VU Research Portal High Resolution Screening of biologically active compounds and metabolites Kool, J. 2007 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Kool, J. (2007). High Resolution Screening of biologically active compounds and metabolites. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: [email protected] Download date: 10. Oct. 2021 High Resolution Screening of biologically active compounds and metabolites High Resolution Screening of biologically active compounds and metabolites Jeroen Kool The financial support of Merck Research Laboratories, Organon, the Jurriaanse Stichting and Bester for the publication of this thesis is gratefully acknowledged. Printed by Printpartners Ipskamp Cover: designed by Steven Straatemans and Jeroen Kool © Jeroen Kool, Beverwijk 2007, All rights reserved. No part of this thesis may be reproduced in any form or by any means without the prior written permission from the author. VRIJE UNIVERSITEIT High Resolution Screening of biologically active compounds and metabolites ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. L. M. Bouter, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de faculteit der Exacte Wetenschappen op vrijdag 27 april 2007 om 10.45 uur in de aula van de universiteit, De Boelelaan 1105 door Jeroen Kool geboren te Velsen promotor: prof.dr. N.P.E. Vermeulen copromotoren : dr. J.N.M. Commandeur dr. J.H.N. Meerman “I love it when a plan comes together!” John “Hannibal” Smith (1983) Leescommissie: prof.dr. G.J. de Jong prof.dr. Th. Hankemeier prof.dr. H. Irth dr. M. Honing The investigations described in this thesis were carried out in the Leiden Amsterdam Center for Drug Research (LACDR)/Division of Molecular Toxicology, Department of Chemistry and Pharmacochemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands Contents Chapter 1: General introduction p. 11 Part I: High Resolution Screening of Cytochrome P450 affinities Chapter 2: Development of a novel Cytochrome P450 enzyme affinity detection system coupled on-line to gradient reversed-phase HPLC p. 57 Chapter 3: Development of three parallel Cytochrome P450 enzyme affinity detection systems coupled on-line to gradient reversed-phase HPLC p. 81 Chapter 4: Cytochrome P450 1A2 and 2D6 enzyme affinity detection coupled on-line to gradient reversed- phase HPLC p. 111 Part II: Cytochrome P450 based bioactivation and High Resolution Screening of Estrogen Receptor binding metabolites Chapter 5: Rapid on-line profiling of Estrogen Receptor binding metabolites of tamoxifen p. 137 Chapter 6: On-line formation, separation and Estrogen Receptor affinity screening of Cytochrome P450- derived metabolites of Selective Estrogen Receptor Modulators p. 161 Part III: High Resolution Screening methodology for Glutathione S-Transferase inhibition and for pro- oxidants and antioxidants Chapter 7: On-line biochemical detection of Glutathione S- Transferase Pi inhibitors in complex mixtures p. 191 Chapter 8: An on-line post-column detection system for the rapid detection of reactive oxygen species producing compounds and antioxidants in mixtures p. 217 Part IV: Summary, Conclusions and Future Prospects Chapter 9: Summary, Conclusions and Future Prospects p. 239 Samenvatting p. 255 List of Publications p. 259 Curriculum Vitae p. 263 Nawoord p. 265 Introduction General Introduction Chapter 1 Chapter 1 General Introduction Active compound screening Compound activity screening is common practice in drug discovery and it is often performed in high throughput fashion to facilitate the screening of large numbers of compounds to be processed [1]. Compound screening is usually performed in a broad variety of different assays among which are cellular and biochemical assays. While biochemical assays usually only give information regarding the affinity of compounds towards target macromolecules they are screened for, they can be performed at a much higher throughput with less complexity than cellular assays [2]. On the contrary, cellular (e.g. signal transduction) based assays provide much information about the biological activity of a compound on cells and therefore more closely resemble the effects compounds may exert in the body [3]. A drawback of cellular assays, however, is the cumbersome and continuous production of viable cells, while the target proteins needed in biochemical assays usually can be stored easily and efficiently in freezers until use. This is why initial screening procedures encompass affinity screening to reduce the compound pipeline into smaller libraries with “higher affinity compounds”. After the first screening process of large libraries, the initial hits found in the libraries present a small fraction of the original libraries rendering them more suitable for further cell based screening. While radioligand based affinity assays were the primary assays in the past, the use of radiolabeled tracer molecules and the heterogenicity of the assays render them less suitable for high throughput automatisation than e.g. fluorescence based assays. This resulted in the development of fluorescence based assays in all areas of drug discovery. Examples such as time-resolved fluorescence resonance energy transfer, fluorescence anisotropy, fluorescence correlation spectroscopy, fluorescence fluctuation spectroscopy and normal fluorescence based assays can be named in this regard [4]. Fluorescence based assays are not necessarily only ligand binding assays. Cellular (e.g. functional) assays that are fluorescence based also exist and have great perspectives due to their usual robustness and ease of handling [5, 6]. A primary bottleneck in compound screening is the identification of the compound or compounds that are responsible for biological activities 11 General Introduction Chapter 1 in complex mixtures, such as combinatorial and natural compound libraries. This also holds true for the screening of metabolic mixtures [7]. The activities and/or affinities of metabolites, which usually closely resemble the structure of the mother compound, are extremely important determinants in the final actions of a drug. Cumbersome labour intensive and costly dereplication processes usually have to be performed in order to separate the compounds in the mixtures (usually in 96- to 1536-well plates) before the separated compounds can be resubmitted to the original assays [8, 9]. An example is shown in Figure 1. Figure 1: Active metabolite profiling using an HPLC based dereplication format with 96-well plates. Bioassay-guided detection of active metabolites with characterization by mass spectrometry allows identification of active metabolites in metabolic mixtures. Metabolite mixtures are collected into 96-well plates after separation by HPLC. The fractions are then screened for activity with relevant bioassays (e.g., receptor binding or enzyme inhibition assays). Adapted from Fura et al [7]. Receptor screening technologies [10, 11] do allow affinity screening of individual components, but they are usually not capable of 12 General Introduction Chapter 1 detecting individual compounds in mixtures. Some receptor affinity screening strategies concern the binding of active metabolites to a receptor followed by off-line centrifugal ultrafiltration, thus resulting in the collection of bound ligands and allowing characterization by LC-MS. With this methodology, Lim et al [12] detected three ERα binding mono- hydroxylated metabolites of tamoxifen and N-desmethyl-tamoxifen in rat liver microsomal incubations. Similar methodologies were reported by Sun et al [13]. These technologies, however, lack the ability of efficient trapping of low affinity ligands in the presence of high affinity ligands or in the presence of high concentrations of the parent compound. This major bottleneck could be circumvented by direct on-line post- column connection of the desired bioassays to the separation technique used in the dereplication process (mostly HPLC) [14], now also known as High Resolution Screening (HRS). When performed in parallel with MS measurements, chemical information of the individual compounds could directly be linked to their biological activities [15, 16]. The aim of the present thesis is the evaluation of HRS for active metabolite screening and for screening of affinities towards drug metabolizing enzymes. Therefore, this chapter firstly reviews the evolvement and current state of HRS. Principles and applications are discussed and advantages and drawbacks are evaluated. Then, drug metabolizing enzymes and bioactivation are introduced as these topics are used