Development of efficient cytochrome P450-dependent whole-cell biotransformation reactions for steroid hydroxylation and drug discovery Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes von Tarek Hakki Saarbrücken 2008 Index . Publications resulting from this work I Abbreviations II Abstract IV Zusammenfassung V Summary VI 1. Introduction 1 1.1. Steroid hormones and cytochromes P450 1 1.2. Human CYP11B1 and CYP11B2 6 1.2.1. General aspects 6 1.2.2. Physiological role of CYP11B1 and CYP11B2 7 1.2.3. Differences and similarities between CYP11B1 and CYP11B2 11 1.2.4. CYP11B1 and CYP11B2 modelling 11 1.2.5. CYP11B1 and CYP11B2 as drug targets 13 1.2.6. General requirements for the development of CYP11B2 inhibitors 15 1.2.7. Heterologous expression of CYP11B1 and CYP11B2 in stable cell 16 cultures 1.2.8. Heterologous expression of CYP11B1 and CYP11B2 in yeast 16 1.2.9. Inhibitors of CYP11B1 and CYP11B2 17 1.3. Fission yeast Schizosaccharomyces pombe as a model system 18 1.4. Biotechnological applications of the 11β-Hydroxylases 20 1.5. Aim of the work 21 2. Materials & Methods 23 2.1. Materials 23 2.1.1. Microorganism growth media 23 2.1.1.1. Growth media for Escherichia coli (E. coli) 23 2.1.1.2. Growth media for Schizosaccharomyces pombe (S. pombe) 24 2.1.2. Microorganisms 26 2.1.3. Plasmids 26 2.1.4. Oligonucleotides 29 2.1.5. Library of pharmacologically active compounds (LOPAC) 29 2.1.6. Mega Block plates 30 2.2. Methods 31 2.2.1. Molecular biology methods 31 Index . 2.2.1.1. pNMT1- TOPO cloning 31 2.2.1.2. Amplification of the human AdR 32 2.2.1.3. DNA electrophoresis and manipulation 33 2.2.1.4. DNA restriction and ligation 33 2.2.1.5. Plasmid purification and DNA sequencing 33 2.2.2. Microbiology methods 34 2.2.2.1. E. coli cultivation and transformation 34 2.2.2.2. S. pombe cultivation and transformation 34 2.2.2.3. ura4 gene disruption in S. pombe 35 2.2.3. Biochemical methods 36 2.2.3.1. Subcellular fractionation and protein preparation from S. pombe 36 2.2.3.2. SDS (Sodium dodecylsulfate) polyacrylamid gel electrophoresis and gel 37 blotting 2.2.3.3. Immunologic detection of proteins 37 2.2.4. Steroid hydroxylation assays 39 2.2.4.1. Bioconversion assay in Erlenmeyer flasks 39 2.2.4.2. Bioconversion in modified 1.5 ml tubes 39 2.2.4.3. Steroid extraction 40 2.2.4.4. Steroid analysing methods 41 2.2.4.4.1. High performance liquid chromatography (HPLC) 41 2.2.4.4.2. High performance thin layer chromatography (HPTLC) 41 2.2.4.5. Measuring of steroid bioconversion 42 2.2.4.6. Measuring of the inhibition of steroid bioconversion (Determination of the 42 IC50 values) 2.2.5. Structure activity relationship (SAR) study 43 2.2.6. Statistical analysis 44 2.2.6.1. Descriptive statistics (Measures of variation) 44 2.2.6.2. Statistical tests 44 2.2.6.2.1. t-test for independent samples 44 2.2.6.2.2. Correlation 45 2.2.6.2.3. Z'-Factor of assay 45 3. Results 47 3.1. Optimisation of a steroid hydroxylation assay for the 96-well plate format 47 Index . 3.1.1. Steroid bioconversion assay in modified 1.5 ml tubes (tip-tube format) 47 3.1.2. Steroid bioconversion assay in 96-well plate 49 3.2. Coexpression of the corresponding redox partners in CYP11B1- expressing fission yeast Schizosaccharomyces pombe 60 3.2.1. The Coexpression of AdR and Adx through two expression vectors 60 (Strategy I) 3.2.1.1. Ura4 gene disruption in S. pombe (SZ1) and the characterisation of the 61 new strain 3.2.1.2. Construction of AdR expressing vector (pTH1) 63 3.2.2. Construction of an AdR+Adx expressing vector pTH2 (Strategy II) 63 3.2.3. Coexpression of Adx and AdR in fission yeast 65 3.2.4. The 11β-hydroxylation activity of CYP11B1 in the new recombinant 68 fission yeast strains 3.2.4.1. Comparison of biotransformation activity of CYP11B1-expressing strains after coexpressing of the corresponding redox partners 69 3.2.4.2. Quantification of hydrocortisone production in the novel strain TH75 71 3.2.4.2.1. Optimisation of the biotransformation parameters to a achieve a high conversion rate 71 3.2.4.2.2. Hydrocortisone production efficiency in the fission yeast strain TH75 72 3.3. Development of a cell-based high throughput screening system for the discovery of human aldosterone synthase inhibitors 74 3.3.1. Automated screening technology plate-format 74 3.3.2. Optimisation of the screening assay parameters to get detectable conversion/inhibition response 76 3.3.3. Proof of principle 81 3.3.4. Validation of the new CYP11B2 inhibitors identified during the screening 88 assay 3.3.4.1. Toxicity in fission yeast 88 3.3.4.2. Determination of the IC50 values against CYP11B2 and CYP11B1 89 4. Discussion and Outlook 95 4.1. Optimisation of steroid hydroxylation assay for the 96-well plate format 95 4.2. Coexpression of redox partners in CYP11B1 expressing fission yeast 96 Schizosaccharomyces pombe Index . 4.3. The development of a cell-based high throughput screening system for the 100 discovery of human aldosterone synthase inhibitors 4.4. Testing of a library of pharmacologically active compounds using the 103 developed screening system 5. References 111 6. Appendix 128 6.1. Contributions to international meetings 128 6.2. Index of Figures 129 6.3 Index of Tables 132 6.4. Materials and Methods 133 6.4.1. Stock solutions for EMM medium 133 6.4.2. Oligonucleotides 134 6.4.3. Library of pharmacologically active compounds 136 6.4.4. Liquid class programs 165 7. Acknowledgment 167 Curriculum Vitae Publications resulting from this work I Publications resulting from this work A. Manuscripts: 1. Derouet-Hümbert, E., Dragan, C. A., Hakki, T. and Bureik, M., 2007. ROS production by adrenodoxin does not cause apoptosis in fission yeast. Apoptosis. 12, 2135-2142. 2. Hakki, T. and Bernhardt, R., 2006. CYP17- and CYP11B-dependent steroid hydroxylases as drug development targets. Pharmacol Ther. 111, 27-52. 3. Hakki, T., Zearo, S., Dragan, C. A., Bureik, M. and Bernhardt, R., 2008. Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe. J Biotechnol. 133, 354-359. 4. Hakki, T., Hübel, K., Waldmann, H. and Bernhardt, R., in preparation. The development of high throughput screening system for the discovery of human aldosterone synthase (CYP11B2) inhibitors. 5. Petric, S., Hakki, T., Bernhardt, R., Cresnar, B. in preparation. Characterization and expression of progesterone-inducible cytochrome P450 genes in the zygomycete fungus Rhizopus oryzae. B. Patent application Tarek Hakki1, Rita Bernhardt 1, Matthias Bureik1, Katja Hübel2, Herbert Waldmann2. Vier neue und spezifische Inhibitoren der humanen Aldosteronsynthase (Submitted) 1Institute of Biochemistry, P. O. Box: 151150, Saarland University, D-66041 Saarbrücken, Germany 2Max Planck Institute of Molecular Physiology, Otto-Hahn-Str, 11, D-44227 Dortmund, Germany Abbreviations II Abbreviations 18-OH-B 18-hydroxycorticosterone 5-FOA 5 -fluoroorotic acid A Area ACE Angiotensin-converting enzyme ACTH Adrenocorticotropic hormone AdR Adrenodoxin reductase Adx Adrenodoxin AdxD113Y Adx substitution mutant containing Tyr instead of Asp at position 113 AdxS112W Adx substitution mutant containing Trp instead of Ser at position 112 AdxWT Adrenodoxin wild type AGS Adrenogenital syndrome Aldo Aldosterone ampR Ampicillin resistance gene arh1 Fission yeast ferredoxin reductase ars1 Autosomal replicating sequence B Corticosterone CAH Congenital adrenal hyperplasia cinh Inhibitor concentration CMO Corticosterone methyl oxidase CPR Cytochrome P450 reductase CRH Corticotropin-relasing hormone CYP11B1 Steroid 11β-hydroxylase, cytochrome P450c11 CYP11B2 Aldosterone synthase, cytochrome P450c11Aldo Da Dalton DBH Dopamine β- hydroxylase DHEA Dehyhdroepiandrosterone DITIs Disposable Tips DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DOC 11-deoxycorticosterone DTE 1,4-Dithioerythritol EMM Edinburgh minimal medium EPHESUS The Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study ET Electron transfer EtOH Ethanol etp1fd Adrenodoxin-like ferrodoxin F Cortisol (Hydrocortisone) f Correction factor FAD Flavine adenine dinucleotide FH-I Familial hyperaldosteronism type I Abbreviations III FMN Flavine mononucleotide GRA Glucocorticoid-remediable aldosteronism GSH Glucocorticoid-suppressible hyperaldosteronism HPLC High performance liquid chromatography HPTLC High performance thin-layer chromatography HTS High throughput screening system IC50 Concentration of inhibitor that gives 50% inhibition INH(P) The inhibition of the product production Iradio Intensity of the radioactive signal IST Internal standard IZA Inner zone antigen kDa kilodalton LB Luria-Bertani LiAc Lithium acetate LOPAC Library of pharmacologically active compounds MAO Monoamine oxidase MeOH Methanol NaAc Sodium acetate NADPH Nicotinamide adenine dinucleotide phosphate NC Negative control PC Positive control PEG Polyethylene glycol PMSF Phenyl methyl sulfonyl fluoride PNMT Phenylethanolamine N-methyltransferase pNMT no message with thiamine promoter RALES Randomized Aldosterone Evaluation Study trial RNA Ribonucleic acid ROI Region of interest RSS 11-deoxycortisol RT Room temperature S. cerevisiae Saccharomyces cerevisiae S. pombe Schizosaccharomyces pombe SAR Structure-activity relationship SCC Side-chain cleavage SDH Steroid dehydrogenases SDS-PAGE Sodium dodecylsulfate polyacrylamid gel electrophoresis SE Standard error of the mean TH Tyrosine hydroxylase YEA Yeast extract and supplements Abstract IV Abstract Cytochromes P450 play a vital role in the steroid biosynthesis in the human adrenal gland, e.g. the production of hydrocortisone and aldosterone by CYP11B1 and CYP11B2, respectively. The steroid hydroxylases of the CYP11B family are important targets for drug development. Since they are very closely related, the discovery of selective inhibitors has been a focus of interest.