Biomedical subjects

C A Laughton

Publications and source records attributed to C A Laughton.

44 records · Page 3Linked to original sources

PubMed1990

Interaction of berenil with the tyrT DNA sequence studied by footprinting and molecular modelling. Implications for the design of sequence-specific DNA recognition agents.

We have developed a technique of partially-restrained molecular mechanics enthalpy minimisation which enables the sequence-dependence of the DNA binding of a non-intercalating ligand to be studied for arbitrary sequences of considerable length (greater than = 60 base-pairs). The technique has been applied to analyse the binding of berenil to the minor groove of a 60 base-pair sequence derived from the tyrT promoter; the results are compared with those obtained by DNAse I and hydroxyl radical footprinting on the same sequence. The calculated and experimentally observed patterns of binding are in good agreement. Analysis of the modelling data highlights the importance of DNA flexibility in ligand binding. Further, the electrostatic component of the interaction tends to favour binding to AT-rich regions, whilst the van der Waals interaction energy term favours GC-rich ones. The results also suggest that an important contribution to the observed preference for binding in AT-rich regions arises from lower DNA perturbation energies and is not accompanied by reduced DNA structural perturbations in such sequences. It is therefore concluded that those modes of DNA distortion favourable to binding are probably more flexible in AT-rich regions. The structure of the modelled DNA sequence has also been analysed in terms of helical parameters. For the DNA energy-minimised in the absence of berenil, certain helical parameters show marked sequence-dependence. For example, purine-pyrimidine (R-Y) base pairs show a consistent positive buckle whereas this feature is consistently negative for Y-R pairs. Further, CG steps show lower than average values of slide while GC steps show lower than average values of rise. Similar analysis of the modelling data from the calculations including berenil highlights the importance of DNA flexibility in ligand binding. We observe that the binding of berenil induces characteristic responses in different helical parameters for the base-pairs around the binding site. For example, buckle and tilt tend to become more negative to the 5'-side of the binding site and more positive to the 3'-side, while the base steps at either side of the centre of the site show increased twist and decreased roll.

Amidines↗

PubMed1990

Crystallographic and molecular modeling studies on 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione and its butyl analogue, inhibitors of mammalian aromatase. Comparison with natural substrates: prediction of enantioselectivity for N-alkyl derivatives.

Inhibitors of the cytochrome P450 enzyme aromatase, which is involved in the biosynthesis of estrogens from androgens, are of proven utility in the treatment of hormone-dependent breast cancer. The determination of the crystal structure of one such inhibitor, 3-ethyl-3-(4-pyridyl)piperidine-2,6-dione (2) and its 3-butyl analogue (3) is described. In the absence of three-dimensional structural information for the enzyme, conformational analysis and comparison with natural substrates has been performed in order to define possible "active" conformations. The enhanced inhibitory activity of 3 may be linked to hydrophobic interactions between the side chain and that portion of the enzyme that normally interacts with the B and C rings of a steroid substrate. Information gained from this study and previous studies by other workers has been combined in order to produce a hypothesis to explain the pattern of activity of N(1)-alkyl derivatives of 2. The successful application of this hypothesis to the prediction of the relative aromatase inhibitory activities of the two enantiomers of the N-octyl derivative (4) is described.

Aminoglutethimide↗

PubMed1990

Inhibitors of the P450 enzymes aromatase and lyase. Crystallographic and molecular modeling studies suggest structural features of pyridylacetic acid derivatives responsible for differences in enzyme inhibitory activity.

Derivatives of 4-pyridylacetic acid are known to be inhibitors of the cytochrome P450 enzymes aromatase and lyase (17 alpha-hydroxylase/C17-20lyase), and are therefore of interest in the treatment of hormone dependent breast and prostate cancers. We report the determination of the crystal structure of one such derivative, the 4-tert-butyl cyclohexyl ester, and molecular modeling studies on two related inhibitors, the cyclohexyl ester and its alpha-methyl derivative. These latter two compounds show a marked difference in their relative activities against aromatase and lyase. Two models are proposed for the interaction of these molecules with the target enzymes on the basis of their ability to adopt conformations that partially mimic steroid substrates. From these models an explanation can be advanced for the fact that, compared with the unmethylated analogue, the (racemic) alpha-methylated compound is seven times poorer as an inhibitor of aromatase but seven times better as an inhibitor of lyase. The model proposed for binding to aromatase places the alpha-carbon of the ester group in the position occupied by C(2) of steroid substrates. In contrast, that proposed for binding to lyase places this atom in the position occupied by C(17) of steroid substrates. The introduction of steric bulk at C(2) is known to be unfavorable for aromatase inhibition, while its introduction at C(17) may lead to a better mimicry of the steroid D-ring and so improve lyase inhibition.

Acetates↗

PubMed1990

Molecular-mechanics modelling of drug-DNA structures; the effects of differing dielectric treatment on helix parameters and comparison with a fully solvated structural model.

This study analyses the influence that the nature of the dielectric constant has on the final structures obtained from in vacuo molecular mechanics calculations on a drug-DNA complex and compares these structures with the energy minimised complex including explicit solvent molecules. Minimisations have been performed on a proflavine-decanucleotide structure, where the drug was intercalated at the d(CpG) site of the d(GpApTpApCpGpApTpApC) decamer duplex, using two expressions for the dielectric constant: a distance-independent, epsilon ij = EPS, and a distance-dependent, epsilon ij = EPS*Rij, form and for values of EPS from 1 to 8. Significantly different structures are obtained for the distance-independent and the distance-dependent expressions of the dielectric constant. The use of a distance-independent dielectric constant leads to distorted structures, which are very sensitive to slight changes in the value of EPS. The use of a distance-dependent dielectric constant leads to less distorted and more stable structures. The effects on helical parameters are analysed in detail. The structures obtained for different values of EPS (within the distance-dependent formalism) seem to converge for values of EPS equal to 4 or greater. Based on these results a distance-dependent form of the dielectric with an EPS value of 4 is recommended in order to produce reliable refined nucleic acid structures by molecular mechanics. These conclusions have been supported by molecular-mechanics minimisation of the same structure with the inclusion of explicit water molecules and counter-ions.

Base Composition↗

PubMed1990

Structural studies on bio-active compounds. Part XIV. Molecular modelling of the interactions between pentamidine and DNA.

Molecular mechanics modelling was carried out on the antimicrobial aromatic diamidine, pentamidine, bound to the minor groove of several AT-rich DNA octamers. The pentamidine molecule was found to span four base pairs, with its highly charged amidinium groups forming hydrogen bonds to the O2 of thymine or the N3 of adenine, but not to the backbone phosphate groups. The mean binding energy of the pentamidine-DNA complexes was -52 kcal/mol, of the same order as that of berenil-DNA complexes. There was no significant energy difference between those models which contained GC base pairs and those which did not. The presence of bound pentamidine resulted in some distortion of helix geometry involving helix opening towards the minor groove in most cases and a decrease in the number of residues per turn.

Base Sequence↗

PubMed1989

Sterically hindered analogues of diacylglycerols. Synthesis, binding to the phorbol ester receptor and metabolism in A549 human lung carcinoma cells.

The 5 following compounds were synthesized in order to investigate the relationship between structure and ability of glyceride-type molecules to bind to the phorbol ester receptor: one dioctanoyl cyclohexane-1,2,4-triol, 2 isomeric methyl analogues of 1,2-dioctanoyl-sn-glycerol (diC8), one dimethyl and one cyclohexyl analogue of diC8. Their ability to compete with 3H-labelled phorbol-12,13-dibutyrate [( 3H]PDBu) for specific binding sites in intact A549 human-derived lung carcinoma cells and in a cytosolic cell extract was compared with that of diC8 and 12-O-tetradecanoylphorbol-13-acetate (TPA). The affinity of diC8 for the phorbol ester receptor was much weaker than that of TPA. The analogues in turn were less able than diC8 to compete with [3H]PDBu for receptor sites. Like diC8 and unlike TPA, the synthesized compounds inhibited cell growth only at those concentrations at which cytotoxicity was also apparent. DiC8 and its methyl and dimethyl derivates, but not the cyclohexyl derivative or the cyclohexanetriol diester, were metabolically removed from cellular incubates as measured by gas liquid chromatography. The results suggest that the binding of glyceride-type molecules to the phorbol ester receptor exhibits stringent specificity and that the design of novel potent agonists of phorbol esters might require the placement of the molecular features of diacylglycerols important for biological activity into a molecular framework which is more complicated than glycerol.

Caenorhabditis elegans Proteins↗

PubMed1989

Studies on bioactive compounds. 13. Synthesis and lack of growth-inhibitory properties of cyclohexane-1,2,4-triol 1,2-diesters, which resemble ring C of the phorbol ester molecule.

It has been suggested that ring C of biologically active phorbol esters is an essential structural feature of the pharmocophore which confers activity on these compounds. In this study the hypothesis has been tested that compounds which resemble ring C of the phorbol ester molecule mimic the ability of phorbol esters to inhibit cell growth at nontoxic concentrations. All four diastereoisomers of (+/-)-1,2-di-O-octanoylcyclohexane-1,2,4-triol have been prepared from cyclohexen-4-ol and tested for growth-inhibitory and cytotoxic properties. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate inhibited the growth of A549 human lung carcinoma cells by 50% at a concentration of 0.2 nM and exerted cytotoxicity at concentrations of greater than 1 microM. Diacylglycerols are the physiological ligands and activators of protein kinase C, the receptor via which phorbol esters are thought to mediate their effects. The diacylglycerols 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol and the cyclohexanetriol diesters inhibited the growth of A549 cells only at concentrations of 10(-5) to 10(-4) M, at which they were also cytotoxic. A computer-assisted analysis of the goodness of fit between the cyclohexanetriol diesters and ring C of the phorbol moiety revealed possible energetic grounds for conformational dissimilarities. The results suggest that activation of protein kinase C alone is probably not sufficient to reproduce phorbol ester induced growth arrest in A549 cells and that the cyclohexanetriol diesters may lack pivotal elements of the phorbol ester pharmacophore.

Cell Division↗

PubMedDate not supplied

The mysteries of telomere structure and recognition: could radioprobing help?

PURPOSE: Telomeres are specialized DNA-protein complexes found at the ends of eukaryotic chromosomes. In normal somatic cells these become shorter with each cell division and appear to control their replicative lifespan. However almost all tumours show activation of the enzyme telomerase, a specialised reverse transcriptase/DNA polymerase, that can add new telomeric repeats to the ends of chromosomes and this appears to be a key factor in the cell immortalization process. Consequently there is much current interest in the potential for inhibitors of telomere extension in the treatment of cancer. Several groups have found that it is possible to produce inhibitory molecules that target the telomeric repeat (substrate) DNA rather than the telomerase enzyme itself. This is thought to work because it has been found that in vitro, these DNA sequences can fold up into a four-stranded (quadruplex) structure that the drugs recognise and stabilize, but which is not recognised by the enzyme. However, while medicinal chemists continue to base rational design programs on this hypothesis, there is currently very little evidence that these structures form in vivo, and that in vivo the drugs work by binding to them. To have incontrovertible evidence of where and how these telomerase inhibitors and DNA interact is therefore a pressing concern for a basic understanding of their mechanism of action and effective drug development. MATERIALS AND METHODS: Radioprobing represents a valuable new approach to the study of DNA structures. Recently we have shown through computer simulations of radioprobing that the technique is a remarkably sensitive probe of quite fine details of DNA conformation. Here we report on our simulations of the binding of a radiolabelled telomerase inhibitor, related to a class of novel inhibitors under development at Nottingham, to a variety of possible structures for telomeric DNA. RESULTS AND CONCLUSIONS: The predicted cleavage patterns prove to be very sensitive to the DNA structure, and the mode of binding of the drug. These results suggest that radioprobing experiments should be able to provide unambiguous evidence as to the 'true' nature of the telomere-drug complexes, and so aid the rational design programme.

Animals↗