James R. Halpert

Learn More
The individual members of a homologous series of phenoxazone ethers related to ethoxyresorufin were O-dealkylated, and the parent compound phenoxazone was ring-hydroxylated, each at different rates with hepatic microsomes of untreated rats. A structure-activity relationship (SAR) was plotted, relating the rate of O-dealkylation to the length and type of the(More)
CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the(More)
Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure-activity study from another laboratory using various effector-substrate(More)
The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority(More)
The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s; P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were(More)
Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology(More)
Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that oxidation of MDZ by(More)
The hydrophobic membrane-spanning domain in four cytochromes P450 2B was removed (Delta3-21) and several positive charges were substituted at the N-terminus to increase expression and solubility. Histidine residues were appended to the C-terminus to simplify purification. The truncated proteins were highly expressed in Escherichia coli, could be released(More)
Phenylalanine and/or tryptophan scanning mutagenesis was performed at 15 sites within CYP3A4 proposed to be involved in substrate specificity or cooperativity. The sites were chosen on the basis of previous studies or from a comparison with the structure of P450(eryF) containing two molecules of androstenedione. The function of the 25 mutants was assessed(More)
The genetic component of the inter-individual variability in CYP3A4 activity has been estimated to be between 60% and 90%, but the underlying genetic factors remain largely unknown. A study of 213 Middle and Western European DNA samples resulted in the identification of 18 new CYP3A4 variants, including eight protein variants. A total of 7.5% of the(More)