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D Tew

Publications and source records attributed to D Tew.

17 recordsLinked to original sources
PubMed2003

Copper modulation of ion channels of PrP[106-126] mutant prion peptide fragments.

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.

Copper↗
PubMed2001

Azepanone-based inhibitors of human and rat cathepsin K.

The synthesis, in vitro activities, and pharmacokinetics of a series of azepanone-based inhibitors of the cysteine protease cathepsin K (EC 3.4.22.38) are described. These compounds show improved configurational stability of the C-4 diastereomeric center relative to the previously published five- and six-membered ring ketone-based inhibitor series. Studies in this series have led to the identification of 20, a potent, selective inhibitor of human cathepsin K (K(i) = 0.16 nM) as well as 24, a potent inhibitor of both human (K(i) = 0.0048 nM) and rat (K(i,app) = 4.8 nM) cathepsin K. Small-molecule X-ray crystallographic analysis of 20 established the C-4 S stereochemistry as being critical for potent inhibition and that unbound 20 adopted the expected equatorial conformation for the C-4 substituent. Molecular modeling studies predicted the higher energy axial orientation at C-4 of 20 when bound within the active site of cathepsin K, a feature subsequently confirmed by X-ray crystallography. Pharmacokinetic studies in the rat show 20 to be 42% orally bioavailable. Comparison of the transport of the cyclic and acyclic analogues through CaCo-2 cells suggests that oral bioavailability of the acyclic derivatives is limited by a P-glycoprotein-mediated efflux mechanism. It is concluded that the introduction of a conformational constraint has served the dual purpose of increasing inhibitor potency by locking in a bioactive conformation as well as locking out available conformations which may serve as substrates for enzyme systems that limit oral bioavailability.

Administration, Oral↗
PubMed2001

Cyclic ketone inhibitors of the cysteine protease cathepsin K.

Cathepsin K (EC 3.4.22.38), a cysteine protease of the papain superfamily, is predominantly expressed in osteoclasts and has been postulated as a target for the treatment of osteoporosis. Crystallographic and structure--activity studies on a series of acyclic ketone-based inhibitors of cathepsin K have led to the design and identification of two series of cyclic ketone inhibitors. The mode of binding for four of these cyclic and acyclic inhibitors to cathepsin K is discussed and compared. All of the structures are consistent with addition of the active site thiol to the ketone of the inhibitors with the formation of a hemithioketal. Cocrystallization of the C-3 diastereomeric 3-amidotetrahydrofuran-4-one analogue 16 with cathepsin K showed the inhibitor to occupy the unprimed side of the active site with the 3S diastereomer preferred. This C-3 stereochemical preference is in contrast to the X-ray cocrystal structures of the 3-amidopyrrolidin-4-one inhibitors 29 and 33 which show these inhibitors to prefer binding of the 3R diastereomer. The 3-amidopyrrolidin-4-one inhibitors were bound in the active site of the enzyme in two alternate directions. Epimerization issues associated with the labile alpha-amino ketone diastereomeric center contained within these inhibitor classes has proven to limit their utility despite promising pharmacokinetics displayed in both series of compounds.

Animals↗
PubMed2001

The role of strand 1 of the C beta-sheet in the structure and function of alpha(1)-antitrypsin.

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C beta-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant alpha(1)-antitrypsin Pittsburgh mutant (P1 = Arg). Mutation of the P10' position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7' and P9' caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6' and P8' positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6' and P8' residues of the C beta-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6' and P8' mutants of alpha(1)-antitrypsin had melting temperatures 14 degrees lower than wild-type alpha(1)-antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.

Antithrombins↗
PubMed1999

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution.

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.

Animals↗
PubMed1998

ATP-Citrate lyase as a target for hypolipidemic intervention. 2. Synthesis and evaluation of (3R,5S)-omega-substituted-3-carboxy-3, 5-dihydroxyalkanoic acids and their gamma-lactone prodrugs as inhibitors of the enzyme in vitro and in vivo.

A series of (3R,5S)-omega-substituted-3-carboxy-3, 5-dihydroxyalkanoic acids have been synthesized and evaluated as inhibitors of the recombinant human form of ATP-citrate lyase. The best of these have Ki's in the 200-1000 nM range. As the corresponding thermodynamically favored gamma-lactone prodrugs, a number of compounds are able to inhibit cholesterol and fatty acid synthesis in HepG2 cells and reduce plasma triglyceride levels in vivo. The best of these, compound 77, is able to induce clear hypocholesterolemic and hypotriglyceridaemic responses when administered orally to rat and dog. These results provide evidence to support the hypothesis that compounds which inhibit ATP-citrate lyase have the potential to be a novel class of hypolipidemic agent, which possess combined hypocholesterolemic and hypotriglyceridemic activities.

ATP Citrate (pro-S)-Lyase↗
PubMed1998

The role of ATP citrate-lyase in the metabolic regulation of plasma lipids. Hypolipidaemic effects of SB-204990, a lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076.

ATP citrate (pro-S)-lyase (EC 4.1.3.8), a cytosolic enzyme that generates acetyl-CoA for cholesterol and fatty acid synthesis de novo, is a potential target for hypolipidaemic intervention. Here we describe the biological effects of the inhibition of ATP citrate-lyase on lipid metabolism in Hep G2 cells, and plasma lipids in rats and dogs, by using SB-204990, the cell-penetrant gamma-lactone prodrug of the potent ATP citrate-lyase inhibitor SB-201076 (Ki=1 microM). Consistent with an important role of ATP citrate-lyase in the supply of acetyl-CoA units for lipid synthesis de novo, SB-204990 inhibited cholesterol synthesis and fatty acid synthesis in Hep G2 cells (dose-related inhibition of up to 91% and 82% respectively) and rats (76% and 39% respectively). SB-204990, when administered orally to rats, was absorbed into the systemic circulation; pharmacologically relevant concentrations of SB-201076 were recovered in the liver. When administered in the diet (0.05-0. 25%, w/w) for 1 week, SB-204990 caused a dose-related decrease in plasma cholesterol (by up to 46%) and triglyceride levels (by up to 80%) in rats. This hypolipidaemic effect could be explained, at least in part, by a decrease (up to 48%) in hepatic very-low-density lipoprotein (VLDL) production as measured by the accumulation of VLDL in plasma after injection of Triton WR-1339. SB-204990 (25 mg/kg per day) also decreased plasma cholesterol levels (by up to 23%) and triglyceride levels (by up to 38%) in the dog, preferentially decreasing low-density lipoprotein compared with high-density lipoprotein cholesterol levels. Overall these results are consistent with the concept that ATP citrate-lyase is an important enzyme in controlling substrate supply for lipid synthesis de novo and a potential enzyme target for hypolipidaemic intervention.

ATP Citrate (pro-S)-Lyase↗
PubMed1998

Antioxidant effects of propofol in human hepatic microsomes: concentration effects and clinical relevance.

Propofol is known to possess antioxidant properties. There is controversy regarding the mechanisms by which the drug produces its antioxidant effects and the significance of these effects in relation to plasma concentrations of propofol in clinical practice. We studied the effects of increasing concentrations of Intralipid, propofol, butylated hydroxytoluene (BHT) and a vitamin E analogue (Trolox C) in 0.9% saline on non-enzymic and enzymic lipid peroxidation in human hepatic microsomes, and on concentrations of antioxidant enzymes in a Hep G2 cell line. Propofol showed significant inhibition of lipid peroxidation, but was less potent than BHT or Trolox C. IC50 values for non-enzymic and enzymic lipid peroxidation were mean 9.47 (SD 0.86) and 7.39 (0.84) mumol litre-1 for propofol, 1.30 (0.57) and 0.32 (0.02) mumol litre-1 for BHT and 2.34 (0.68) and 0.35 (0.04) mumol litre-1 for Trolox C, respectively. The antioxidant activities of propofol were substantially retained in the presence of up to 30 g litre-1 of human serum albumin. Propofol at concentrations of up to 100 mumol litre-1 had no significant effect on the activities of antioxidant enzymes. Clinically relevant concentrations of propofol produced significant inhibition of both enzymic and non-enzymic lipid peroxidation in hepatic microsomal preparations, possibly as a result of accumulation in lipophilic environments. Measurement of antioxidant effects of drugs in aqueous media may have little relevance to their effects in protecting against lipid peroxidation in biological systems.

Anesthetics, Intravenous↗
PubMed1996

ATP-citrate lyase as a target for hypolipidemic intervention. Design and synthesis of 2-substituted butanedioic acids as novel, potent inhibitors of the enzyme.

ATP-citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. Inhibitors of the enzyme represent a potentially novel class of hypolipidemic agent, which are anticipated to have combined hypocholesterolemic and hypotriglyceridemic properties. A series of 2-substituted butanedioic acids have been designed and synthesized as inhibitors of the enzyme. The best compounds, 58, 68, 71, 74 have reversible Ki's in the 1-3 microM range against the isolated rat enzyme. As representative of this compound class, 58, has been shown to exert its inhibitory action through a mainly competitive mechanism with respect to citrate and a noncompetitive one with respect to CoA. None of the inhibitors were able to inhibit cholesterol and/or fatty acid synthesis in HepG2 cells. This has been attributed to the adverse physicochemical properties of the molecules leading to a lack of cell penetration. Despite this, a lead structural class of compound has been identified with the potential for modification into potent, cell-penetrant, and efficacious inhibitors of ATP-citrate lyase.

ATP Citrate (pro-S)-Lyase↗
PubMed1993

Autoantibodies to hepatic microsomal carboxylesterase in halothane hepatitis.

Halothane hepatitis can be life-threatening, and this severe adverse reaction may arise via an immune process. We have detected autoantibodies to purified human liver microsomal carboxylesterase in sera of 17 out of 20 patients with halothane hepatitis (85%) but not in 9 halothane-exposed controls and in only 2 (at low levels) of 33 patients with liver disease due to other causes. Immunohistochemical studies localised the carboxylesterase predominantly to the centrilobular region of liver sections, which is consistent with the area affected by halothane hepatitis. Human hepatic microsomal carboxylesterase is a target antigen in halothane hepatitis, and an immune response to this protein may be involved in the liver damage observed.

Adult↗
PubMed1988

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin.

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.

Animals↗
PubMed1988

Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX.

Catalytic turnover of sodium azide by horseradish peroxidase, which produces the azidyl radical, results in inactivation of the enzyme with KI = 1.47 mM and kinact = 0.69 min-1. Inactivation of 80% of the enzyme requires approximately 60 equiv each of NaN3 and H2O2. The enzyme is completely inactivated by higher concentrations of these two agents. meso-Azidoheme as well as some residual heme are obtained when the prosthetic group of the partially inactivated enzyme is isolated and characterized. Reconstitution of horseradish peroxidase with meso-azidoheme yields an enzyme without detectable catalytic activity even though reconstitution with heme itself gives fully active enzyme. The finding that catalytically generated nitrogen radicals add to the meso carbon of heme shows that biological meso additions are not restricted to carbon radicals. The analogous addition of oxygen radicals may trigger the normal and/or pathological degradation of heme.

Azides↗
PubMed1981

Relationship of raised platelet IgG in thrombocytopenia to total platelet protein content.

Platelet-associated IgG (PA IgG) of washed platelets of 20 normal controls and 45 thrombocytopenic patients was measured by radioimmunoassay and related to total platelet protein content. Mean values (+/- 2 SD) for 20 normals were 4.4 +/- 3.5 fg IgG per platelet (fg IgG/pl), 2.1 +/= 1.0 pg protein per platelet (pg protein/pl) and a ratio of IgG per g platelet protein of 2.1 +/- 1.8 mg IgG/g protein. Patients could be divided into two groups: Group I (31) with normal IgG/g protein ratios and either normal (16) or raised (15) fg IgG/pl, and Group II (14) with both fg IgG/pl and IgG/g protein raised. Of the 21 patients with idiopathic thrombocytopenia (ITP), 13 were in Group I (seven with raised and six with normal fg IgG/pl), and eight in Group II. There was significant correlation between PA IgG (fg IgG/pl) and platelet protein for the entire Group I (r = 0.95, P less than 0.001). The ITP patients in this group showed a similar correlation (r = 0.77, P less than 0.001), suggesting that raised PA IgG levels in Group I patients may be a result of protein-rich platelets. 60-90% of PA IgG was found in the soluble fraction of lysates of platelets from both normals and patients. Thus, it is either non-specifically associated with platelets or is antibody directed towards easily solubilized platelet antigens. Nine thrombocytopenic patients without immune disorders had raised PA IgG. These results question the assumption that raised PA IgG always indicates specific antiplatelet antibody.

Blood Platelets↗
PubMedDate not supplied

Crystallization and preliminary X-ray diffraction studies of human cytochrome P450 reductase.

The two functional domains of a cloned human fibroblast NADPH-cytochrome P450 reductase have been expressed in Escherichia coli and purified on the milligram scale for crystallization studies. One domain contains the cofactor FMN-binding site and the other contains the binding sites for cofactor FAD and substrate NADPH. Crystals of both domains have been obtained by the microbatch method. The crystals of the FMN domain belong to the monoclinic space group P21, with unit cell dimensions of a = 39.3 A, b = 51.5 A, c = 47.8 A, and beta = 105.7 degrees and have one molecule in the asymmetric unit. Diffraction data up to 2.3 A were collected with a merging residual on intensity of 9.3%. The crystals of the FAD/NADPH domain belong to the ortho-rhombic space group P212121 with unit cell dimensions of a = 55.9 A, b = 58.6 A, c = 131.1 A and have one molecule in the asymmetric unit. Diffraction data up to 2.6 A were collected with a merging residual on intensity of 8.0%.

Amino Acid Sequence↗