Detection of covalent adducts to cytochrome P450 3A4 using liquid chromatography mass spectrometry.
Protein covalent labeling can be an undesirable property of compounds being studied in drug discovery programs. Identifying such compounds relies on the use of radiolabeled material, which requires an investment in time and resources not typically expended until later in the discovery process. We describe the detection of covalent adducts to cytochrome P450 3A4, the most abundant and important P450 from a human and drug discovery viewpoint, using liquid chromatography mass spectrometry. The technique is illustrated using L-754,394 and 6',7'-dihydroxybergamottin, two known inhibitors of P450 3A4. Mass spectrometry of the intact apoprotein as well as the adducted protein is demonstrated. Such methodology may provide the means for screening compounds for covalent protein binding without the use of a radiolabel. It also provides direct information about mechanism-based inhibitors in terms of extent, stoichiometry, and nature of the adduct(s) (mass shift). This information may provide a means for understanding the mechanism of covalent labeling earlier in a drug discovery environment.
Bateman
Kevin P
KP
Department of Medicinal Chemistry, Merck Frosst Canada and Co., 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada. Kevin_Bateman@merck.com
Baker
Jennifer
J
Wilke
Mark
M
Lee
Justin
J
Leriche
Tammy
T
Seto
Carmai
C
Day
Stephen
S
Chauret
Nathalie
N
Ouellet
Marc
M
Nicoll-Griffith
Deborah A
DA
D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D015195:Drug Design; D011564:Furocoumarins / Q000737:chemistry / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D007189:Indans / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D011564:Furocoumarins; D007189:Indans; C094644:L 754394; D010879:Piperazines; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C104774:6',7'-dihydroxybergamottin