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D F Lewis

Publications and source records attributed to D F Lewis.

At least 37 records · Page 2Linked to original sources
PubMed2000

On the recognition of mammalian microsomal cytochrome P450 substrates and their characteristics: towards the prediction of human p450 substrate specificity and metabolism.

The characteristics of mammalian microsomal P450 xenobiotic substrates are described, particularly with reference to the major P450 isoforms associated with drug metabolism in humans. It is further reported that a relatively small number of molecular, electronic, and physico-chemical properties are required to discriminate between chemicals that exhibit specificity for human P450 isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. Molecular templates of superimposed substrates are shown to be complementary with the putative active sites of the relevant enzymes, thus enabling a possible prediction of P450 specificity from structure. Factors contributing to metabolic clearance and binding affinity are also discussed, and methods for their calculation are described.

Animals↗
PubMed2000

Structural characteristics of human P450s involved in drug metabolism: QSARs and lipophilicity profiles.

The factors with human P450 substrate selectivity are reviewed, together with extensive tabulation of quantitative relationships between structure and activity for many species of P450 substrates, inducers and inhibitors. In addition, the physicochemical characteristics (logP and pK(a) values) of heptic microsomal P450 substrates are collated, such that comparisons can be made on the grounds of compound lipophilicities.

Animals↗
PubMed2000

Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette.

A capillary array electrophoresis DNA sequencer is reported based on a micromachined sheath-flow cuvette as the detection chamber. This cuvette is equipped with a set of micromachined features that hold the capillaries in precise registration to ensure uniform spacing between the capillaries, in order to generate uniform hydrodynamic flow in the cuvette. A laser beam excites all of the samples simultaneously, and a microscope objective images fluorescence onto a set of avalanche photodiodes, which operate in the analog mode. A high-gain transimpedance amplifier is used for each photodiode, providing high duty-cycle detection of fluorescence.

DNA↗
PubMed2000

Placental transfusion: umbilical cord clamping and preterm infants.

OBJECTIVE: To investigate the clinical effects of early versus late cord clamping in preterm infants. STUDY DESIGN: A total of 32 premature infants were prospectively randomized. The following parameters were measured: Initial spun hematocrit (Hct), hemoglobin (Hgb), red blood cell (RBC) counts, frequency of blood transfusions, peak serum bilirubin, mean blood pressure (MBP), oxygen index, intraventricular hemorrhage, and significant patent ductus arteriosus (PDA). RESULTS: Over the 4-week study period, the delayed cord clamping (DCC) group exhibited a decrease in the frequency of blood transfusion (p < 0.001) and also a decrease in albumin transfusions over the first 24 hours (p < 0.03). MBP in the first 4 hours was higher in the DCC group (p < 0.01), and there were statistically significant increases in Hct (21%), Hgb (23%), and RBC count (21%) compared with the early cord clamping group. The risks of patent ductus arteriosus, hyperbilirubinemia, or intraventricular hemorrhage were similar in both groups. Late clamping of the umbilical cord had little or no effect on the oxygen index. CONCLUSION: DCC significantly reduced the requirement for blood and albumin transfusion. It also increased the initial Hct, RBC count, Hgb levels, and MBP.

Blood Component Transfusion↗
PubMed2000

Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform.

1. The construction of a three-dimensional model of human CYP2E1 is reported. It is based on homology with the haemoprotein domain of the unusual bacterial P450, CYP102, which is of known crystal structure. 2. Interactive docking of a number of human CYP2E1 substrates is consistent with their known positions of CYP2E1-mediated metabolism, where specific interactions with key active site amino acid side-chains appear to rationalize the binding and orientation of substrate molecules. 3. Amino acid residues within the putative active site of human CYP2E1, including those associated with the binding of substrates and inhibitors, are shown to correspond with those identified by site-directed mutagenesis experiments conducted on CYP2 family isoforms, and they are known to affect substrate metabolism regioselectivity. 4. Consequently, it was found that the CYP2E1 active site exhibits complementarity with the structural characteristics of known substrates and inhibitors of this enzyme, including their relatively low molecular weights and disposition of hydrogen bond-forming groups.

Amino Acid Sequence↗
PubMed2000

Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase.

A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R.

Aerobiosis↗
PubMed2000

Modelling human cytochromes P450 for evaluating drug metabolism: an update.

Cytochrome P450 (CYP) enzymes represent the major catalysts for the Phase 1 metabolism of drugs and other xenobiotics in Mammalia, including Homo sapiens. There is considerable current interest in evaluating and, consequently, predicting the metabolic fate of new chemical entities (NCEs) via modelling molecular interactions with P450 constructs, such that sites of metabolism, particular CYP involvement and binding affinities, can be estimated. This paper focuses on the principles for homology modelling of typical enzyme-substrate interactions within the putative active sites of major P450s associated with drug metabolism in man. It also represents an update on previously published work in this journal /1/.

Cytochrome P-450 Enzyme System↗
PubMed1999

A multiple-capillary electrophoresis system for small-scale DNA sequencing and analysis.

A five-capillary system has been developed for DNA sequencing and analysis. The post-column fluorescence detector is based on a sheath-flow cuvette. The instrument provides uniform and continuous illumination of the samples. The cuvette virtually eliminates cross-talk in the fluorescence signal between capillaries. Discrete single-photon counting avalanche photodiodes provide high efficiency light detection. The instrument has detection limits (3sigma) of 130 +/- 30 fluorescein molecules injected onto each capillary. Over 650 bases of sequence at 98.8% accuracy were generated in 100 min at 50 degrees C from M13mp18. Separation and detection of short tandem repeats proved efficient and accurate with the use of internal standards for direct comparison of migration times between capillaries.

Adult↗
PubMed1999

Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102.

Molecular modelling of a number of CYP1 family enzymes from rat, plaice and human is described based on amino acid sequence homology with the haemoprotein domain of CYP102, a unique bacterial P450 of known structure. The interaction of various substrates and inhibitors within the putative active sites of rat CYP1A1, human CYP1A2, a fish CYP1 enzyme CYP1A6 (from plaice) and human CYP1B1, is shown to be consistent with P450-mediated oxidation in each example or, in the case of inhibitors, mechanism of inhibition. It is reported that relatively small changes between the enzymes' active site regions assist in the rationalization of CYP1 enzyme preferences for particular substrate types, and a template of superimposed CYP1A2 substrates is shown to fit the putative active site of the human CYP1A2 enzyme.

Animals↗
PubMed1999

Molecular modelling of the human cytochrome P450 isoform CYP2A6 and investigations of CYP2A substrate selectivity.

(1) The generation of a homology model of CYP2A6, the major catalyst of human hepatic coumarin 7-hydroxylase activity, involves the use of the recently published substrate-bound CYP102 crystal structure as a template. (2) A substantial number of structurally diverse CYP2A6 substrates are found to dock satisfactorily within the putative active site of the enzyme, leading to the formulation of a structural template (or pharmacophore) for CYP2A6 specificity/selectivity. (3) The CYP2A6 model is consistent with available evidence from site-directed mutagenesis studies carried out on CYP2A subfamily isoforms, and enables some explanation of species differences in CYP2A-mediated metabolism of certain substrates. (4) Quantitative structure-activity relationship (QSAR) analysis of CYP2A5 (the mouse orthologue) mutants yields statistically significant correlations between various properties of amino acid residues and coumarin 7-hydroxylase activity.

Amino Acid Sequence↗
PubMed1999

A randomized clinical trial of daily nonstress testing versus biophysical profile in the management of preterm premature rupture of membranes.

OBJECTIVE: Our purpose was to evaluate the ability of 2 different antepartum testing modalities to predict infectious morbidity in patients with preterm premature rupture of membranes. STUDY DESIGN: During a 36-month period, patients with preterm premature rupture of membranes (at 23 to 34 weeks of gestation) were randomly assigned to either a daily nonstress test or a biophysical profile, after a 24-hour observational period. We used the original scoring system of Manning et al for the biophysical profile, with a score of </=6 considered abnormal. Nonstress test results were considered abnormal if the test was nonreactive or if the patient had late decelerations or significant variable decelerations; abnormal results led to further evaluation with a biophysical profile. Results of the last test before delivery were evaluated to determine whether infectious complications had been predicted. RESULTS: One hundred thirty-five patients were enrolled in the study. Demographics, pregnancy characteristics, and neonatal outcomes were similar. Neither the daily nonstress test nor the daily biophysical profile had good sensitivity for predicting infectious complications (39.1% and 25.0%, respectively). However, both had good specificity (84.6% and 92.6%, respectively). Positive and negative predictive values were 52.9% and 75.9%, respectively, for the daily nonstress test and 66.7% and 68.4%, respectively, for the daily biophysical profile. Cost was significantly higher in the daily biophysical profile group. Nonstress testing of patients at <28 weeks' gestation generally required a backup biophysical profile. CONCLUSION: Neither the daily nonstress test nor the daily biophysical profile had good sensitivity for predicting infectious complications after preterm premature rupture of membranes.

Adult↗
PubMed1999

Homology modelling of human cytochromes P450 involved in xenobiotic metabolism and rationalization of substrate selectivity.

Molecular modelling of human cytochrome P450 (CYP) isoforms is described, based on amino acid sequence homology with a unique bacterial P450 (CYP102) of known crystal structure. It is found that for the human hepatic P450s involved in the metabolism of xenobiotics, ie. CYPIA2, CYP 1A6, CYP2B6, CYP2C9, CYP2C 19, CYP2D6, CYP2E1 and CYP3A4, there is a satisfactory agreement between specific substrate characteristics and topographical features of the putative active sites, including complementarity with key amino acid residues in the P450 haem environments. A combination of homology model interactions with substrates and certain molecular properties of the compounds themselves provides a methodology for the evaluation of potential P450 selectivity in new chemical entities (NCEs).

Amino Acid Sequence↗
PubMed1999

Molecular modelling of CYP4A subfamily members based on sequence homology with CYP102.

1. Homology modelling of various members of the CYP4A subfamily based on the CYP102 template structure is reported. 2. The binding interactions of specific substrates to the CYP4A forms from rat (CYP4A1), human (CYP4A11) and rabbit (CYP4A4) are shown to be consistent with experimental evidence regarding regioselectivity of metabolism. 3. The differences in substrate specificity between CYP4A1, CYP4A11 and CYP4A4 towards fatty acids and prostaglandins respectively are rationalized in terms of variations in active site amino residues.

Animals↗
PubMed1999

Molecular modelling of CYP2B6, the human CYP2B isoform, by homology with the substrate-bound CYP102 crystal structure: evaluation of CYP2B6 substrate characteristics, the cytochrome b5 binding site and comparisons with CYP2B1 and CYP2B4.

1. Molecular modelling studies of CYP2B isoforms from rat (CYP2B1), rabbit (CYP2B4) and man (CYP2B6) are reported, with particular emphasis on substrate interactions with the human CYP2B isoform, CYP2B6. 2. The findings represent an advance on our previous study that focused primarily on the rat CYP2B isoform, CYP2B1, and involved homology modelling with substrate-free CYP102. 3. The current work utilizes the recently published substrate-bound CYP102 crystal structure as a template for construction of the CYP2B subfamily isoforms and shows, in particular, that known CYP2B6 substrate specificity and regioselectivity can be rationalized by putative active site interactions.

Amino Acid Sequence↗
PubMed1999

Interleukin-8 stimulates placental prostacyclin production in preeclampsia.

PROBLEM: Our purpose was to determine placental interleukin (IL)-8 production and its correlation with the prostacyclin production in normal and preeclamptic pregnancies and to evaluate the beneficial effect of IL-8 on prostacyclin production. METHOD OF STUDY: We determined 1) the in vitro production of IL-8 and prostacyclin by placental villous tissues from normal and preeclamptic pregnancies and 2) the production of prostacyclin by villous tissues from preeclampsia treated with recombinant human IL-8 (rhIL-8). IL-8 levels were measured by enzyme-linked immunosorbent assay and prostacyclin by radioimmunoassay of 6-keto PGF1alpha, the stable metabolite of prostacyclin. RESULTS: 1) Placental production of IL-8 and 6-keto PGF1alpha were significantly less in preeclampsia than in normal pregnancies, P<0.05. 2) Placental production of 6-keto PGF1alpha and IL-8 was significantly correlated in preeclampsia, P<0.01. 3) Placental tissues treated with IL-8 exhibited a concentration-dependent increase in 6-keto PGF1alpha production. CONCLUSIONS: Placental tissues from preeclampsia produce significantly less IL-8 than tissues from normal pregnancies, which correlates with decreased prostacyclin production. IL-8 improves placental prostacyclin production in preeclampsia.

6-Ketoprostaglandin F1 alpha↗
PubMed1999

Cytochrome P450 substrate specificities, substrate structural templates and enzyme active site geometries.

The structural characteristics of human cytochrome P450 substrates are outlined in the light of extensive studies on P450 substrate specificity. Templates of superimposed substrates for individual P450 isozymes are shown to fit the corresponding enzyme active sites, where contacts with specific amino acid residues appear to be involved in the interaction with each structural template. Procedures leading to the evaluation of likely P450 specificity, binding affinity and rate of metabolism are described in the context of key examples in which molecular modelling appears to rationalize experimentally observed findings.

Animals↗
PubMed1999

Molecular modelling of lanosterol 14 alpha-demethylase (CYP51) from Saccharomyces cerevisiae via homology with CYP102, a unique bacterial cytochrome P450 isoform: quantitative structure-activity relationships (QSARs) within two related series of antifungal azole derivatives.

The construction of a three-dimensional molecular model of the fungal form of cytochrome P450 (CYP51) from Saccharomyces cerevisiae, based on homology with the haemoprotein domain of CYP102 from Bacillus megaterium (a unique bacterial P450 of known crystal structure) is described. It is found that the endogenous substrate, lanosterol, can readily occupy the putative active site of the CYP51 model such that the known mono-oxygenation reaction, leading to C14-demethylation of lanosterol, is the preferred route of metabolism for this particular substrate. Key amino acid contacts within the CYP51 active site appear to orientate lanosterol for oxidative attack at the C14-methyl group, and the position of the substrate relative to the haem moiety is consistent with the phenyl-iron complexation studies reported by Tuck et al. [J. Biol. Chem., 267, 13175-13179 (1992)]. Typical azole inhibitors, such as ketoconazole, are able to fit the putative active site of CYP51 by a combination of haem ligation, hydrogen bonding, pi-pi stacking and hydrophobic interactions within the enzyme's haem environment. The mode of action of azole antifungals, as described by the modelling studies, is supported by quantitative structure-activity relationship (QSAR) analyses on two groups of structurally related fungal inhibitors. Moreover, the results of molecular electrostatic isopotential (EIP) energy calculations are compatible with the proposed mode of binding between azole antifungal agents and the putative active site of CYP51, although membrane interactions may also have a role in the antifungal activity of azole derivatives.

Amino Acid Sequence↗