Characterization of HKI-272 covalent binding to human serum albumin
| Autor: | Abdul Mutlib, Appavu Chandrasekaran, Robert Espina, Xiao Xian Li-Chan, Peter M. Horwatt, Lin Deng, Jim Atherton, JoAnn Scatina, Steven Ross, Aram Oganesian, Linning Yu, Rasmy Talaat, Syed Ahmad, Jianyao Wang, Susan Lockhead |
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| Rok vydání: | 2010 |
| Předmět: |
Chemistry
Pharmaceutical Serum albumin Pharmaceutical Science Mass spectrometry Peptide Mapping Adduct Residue (chemistry) Radioligand Assay medicine Humans Amino Acid Sequence Carbon Radioisotopes Serum Albumin Pharmacology Gel electrophoresis Chromatography Binding Sites biology Molecular mass Chemistry Human serum albumin Covalent bond biology.protein Quinolines Peptides medicine.drug |
| Zdroj: | Drug metabolism and disposition: the biological fate of chemicals. 38(7) |
| ISSN: | 1521-009X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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