Mutational Studies on 17β-HSD14, Serial Synchrotron X-ray Crystallography, Solubility Enhancement using Cyclodextrins and Fragment-Based Drug Discovery Multiple Blocks to Pave the Road of Drug Design
The first topic of this thesis (Chapter 2) presents a mutational study performed on 17β-hydroxysteroid dehydrogenase type 14 (17β-HSD14) S205 variant. Five different mutations were done with respect to five amino acids which are believed to have an essential role in the enzyme activity and assembly....
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|The first topic of this thesis (Chapter 2) presents a mutational study performed on 17β-hydroxysteroid dehydrogenase type 14 (17β-HSD14) S205 variant. Five different mutations were done with respect to five amino acids which are believed to have an essential role in the enzyme activity and assembly. The five variants are: His93Ala, Gln148Ala, Lys158Ala, Tyr253Ala and Cys255Ala. The mutated amino acids are located in the active site of the enzyme (His93, Gln148 and Lys158) or on a flexible loop of the enzyme, which is located above the active site (Tyr253 and Cys255). X-ray crystallography is the method utilized in this study to obtain a 3D crystal structure of each variant. A non-steroidal potent 17β-HSD14 inhibitor (inhibitor 1) has been crystallized in complex with each variant, that has been used to verify the binding capability of the mutated enzyme. Enzymatic assays have been performed with each variant to compare the activity of each one. Estrogen (estradiol) and androgen (5-diol) have been used as a substrate in the enzyme kinetics assay with NAD⁺ as a cofactor.
The second part of this thesis (Chapter 3) is focused on a new crystal sample holder (the Roadrunner I chip) which is used in Serial Synchrotron X-ray Crystallography (SSX). The Roadrunner I chip is a micro-patterned sample holder from single crystalline silicon (waiver technology) with micropores. The aim of using the Roadrunner I chip is to have a sample holder that can present hundreds to thousands of crystals to the high intensity PETRA III beam line P11 (DESY – Hamburg) without interfering with the diffraction pattern. In this study, Thermolysin (TLN) is the protein used to test the limit of this new method. Thermolysin crystals were grown, washed, soaked and frozen at cryogenic temperature without removing them from the chip. Data sets were collected of TLN crystals while they are located on the chip. The experimental part of this study was performed at Deutsches Electronen-Synchrotron (DESY), Hamburg at PETRA III P11 beamline in collaboration with associated laboratories at the facility.
The third part of this thesis (Chapter 4) discusses cyclodextrins (CDs) and their ability to enhance hydrophobic compounds solubility in aqueous solutions. The targeted protein in this study is 17β-HSD14. Many compounds were assembled for this study, such as a fluorine-compound library, hydrophobic drugs and sex hormones. The aim of this study is to obtain a compound that binds to the enzyme by introducing it as a compound/CD complex. Most of the compounds used in this study have already been tested with 17β-HSD14 without the use of CDs, but due to their low solubility it was not possible to introduce them in crystallization samples of the enzyme. The data obtained from this study show the effect of the compound/CD complex, as it is introduced to the enzyme via co-crystallization method.
The fourth part of this thesis (Chapter 5) focuses on a fragment screening. A 96-fragment library is screened against trypsin using X-ray crystallography. This study focuses on the difference of hits and partial hits obtained from the fragment screening. Fragment screening has been performed on two trypsin crystal form (trigonal and orthorhombic). The data obtained from this study show the different results from each screen and how the crystal form and the fragment delivery method influence the hit ratio. Many aspects were considered in this study, such as the difference in electron density, volume of the binding pocket, anomalous peaks and water channels.