High Resolution Screening of biologically active compounds and metabolites

High Resolution Screening of biologically active compounds and metabolites Jeroen Kool Biotransformation enzymes play a crucial role in the metabolism of both endogenous compounds and xenobiotics. Usually, the detoxication of these compounds by biotransformation enzymes results in harmless metabolites, which are more readily excreted from the body due to their enhanced hydrophilicity. In some cases, however, biotransformation does not yield metabolites that are less toxic than the parent compounds but rather more toxic. Metabolites may be chemically reactive metabolites or intermediates that react with proteins, RNA or DNA or metabolites that show high affinity towards enzymes, receptors or other macromolecules. Cytochromes P450 (CYPs) are the most important phase I metabolism enzymes and play a central role in oxidative metabolic reactions of drugs and other xenobiotics. Affinity of drugs and metabolites towards CYPs can result in unwanted drug-drug interactions (DDIs) at the level of drug metabolism. Phase II metabolism can also result in adverse drug reactions. The phase II metabolic enzymes, among which are the glucuronosyl transferases (UGTs), sulfotransferases (STs) and glutathione S-transferases (GSTs), add substituents to functional groups in compounds usually resulting in strongly increased hydrophilicity and facilitated excretion. Of these phase II metabolic enzymes, GSTs are the most important enzymes catalyzing the detoxication of electrophilic compounds by conjugation to glutathione (GSH). As metabolism of drugs can thus result in the formation of pharmacologically active, drug-drug interacting and/or reactive metabolites and metabolites causing toxicological effects, it renders them essential for early consideration in drug discovery and development programs. The screening process of compounds often employs High Throughput Screening (HTS) methodologies. However, most HTS-methodologies used as yet do not allow the identification of individual ligands in mixtures. Mixtures have to be separated chromatographically before affinity screening of individual compounds can occur. Several years ago, a novel on-line High Resolution Screening (HRS) concept was developed that enabled the identification of individual ligands in complex mixtures. HRS is based on continuous-flow biochemical detection assays coupled on-line to HPLC. Until now, bio-affinity detection systems have been described for e.g. the estrogen receptor, phosphodiesterases, acetylcholine esterases and angiotensin converting enzymes. This thesis describes the use of HRS for screening of biologically active metabolites and for screening CYP and GST affinity of individual compounds in mixtures. The general introduction (Chapter 1) of this thesis focuses on the background, applications and the present status of the HRS concept as well as on drug metabolism enzymes, bioactivation and receptor- and enzyme targets in drug discovery. Part I of this thesis deals with the development and validation of newly developed HRS-methodologies for the interaction of drugs and drug metabolites with CYPs. Part II describes the use of HRS-methodologies for estrogen receptor (ER) affinity screening of individual metabolites in metabolic mixtures. Moreover, a CYP-containing bioreactor unit, developed in a concomitant with Van Liempd et al, was coupled on-line to HRS and used for fully automated generation and subsequent HRS screening of metabolic mixtures for ER affinity. The automated HRS-technology allows the rapid and efficient monitoring of parent compounds as well as their metabolites in terms of metabolic stability and bioactivity. Part III of this thesis describes a novel HRS-methodology for the screening of affinities to GSH-S-Transferases (GSTs). Non-specific and specific GST ligands and substrates in mixtures could individually be detected with this HRS-methodology. Another important aspect of metabolism is the formation of reactive oxygen species (ROS). Part III also describes a HRS-methodology for the measurement of ROS resulting from individual compounds in mixtures. This HRS-methodology is capable of simultaneously identifying antioxidants in these mixtures. Part IV provides a summary and the conclusions of the work described in this thesis. Some future prospects are also given.