the P450 CYPeryF as a template. After energy minimisation followed by molecular dynamics simulation the refined model exhibits reasonable protein geometry and a good protein folding quality. For evaluation of protein stability the structure was subjected to molecular dynamics in a waterbox under almost physiological conditions using the GROMACS program. The protein structure and folding remains stable even after 300 ps of free molecular dynamics simulation. The calculation of interaction fields employing the program GRID was used to characterise the active site of the protein. Subsequent docking studies with the natural substrate pregnenolone and further molecular dynamics of the protein-substrate-complexes enabled us to propose a putative binding-site for the physiological substrates.

Amino Acid Sequence↗
PubMed2001

Investigations on inhibitors of human 17 alpha-hydroxylase-17,20-lyase and their interactions with the enzyme. Molecular modelling of 17 alpha-hydroxylase-17,20-lyase, Part II.

New methods in treatment of hormone-dependent diseases like prostate or breast cancer have become a major subject in medical and pharmaceutical research. Because of the direct correlation of cancer growth and hormone concentration, inhibition of hormone biosynthesis presents a promising strategy in cancer therapy. The key enzyme in androgen biosynthesis is the 17 alpha-hydroxylase-17,20-lyase a cytochrome P450 system, which specifically converts gestagens to androgens. Because the 3D-structure of the enzyme is still unknown most recently a ligand-based design was used to gain deeper insights into protein structure and function. In this paper we present molecular modelling studies on compounds acting as competitive inhibitors of the human 17 alpha-hydroxylase-17,20-lyase. The compounds developed by Hartmann et al. belong to two different structural classes and show a wide range of inhibitory potency. The physico-chemical properties of the molecules were investigated and compared by studying structural flexibility and by calculating molecular interactions fields. The superimposition of all inhibitors in a low energy conformation yielded in the common pharmacophore. In the second part of the paper individual inhibitors were docked into the active site of the enzyme model of CYP17 developed in our group. The dynamic behaviour and stability of the protein-inhibitor-complexes was studied. The protein ligand interactions observed in course of the molecular dynamics simulations correspond well with the experimental data.

Computer Simulation↗
PubMed2000

Construction of a full three-dimensional model of the transpeptidase domain of Streptococcus pneumoniae PBP2x starting from its Calpha-atom coordinates.

A new method is described for generating all-atom protein structures from Calpha-atom information. The method, which combines both local structural trace alignments and comparative side chain modeling with ab initio side chain modeling, makes use of both the virtual-bond and the dipole-path methods. Provided that 3D structures of structurally and functionally related proteins exist, the method presented here is highly suitable for generating all-atom coordinates of partly solved, low-resolution crystal structures. Particularly the active site region can be modeled accurately with this procedure, which enables investigation of the binding modes of different classes of ligands with molecular dynamics simulations. The method is applied to the trace of Streptococcus pneumoniae, in order to construct an all-atom structure of the transpeptidase domain. Since after generation of full coordinates of the transpeptidase domain the structure had been solved to 2.4 A resolution, new X-ray coordinates for the worst modeled loop (residues T370 to M386; 17 out of a total number of 351 residues constituting the transpeptidase domain) were incorporated, as kindly provided by Dr. Dideberg. The structure was relaxed with molecular dynamics simulations and simulated annealing methods. The RMS deviation between the 144 aligned Calpha-atoms and the corresponding ones in the originally solved 3.5 A resolution crystal structure was 0.98. The 351 Calpha-atoms of the whole transpeptidase domain of the final model showed an RMS deviation of 1.58. The Ramachandran plot showed that 79.3% of the residues are in the most favored regions, with only 1.0% occurring in disallowed regions. The model presented here can be used to investigate the three-dimensional influences of mutations around the active site of PBP2x.

Amino Acid Sequence↗
PubMed1999

Relaxant activity in rat aorta and trachea, conversion to a muscarinic receptor antagonist and structure-activity relationships of new K(ATP) activating 6-varied benzopyrans.

To characterize ATP-sensitive channels (K(ATP) channels) benzopyrans with different substituents at position 6 were synthesized as new K(ATP)-activators. Their relaxant potencies were determined in rat aorta and trachea. In aorta, pEC50-values (-log, M) ranged from 7.37 to 5.43; in trachea, pEC50-values were 0.3 to 0.8 log units lower. Functional data were compared with binding data obtained in calf tracheal cells using the cyanoguanidine [3H]P1075 (N-cyano-N'-1,1-dimethyl[2,3(n)-3H]propyl)-N11-(3-pyridinyl)guanidine) as radioligand. A high correlation (r = 0.96) between pEC50- and pKD-values indicated that tracheal relaxation produced by benzopyrans is mediated via K(ATP) channels without signal amplification. The permanently charged trimethylammonium derivative designed as a probe for the membrane site of action completely lost its affinity for K(ATP) channels, but converted to an antagonist for muscarinic acetylcholine receptors (pK(B) = 6.12+/-0.10), as confirmed in radioligand binding studies (pK(D) = 5.77+/-0.04). Structure-activity analyses revealed that the 6-substituent influences biological activity by a direct receptor interaction of its own and not indirectly by withdrawing electrons from the benzopyran nucleus. The variance of the biological activity is primarily determined by electrostatic properties, but desolvation energies additionally contribute.

Animals↗
PubMed1999

Skin penetration and metabolism of topical glucocorticoids in reconstructed epidermis and in excised human skin.

PURPOSE: To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate (PC) and the fluorinated monoester betamethasone 17-valerate (BM17V) their metabolism in human keratinocytes and fibroblasts as well as their permeation and biotransformation in reconstructed epidermis and excised human skin was compared. Special attention was given to the 17-monoesters because of their high receptor affinity and antiproliferative effects. METHODS: Glucocorticoid penetration was determined using Franz diffusion cells, quantifying metabolite concentrations by HPLC. Chemical stability and reactivity of the monoesters was determined by molecular modeling analysis. RESULTS: PC accumulated in the stratum corneum. A considerable amount of penetrating PC was hydrolyzed by viable keratinocytes to prednisolone 17-ethylcarbonate (PI7EC), P17EC permeated the skin very rapidly when compared to BM17V. Overall P17EC concentrations in viable tissue were low. Inside of the acceptor fluid, but not within the tissue, P17EC was converted to the more stable prednisolone 21-ethylcarbonate (P21EC). CONCLUSIONS: The inactivation of highly potent, but also cell toxic, 17-monoesters to almost inactive 21-congeners seen with isolated cell monolayers appears less important in the skin. In vitro determination of the dermal 17-monoesters concentrations may allow the prediction of the atrophogenic risk in man. BM17V levels exceeding P17EC concentration about 6-fold may contribute to its lower tolerance when compared to PC.

Administration, Topical↗
PubMed1998

Modified cyclodextrins as chiral selectors: molecular modelling investigations on the enantioselective binding properties of heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-beta-cyclodextri n.

Molecular modelling methods have been used to investigate the enantioselective binding properties of chiral dihydrofuranones on heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-beta-cyclod extrin in capillary gas chromatography. A conformational analysis of the modified beta-cyclodextrin was performed using annealed molecular dynamics. With the program GRID the molecular interaction potential for each of the received energetically reasonable structures of the beta-cyclodextrin and the dihydrofuranones was evaluated using different probe groups. The results of these computations have been used as starting points for constructing geometrically reasonable host-guest complexes between the beta-cyclodextrin and the dihydrofuranones. The subsequently performed molecular dynamics simulations yielded different complex states reflecting the conformational flexibility of the diastereomeric complexes. Considering the evaluated interaction energy between the beta-cyclodextrin and the dihydrofuranones as a measure of complex stability the results are in close agreement with the experimentally determined elution sequences. The methodology for the construction of the interaction model used in this study is capable of simulating the experimental data. We believe that it may serve as a basis for predictions of hitherto unknown elution sequences at modified cyclodextrins.

Carbohydrate Sequence↗
PubMed1998

Construction of a model of the Candida albicans lanosterol 14-alpha-demethylase active site using the homology modelling technique.

On the basis of all hitherto known P450 X-ray structures and applying standard homology modelling procedures a three-dimensional model of the lanosterol-14 alpha-demethylase active site was constructed. The modelled active site nicely hosts the natural substrate lanosterol and the substrate-enzyme complex displayed stability in a 70 ps molecular dynamics simulation. The importance of Thr 122 of lanosterol 14 alpha-demethylase for hydrogen bond formation with the 3-hydroxyl group of lanosterol was found to be a characteristic feature of the interaction geometry.

Binding Sites↗
PubMed1998

Molecular modelling investigation of wild-type and the R528H mutated segment IIS4 of human L-type voltage-gated calcium channels.

A molecular modelling study was performed in order to investigate the pathologically modified properties of L-type voltage-gated calcium channels caused by the arginine-to-histidine mutation at position 528 (R528H) in segment IIS4. For an appropriate consideration of the ionization state the finite difference Poisson-Boltzmann method was applied to compute the apparent pKa values of all titratable residues using standard conditions and an explicit lipophilic environment, respectively. Restrained molecular dynamics simulations were carried out for the alpha-helical transmembrane segments of the wild-type and the R528H mutant to explore their conformational behaviour. While both structures showed almost the same side-chain flexibility around the conserved residues, only the mutant partially formed a hydrogen bond from H528 to R531 during dynamics simulations. This local interaction not only causes a lower mobility of the directly involved residues but also leads to a global distortion of all positively charged amino acids of the mutant. Mostly affected is the side-chain of R534 that is shifted about 61 degrees closer to R531 and about 5 A in the direction of the cytoplasm. Subsequent examination of the molecular characteristics of this putative voltage sensor of the channel revealed considerable variations with regard to hydrogen bonding and electronic properties. Most obvious are the dramatic loss of a strong positive molecular electrostatic potential and the reduced hydrogen donor activities around position 528 of the mutant. How these results may be interpreted in relation to an enhanced inactivation rate is discussed, considering earlier findings at homologous voltage-gated potassium and sodium channels.

Amino Acid Sequence↗
PubMed1998

Development of a binding site model for histamine H3-receptor agonists.

On the basis of molecular modelling studies the structural and conformational requirements for receptor affinity and activity of histamine H3-receptor agonists were studied. It was shown that the known H3-receptor agonists can be fitted accurately into a common pharmacophoric pattern. Using the YAK pseudoreceptor approach an amino acid model for the H3-receptor agonist binding site was generated which reflects binding properties and biological data of the investigated agonists. The postulated binding site model was validated by predicting biological data for four structures not considered in model construction. The amino acid positions of the pseudoreceptor were found to be in good agreement with calculated GRID interaction fields for the investigated histamine H3-receptor agonists.

Binding Sites↗
PubMed1998

Pharmacophore and pseudoreceptor modelling of class Ib antiarrhythmic and local anaesthetic lidocaine analogues.

A molecular modelling study was carried out in order to investigate the molecular binding behaviour of antiarrhythmically and local anaesthetically active aminoacylanilide derivatives from the lidocaine type at their specific sodium channel binding site. An examination of relevant X-ray structures and of results derived from systematic and random search conformational analyses yielded information about the spatial requirements of these sodium channel blocking compounds. Common structural elements in combination with their non-covalent interaction potentials were used to generate a rational pharmacophore model. To further support and refine this model an atomistic pseudoreceptor of the Na+ channel binding site was constructed using a training set of eight well-defined lidocaine homologues. With the final pseudoreceptor, composed of tyrosine, phenylalanine, serine, valine and three isoleucine residues, it was possible to correlate experimental versus calculated dissociation constants of the training set with a correlation coefficient of 0.98. To test the accuracy of this model, the affinities of three additional compounds, not used for pseudoreceptor modelling, were predicted. After free relaxation within the binding cavity using a Monte-Carlo minimization the test set yielded a RMS error in the prediction of 0.039 kcal/mol corresponding to an uncertainty factor of 1.06. In addition, this hypothetical receptor model provides evidence for an exceptional binding mode of the lidocaine metabolite glycinexylidide (GX) which could explain its low binding affinity and thereby possibly the minor physiological effects with respect to lidocaine.

Amino Acid Sequence↗
PubMed1997

The development of nonpeptide angiotensin II receptor antagonists: a success story.

The modulation of the renin-angiotensin system continues to be an important target in drug design. The development of nonpeptide angiotensin II receptor antagonists offers a new tool in treatment of hypertension. Starting from benzimidazole lead structures detailed structure-activity analyses, molecular modeling methods, and dedicated syntheses led to the development of potent, orally active antihypertensive drugs.

Angiotensin II↗
PubMed1995

Band-shifting through polypeptide beta-sheet structures in the cyanine UV-Vis spectrum.

If oxa- or thiacarbocyanine is introduced into an aqueous poly-L-lysine (PL) solution in a concentration higher than that of aggregation, then a shift of the absorption band of the cyanine monomer (M) can be observed in the UV-Vis spectrum, provided that the PL has a beta-sheet conformation. Other polypeptide aggregates with a high beta-sheet content exhibit this effect as well, whereas for PL with an alpha-helix conformation no spectral shift is observed. The force-field optimized molecular models and the calculated interaction energies prove that the beta-sheet interacts significantly more intensively with the cyanine than the alpha-helix does. The quantum chemically calculated highest occupied and lowest unoccupied molecular orbital (HOMO-LUMO) energies of the cyanines and cyanine beta-sheet polypeptide complexes predict a M-shift to bathochromic frequencies in agreement with experimental findings. In the case of the measured M-shift to hypsochromic frequencies, the shift appears to be influenced by the presence of cyanine J-aggregates. The results open the way for a fast and simple method to identify polypeptide beta-sheet structures in biological and other systems containing polypeptides by using cyanine as a sensor.

Carbocyanines↗
PubMed1994

Conformational analysis and receptor modelling of m1 and m2 selective antagonists.

On the basis of an elaborate conformational analysis of m1- and m2-selective antagonists a respective pharmacophore was deduced. This then was introduced into models of the two muscarinic receptor subtypes. These models were constructed starting from bacteriorhodopsin as a template in accordance with alignment data and mutation experiments. The validity of the interaction geometries between ligands and receptor subtypes is supported by a significant correlation between calculated interaction energies and experimentally determined affinity data.

Amino Acid Sequence↗
PubMed1993

A new consistent model explaining structure (conformation)-activity relationships of opiates with mu-selectivity.

Several different classes of opiates such as PET (7-alpha-(1-Hydroxy-1-methyl-3-phenylpropyl)-6,14-endo-ethenotetra - hydronorthebaine), phenazocine, fentanyl, carfentanil, ohmefentanyl, prodine derivatives, methadone and etonitazene have been investigated using the molecular modelling program package SYBYL and the TRIPOS empirical force field. Comparison of the energetically optimized structures and their corresponding molecular electrostatic potentials was used for the development of a new model of conformation-activity relationships of mu-selective opiates. We considered six important spatial positions of these molecules which were assumed to be directly implicated in the interaction with the opiate receptor. We found that these opiates may bind to the receptor with their protonated nitrogen atom assuming one or the other of two different orientations. The obtained results offer new insights into important receptor interactions and the diverse opiate activities of all the compounds examined can be explained in a unified manner. For example, the most active ohmefentanyl stereo-isomer was predicted on the basis of the proposed model.

Analgesics↗