By measuring a diverse compound set, we have obtained statistics around the frequency of limited solubility, fluorescence interference, or atypical kinetics for 2C9 and 2D6

By measuring a diverse compound set, we have obtained statistics around the frequency of limited solubility, fluorescence interference, or atypical kinetics for 2C9 and 2D6. of competitive inhibitors and inactive compounds was analyzed for styles in binding affinity. For CYP2D6, a clear relationship between polar surface area and charge was observed, with the most potent inhibitors using a formal positive charge and a low percent polar surface area. For CYP2C9, no obvious pattern between activity and physicochemical properties could be seen for the group as a whole; however, certain classes of compounds have altered frequencies of activity and atypical kinetics. Introduction The cytochrome P450 enzymes (CYPs) are versatile enzymes that can oxidize a wide variety of hydrophobic compounds. The ability to metabolize a diverse set of substrates is required for the eventual removal of foreign compounds. This versatility is usually accomplished because the enzymes generate highly reactive GPI-1046 species of oxygen, 1C2 have relatively nonspecific substrate binding interactions, and because there is superfamily of CYPs with overlapping substrate selectivities. Three CYPs 3A4, 2D6, and 2C9 are responsible for the microsomal oxidation of a majority of drugs in the human. Since relatively few enzymes are responsible for the metabolism of many different drugs, administration of one drug can result in the inhibition of the metabolism of other co-administered drugs. As a result, inhibition of CYPs by a drug is an important cause of drugCdrug interactions (DDI). In order to avoid dangerous interactions as well as prevent the need for specially designed clinical trials to assess DDI potential, it is advantageous to select clinical candidates that are not high-affinity inhibitors of the major CYPs. For this reason, in vitro screens have been extensively used to measure the affinity of drug candidates to the CYPs. In routine screening protocols, the accurate determination of CYP affinity is usually often hampered by several factors, including compound or metabolite fluorescence in a fluorescent substrate assay, limited compound solubility, and atypical kinetics.3 Atypical or non-MichaelisCMenten kinetics is most likely a result of multiple substrates or effectors simultaneously binding to the CYP. The result is usually non-hyperbolic saturation kinetics for a single substrate, or mixed inhibition kinetics or activation for two substrates.3 Interpretation of atypical kinetics can be complicated. The impact of one molecule around the metabolism of another can vary with different substrates. A molecule may inhibit the metabolism of one substrate and activate the metabolism of another. This suggests that inhibition of a single probe substrate may not properly predict the drug interaction potential of that compound for all those drugs. Although atypical kinetics are most commonly observed for CYP3A4,3C6 they have been reported for other enzymes including CYP2C9,5,7C9 CYP2D6,10C11 and CYP1A2.12 However, the frequency of atypical kinetics for the different P450 isoforms is generally unknown. Here we statement the generation and analysis of inhibition GPI-1046 data over a diverse set of 500 drug-like molecules against recombinant CYP 2C9 and 2D6 enzymes. A method to distinguish common from atypical kinetics is usually presented. By measuring a diverse compound set, we have obtained statistics around the frequency of limited solubility, fluorescence interference, or atypical kinetics for 2C9 and 2D6. In addition to these statistics, the dataset we have collected provides a diverse data set free of compounds with uncertain affinity due to atypical kinetics, which can be utilized for the construction of quantitative structureCactivity relationship (QSAR) models. Methods Compound selection A set of 500 compounds were selected from your Merck sample repository based on two different criteria. First, some well-known generic drugs for which a sample was present in the Merck repository were retrieved, excluding compounds with low purity as determined by mass spectrometry or for which insufficient sample was available. Second, an additional diverse set of Merck proprietary compounds was added, making sure that: 1) the same availability and purity criteria applied, 2) they were drug-like in the sense of Lipinskis rule-of-five,13 and 3) none of them experienced high structural similarity to any of the generic drugs. Enzymology In order to characterize the inhibition (or activation) kinetics of a cytochrome P450 reaction, both single and multiple binding site equations must be used.14 Competitive inhibition occurs when the binding of one compound to GPI-1046 the active site prevents the binding of other substrates, and saturating concentrations of a competitive inhibitor eliminates substrate metabolism. For competitive Sirt6 inhibition, one should use standard hyperbolic inhibition equations that are consistent with 100% activity remaining at zero inhibitor concentration and.