Photochromic agents as tools for protein structure study: lapachenole is a photoaffinity ligand of cytochrome P450 3A4.
Cytochrome P450 3A4 is a drug-metabolizing enzyme of extraordinarily broad substrate specificity. This quality imparts upon the enzyme special importance in understanding its determinants of activity and substrate recognition. Limited successes in P450 3A4 active-site structure studies have been achieved by use of mechanism-based inactivators and photoaffinity ligands. We report here the potential of photochromic agents, compounds with the ability to undergo light-induced, reversible reactions, to be used as effective photoaffinity ligands. Four such compounds of the chromene family were shown by ultraviolet and visible spectroscopy to undergo photoinduced rearrangements to highly conjugated and reactive products in buffered aqueous solution. While some of these intermediates were very long-lived (>12 h, photoactivated lapachenole), others existed for milliseconds in their opened forms (precocene I and 2,2-dimethyl-5,6-benzo-2H-chromene) and were observed by laser flash photolysis. Each of the tricyclic structures studied rapidly underwent Michael addition reactions with the test nucleophile glutathione upon irradiation to form single conjugated products. The smaller precocene I reacted more extensively to form multiple products. These attributes of the chromenes inspired testing of their potential to label cytochrome P450 3A4 in a light-dependent fashion. Access to the protein active site by lapachenole was demonstrated with the molecule's ability to competitively inhibit P450 3A4-mediated oxidative metabolism of midazolam with an IC(50) value of 71 microM. This inhibition became irreversible upon irradiation of the enzyme-ligand complex with ultraviolet light. These results clearly demonstrate that chromenes are effective photoaffinity reagents for the cytochrome P450 superfamily of enzymes and probably other proteins as well.
Gartner
Carlos A
CA
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA. cgartner@hms.harvard.edu
Wen
Bo
B
Wan
Jiandi
J
Becker
Ralph S
RS
Jones
Guilford
G
Gygi
Steven P
SP
Nelson
Sidney D
SD
D000082:Acetaminophen / Q000037:antagonists & inhibitors / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001578:Benzopyrans / Q000737:chemistry* / Q000378:metabolism*; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002021:Buffers; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry; D008024:Ligands; D008874:Midazolam / Q000037:antagonists & inhibitors; D019906:Nuclear Magnetic Resonance, Biomolecular; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism*; D010782:Photolysis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
D001578:Benzopyrans; D016227:Benzoquinones; D002021:Buffers; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D008024:Ligands; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; C498939:lapachenole; C019539:precocene I; D000082:Acetaminophen; C004532:quinone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D005978:Glutathione; D008874:Midazolam