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Research ArticleArticle

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

Jianyao Wang, Xiao Xian Li-Chan, Jim Atherton, Lin Deng, Robert Espina, Linning Yu, Peter Horwatt, Steven Ross, Susan Lockhead, Syed Ahmad, Appavu Chandrasekaran, Aram Oganesian, JoAnn Scatina, Abdul Mutlib and Rasmy Talaat
Drug Metabolism and Disposition July 2010, 38 (7) 1083-1093; DOI: https://doi.org/10.1124/dmd.110.032292
Jianyao Wang
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Xiao Xian Li-Chan
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Jim Atherton
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Lin Deng
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Robert Espina
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Linning Yu
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Peter Horwatt
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Steven Ross
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Susan Lockhead
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Syed Ahmad
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Appavu Chandrasekaran
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Aram Oganesian
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JoAnn Scatina
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Abdul Mutlib
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Rasmy Talaat
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Abstract

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 [14C]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 ε-amine of lysine attacking to the β-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°C for 18 h.

Footnotes

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.110.032292.

  • ABBREVIATIONS:

    HKI-272
    N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide
    LC/MS
    liquid chromatography/mass spectrometry
    PAGE
    polyacrylamide gel electrophoresis
    AA
    amino acid
    HPLC
    high-performance liquid chromatography
    HSA
    human serum albumin
    IS
    internal standard
    TIC
    total ion chromatogram
    XIC
    extract ion chromatogram
    BMS-204352
    (3S)-3-(5-chloro-2-methoxyphenyl)-3-fluoro-6-(trifluoromethyl)-1,3-dihydro-2H-indol-2-one.

  • Received January 18, 2010.
  • Accepted April 12, 2010.
  • Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 38 (7)
Drug Metabolism and Disposition
Vol. 38, Issue 7
1 Jul 2010
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Research ArticleArticle

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

Jianyao Wang, Xiao Xian Li-Chan, Jim Atherton, Lin Deng, Robert Espina, Linning Yu, Peter Horwatt, Steven Ross, Susan Lockhead, Syed Ahmad, Appavu Chandrasekaran, Aram Oganesian, JoAnn Scatina, Abdul Mutlib and Rasmy Talaat
Drug Metabolism and Disposition July 1, 2010, 38 (7) 1083-1093; DOI: https://doi.org/10.1124/dmd.110.032292

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Research ArticleArticle

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

Jianyao Wang, Xiao Xian Li-Chan, Jim Atherton, Lin Deng, Robert Espina, Linning Yu, Peter Horwatt, Steven Ross, Susan Lockhead, Syed Ahmad, Appavu Chandrasekaran, Aram Oganesian, JoAnn Scatina, Abdul Mutlib and Rasmy Talaat
Drug Metabolism and Disposition July 1, 2010, 38 (7) 1083-1093; DOI: https://doi.org/10.1124/dmd.110.032292
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