CYP2C9 is a significant P450 protein responsible for drug metabolism. With the increased use of heterocyclic compounds in drug design, a rapid and efficient pre-drug screening of these potential type II binding compounds is essential to avoid adverse drug reactions. To understand binding modes, we use quinoline-4-carboxamide analogs to study the factors that determine the structure-activity relationships. The results of this study suggest that the more accessible pyridine with the nitrogen para to the linkage can coordinate directly with the ferric heme iron, but this is not seen for the meta or ortho isomers. The π-cation interaction of the naphthalene moiety and Arg 108 residue may also assist in stabilizing substrate binding within the active-site cavity. The type II substrate binding affinity is determined by the combination of steric, electrostatic, and hydrophobicity factors; meanwhile, it is enhanced by the strength of lone pair electrons coordination with the heme iron.

RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.

ABSTRACT:The rhesus monkey (Macaca mulatta) is a primate species used extensively as a preclinical safety species in drug development. In this report, we describe the cloning, expression, and characterization of CYP3A64 (AY334551), a CYP3A4 homolog expressed in rhesus liver. The deduced amino acid sequence was found to be 93% similar to human CYP3A4, 83% similar to human CYP3A5, and identical to the previously reported cynomolgus monkey CYP3A8 Cytochromes P450 (P450s) are a superfamily of enzymes involved in the elimination of a wide variety of chemical xenobiotics including pharmaceuticals, carcinogens, and environmental pollutants (Wrighton and Stevens, 1992). CYP3A4 is the most abundant of these enzymes in humans and is responsible for the biotransformation of nearly 50% of all pharmaceuticals (Guengerich, 1995). Substrates for CYP3A4 include such structurally distinct molecules as testosterone, nifedipine, lidocaine, lovastatin, erythromycin, cyclosporine, diazepam, midazolam, and coumarins.Rhesus monkeys (Macaca mulatta) and cynomolgus monkeys (Macaca fascicularis) are widely used throughout the pharmaceutical industry as preclinical safety species. Much emphasis is placed on the overall drug safety profile and the extrapolated CYP3A metabolic activities of monkeys to corresponding human drug metabolism variables, even though very little information is known about monkey CYP3A enzymes or their metabolic capabilities. There are relatively few reports, compared with rat and human, regarding the specific activity of rhesus and cynomolgus monkey CYP3A enzyme activities, and most of these reports are from purified regenerated systems or liver homogenates and not from recombinantly expressed enzyme systems (Ohta et al., 1989;Ohmori et al., 1993;Ramana and Kohli, 1999;Matsunaga et al., 2002). Although it is believed that monkey CYP3A metabolic capabilities should be similar to those of human CYP3A, there has not been an in-depth investigation of the enzymatic properties of the individual monkey CYP3A isoforms. Therefore, we sought to clone, express, and characterize the major CYP3A4-like drug-metabolizing enzyme from rhesus monkey liver.Based on its predicted homology to human CYP3A4, we cloned the CYP3A4 homolog from rhesus monkeys. The cloned cDNA was expressed using a commonly used insect cell expression system and characterized with multiple probe substrates. Insect cells offer several advantages over other expression systems: 1) they do not require alteration of the P450 coding sequence for expression, 2) they lack endogenous cytochromes P450, and 3) they can be used to make microsomes or coexpressed with NADPH-P450 oxidoreductase (OR) to produce Supersomes (Gonzalez and Korzekwa, 1995;Crespi and Miller, 1999).It is valuable to understand the metabolic capabilities of preclinical animal models to accurately predict safety and clearance profiles of pharmaceutical candidates in development. Therefore, in addition to the evaluation of the rhesus CYP3A64 enzyme activity in relationship Article, publication date, ...