Characterization of HKI-272 covalent binding to human serum albumin.
The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [(14)C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182-195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the epsilon-amine of lysine attacking to the beta-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37 degrees C for 18 h.
Wang
Jianyao
J
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer Inc., Collegeville, Pennsylvania 19426, USA. jackwang100@hotmail.com
Li-Chan
Xiao Xian
XX
Atherton
Jim
J
Deng
Lin
L
Espina
Robert
R
Yu
Linning
L
Horwatt
Peter
P
Ross
Steven
S
Lockhead
Susan
S
Ahmad
Syed
S
Chandrasekaran
Appavu
A
Oganesian
Aram
A
Scatina
Joann
J
Mutlib
Abdul
A
Talaat
Rasmy
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D002250:Carbon Radioisotopes / Q000097:blood; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D006801:Humans; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000378:metabolism; D011804:Quinolines / Q000097:blood*; D011869:Radioligand Assay / Q000379:methods; D012709:Serum Albumin / Q000378:metabolism*
D002250:Carbon Radioisotopes; D010455:Peptides; D011804:Quinolines; D012709:Serum Albumin; C487932:neratinib