Analysis of human CYP3A4 structure-function relationships using photoaffinity labels.

摘要

Cytochrome P450s (CYPs) constitute the most important family of biotransformation enzymes involved in drug metabolism, playing a key role in the disposition of drugs and their pharmacological and toxicological effects. A major emphasis in this work is CYP3A4, which is the most abundant CYP expressed in human liver and is capable of monooxygenating approximately half of all therapeutic agents on the market today. Because of its general importance in drug metabolism and carcinogen bioactivation, elucidation of the key structural elements responsible for substrate recognition and binding leading to oxidation by CYP3A4 is of considerable interest. Studies of cytochrome P450 active site structural components and substrate recognition determinants have commonly included the covalent modification of substrate binding regions with ligands followed by the identification of the adducted peptides. The studies described herein develop a class of benzochromene compounds as a novel type of photoaffinity ligands (PALs) used to probe the P450 active site structures. Development of photochromic agents as tools for protein structure study is discussed in Chapter 2. Photoaffinity labeling of CYP3A4 by lapachenole and subsequent identification of the absolute modification sites by mass spectrometry are described in Chapter 3. Analysis of structure-function relationships of CYP3A4 using select mutants on the modification sites is discussed in Chapter 4. A partial mimicking of the protein labeling effect by a single-point mutant is discussed. Finally, Chapter 5 described a new method to probe the CYP3A4 active site structures using a combination of techniques including cysteine-scanning mutagenesis, photoaffinity labeling and molecular modeling. Overall, the results demonstrate that benzochromene compounds, as a novel type of PALs, can be used in the study of P450 structures. The flexible substrate recognition site (SRS)-1 region serves as a substrate access channel or potentially as a part of CYP3A4 active site, which is directly involved in the initial substrate recognition and binding in CYP3A4. Future investigations are suggested to employ these benzochromene agents in the protein structure study of other P450 isoforms.