79-62-9Relevant articles and documents
Spin equilibrium and O2-binding kinetics of Mycobacterium tuberculosis CYP51 with mutations in the histidine-threonine dyad
Jennings, Gareth K.,Modi, Anuja,Elenewski, Justin E.,Ritchie, Caroline M.,Nguyen, Thuy,Ellis, Keith C.,Hackett, John C
, p. 81 - 91 (2014/05/20)
The acidic residues of the "acid-alcohol pair" in CYP51 enzymes are uniformly replaced with histidine. Herein, we adopt the Mycobacterium tuberculosis (mt) enzyme as a model system to investigate these residues' roles in finely tuning the heme conformation, iron spin state, and formation and decay of the oxyferrous enzyme. Properties of the mtCYP51 and the T260A, T260V, and H259A mutants were interrogated using UV-Vis and resonance Raman spectroscopies. Evidence supports that these mutations induce comprehensive changes in the heme environment. The heme iron spin states are differentially sensitive to the binding of the substrate, dihydrolanosterol (DHL). DHL and clotrimazole perturb the local environments of the heme vinyl and propionate substituents. Molecular dynamics (MD) simulations of the DHL-enzyme complexes support that the observed perturbations are attributable to changes in the DHL binding mode. Furthermore, the rates of the oxyferrous formation were measured using stopped-flow methods. These studies demonstrate that both HT mutations and DHL modulate the rates of oxyferrous formation. Paradoxically, the binding rate to the H259A mutant-DHL complex was approximately four-fold that of mtCYP51, a phenomenon that is predicted to result from the creation of an additional diffusion channel from loss of the H259-E173 ion pair in the mutant. Oxyferrous enzyme auto-oxidation rates were relatively constant, with the exception of the T260V-DHL complex. MD simulations lead us to speculate that this behavior may be attributed to the distortion of the heme macrocycle by the substrate.
Fast and easy in vitro screening assay for cholesterol biosynthesis inhibitors in the post-squalene pathway
Giera, Martin,Ploessl, Florian,Bracher, Franz
, p. 633 - 642 (2008/02/04)
A whole-cell assay for screening cholesterol biosynthesis inhibitors in the post-squalene pathway has been developed. HL 60 cells were incubated for 24 h with test substances. The nonsaponifiable lipids were extracted by means of liquid-liquid extraction using tert-butylmethylether. The raw extracts were purified by dispersive solid phase extraction using a primary-secondary amine material (PSA) and dried using sodium sulphate. The sterols were derivatized using N-trimethylsilylimidazole. GLC/MS analysis was carried out in less than 12.5 min using fast GLC mode. The obtained sterol patterns indicated which enzyme had been inhibited. Specific sterol patterns which reflect the different enzyme inhibitions were obtained using established inhibitors of cholesterol biosynthesis like AY 9944, NB 598, clotrimazole, aminotriazole and DR 258, a Δ24-reductase inhibitor prepared in our working group. For characterizing IC50 values we used sodium 2-13C-acetate and quantified the incorporation of it into cholesterol relative to control levels after the samples had been normalized to their protein content.
Synthesis of 15alpha-fluoro-24,25-dihydrolanosterol as a potential inhibitor and/or mechanistic probe for lanosterol 14alpha-demethylase.
Morisaki,Igata,Yamamoto
, p. 1474 - 1479 (2007/10/03)
As a potential inhibitor and/or mechanistic probe for lanosterol 14alpha-demethylase, 15alpha-fluoro-24,25-dihydrolanosterol was prepared by fluorination of 15alpha-hydroxy-24,25-dihydrolanost-7-en-3beta-yl benzoate with diethylaminosulfur trifluoride, followed by hydrogen chloride-catalyzed isomerization of the delta7 to delta8 and reductive cleavage of the benzoate.
