Use of the steroid derivative RPR 106541 in combination with site-directed mutagenesis for enhanced cytochrome P-450 3A4 structure/function analysis.
RPR 106541 (20R-16alpha,17alpha-[butylidenebis(oxy)]-6al pha, 9alpha-difluoro-11beta-hydroxy-17beta-(methylthio)androst a-4-en-3-one) is an airway-selective steroid developed for the treatment of asthma. Two metabolites produced by human liver microsomes were identified as R- and S-sulfoxide diastereomers based on liquid chromatography/mass spectrometry analysis, proton nuclear magnetic resonance, and cochromatography with standards. Sulfoxide formation was determined to be cytochrome P-450 (CYP) 3A4-dependent by correlation with CYP3A4-marker nifedipine oxidase activity, inhibition by cyclosporin A and troleandomycin, and inhibition of R- (70%) and S- (64%) sulfoxide formation by anti-3A antibody. Expressed CYP2C forms catalyzed RPR 106541 sulfoxidation; however, other phenotyping approaches failed to confirm the involvement of CYP2C forms in these reactions in human liver microsomes. Expressed CYP3A4 catalyzed the formation of the sulfoxide diastereomers in a 1:1 ratio, whereas CYP3A5 displayed stereoselectivity for formation of the S-diastereomer. The high rate of sulfoxidation by CYP3A4 and the blockage of oxidative metabolism at the electronically favored 6beta-position provided advantages for RPR 106541 over other substrates as an active site probe of CYP3A4. Therefore, oxidation of RPR 106541 by various CYP3A4 substrate recognition site (SRS) mutants was assessed. In SRS-4, A305V and F304A showed dramatically reduced rates of R-diastereomer formation (83 and 64% decreases, respectively), but S-diastereomer formation was affected to a lesser extent. A370V (SRS-5) showed decreased formation of the R-sulfoxide (52%) but increased formation of the S-diastereomer. In the SRS-2 region, the most dramatic change in sulfoxide ratios was observed for L210A. In conclusion, the structure of RPR 106541 imposes specific constraints on enzyme binding and activity and thus represents an improved CYP3A4 probe substrate.
Department of Drug Metabolism and Pharmacokinetics, Rhône-Poulenc Rorer, Collegeville, Pennsylvania, USA. Jeffrey.STEVENS@RP-Rorer
J C
JC
Stevens
T L
TL
Domanski
G R
GR
Harlow
R B
RB
White
E
E
Orton
J R
JR
Halpert
D000736:Androstenes / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D004285:Dogs; D006801:Humans; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051381:Rats; D017207:Rats, Sprague-Dawley; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013454:Sulfoxides / Q000378:metabolism
D000736:Androstenes; D065607:Cytochrome P-450 Enzyme Inhibitors; D013454:Sulfoxides; C109111:RPR 106541; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human