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
Badran, Mohammed
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.
Philipps-Universität Marburg
Life sciences
opus:9792
https://doi.org/10.17192/z2021.0095
urn:nbn:de:hebis:04-z2021-00954
Life sciences
Biowissenschaften, Biologie
Das erste Kapitel dieser Dissertation (Kapitel 2) präsentiert eine Mutationsstudie, die an der 17β-Hydroxysteroid-Dehydrogenase Typ 14 (17β-HSD14) S205-Variante durchgeführt wurde. Dafür wurden fünf Aminosäuren mutiert, von denen man annimmt, dass sie eine wesentliche Rolle bei der Enzymaktivität und dem struckturen Aufbau spielen. Die fünf mutierten Aminosäuren sind: His93Ala, Gln148Ala, Lys158Ala, Tyr253Ala und Cys255Ala. Alle mutierten Aminosäuren befinden sich im aktiven Zentrum des Enzyms (His93, Gln148 und Lys158) oder auf der flexiblen Schleife, die sich über dem aktiven Zentrum befindet (Tyr253 und Cys255). Die Röntgenkristallographie ist die Methode der Wahl, die in dieser Studie verwendet wird, um die Kristallstruktur jeder Variante zu erhalten. Ein nicht-steroidal potenter 17β-HSD14-Inhibitor (Inhibitor 1) wurde in einem Komplex mit dem Wildtyp kristallisiert, der zur Überprüfung der Bindungsfähigkeit des mutierten Enzyms verwendet wurde. Mit jeder Variante wurde ein enzymatischer Assay durchgeführt, um die Aktivität der einzelnen Varianten zu vergleichen. Östrogen (Östradiol) und Androgen (5-Diol) wurden als Substrat im Enzymkinetik-Assay mit NAD⁺ als Co-Faktor verwendet.
Der zweite Teil der Arbeit (Kapitel 3) konzentriert sich auf einen neuen Kristallprobenhalter (The Roadrunner I Chip), der in der seriellen Röntgenkristallographie (SSX) eingesetzt wird. Der Roadrunner I-Chip ist ein mikrostrukturierter Probenhalter aus monokristallinem Silizium (Waiver-Technologie) mit Mikroporen. Das Ziel der Verwendung des Roadrunner I-Chips ist es, einen Probenhalter zu haben, der Hunderte bis Tausende von Kristallen dem hochintensiven Röntgenstrahl präsentieren kann, ohne das Beugungsmuster zu stören. In dieser Studie wurde Thermolysin (TLN) als Protein verwendet, um die Grenzen dieser neuen Methode zu testen. Thermolysin-Kristalle wurden bei kryogener Temperatur gezüchtet, gewaschen, in Ligandlösungen eingeweicht und eingefroren, ohne sie vom Chip zu entfernen. Nachdem alle Kristallhandhabungsschritte durchgeführt worden waren, wurden Datensätze von TLN-Kristallen gesammelt, während sie auf dem Chip lokalisiert sind. Der experimentelle Teil dieser Studie wurde am Deutschen Elektronen-Synchrotron (DESY), Hamburg, an der PETRA III P11-Strahllinie in Zusammenarbeit mit assoziierten Labors der Anlage durchgeführt.
Der dritte Teil dieser Arbeit (Kapitel 4) befasst sich mit Cyclodextrinen (CDs) und ihrer Fähigkeit, die Löslichkeit hydrophober Verbindungen in wässrigen Lösungen zu verbessern. Das Zielprotein in dieser Studie ist wieder 17β-HSD14. Viele Verbindungen wurden für diese Studie zusammengestellt, wie z.B. eine Fluorverbindungsbibliothek, hydrophobe Medikamente und Sexualhormone. Das Ziel dieser Studie war es, eine Verbindung zu erhalten, die an das Enzym bindet, indem es als Verbindung/CD-Komplex eingeführt wird. Die meisten der in dieser Studie verwendeten Verbindungen wurden bereits mit 17β-HSD14 ohne die Verwendung von CDs getestet, aber aufgrund ihrer geringen Löslichkeit war es nicht möglich, sie in das Enzym einzubringen. Die aus dieser Studie gewonnenen Daten zeigen die Wirkung des Verbindungs-CD/Komplexes, wie er über die Co-Kristallisationsmethode in das Enzym eingebracht wird.
Der vierte Teil dieser Arbeit (Kapitel 5) konzentriert sich auf das Fragmentscreening. Eine Bibliothek von 96 Fragmenten wurde mittels Röntgenkristallographie gegen Trypsin gescreent. Diese Studie konzentriert sich auf den Unterschied zwischen den durch das Fragmentscreening erhaltenen Treffern und Teiltreffern. Das Fragmentscreening wurde an zwei Kristallformen (trigonal und orthorhombisch) durchgeführt. Die aus dieser Studie gewonnenen Daten zeigen den Unterschied zwischen den einzelnen Screeningergebnissen und den Einfluss der Packung, der Kristallsymmetrie und der Fragmentabgabe auf die Trefferquote. Viele Aspekte wurden in dieser Studie berücksichtigt, wie z.B. der Unterschied in der Elektronendichte, das Volumen der Bindungstasche, anomale Peaks und Wasserkanäle.
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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.
doctoralThesis
Fachbereich Pharmazie
2021-12-02
Inhibitoren im Komplex mit dem Protein Strukturbestimmung Kristallstrukturen 17b-HSD14 Fragment Screening crystallography, Inhibitor-Protein com
monograph
2021-12-02
Badran, Mohammed
Badran
Mohammed
opus:9792
English
https://archiv.ub.uni-marburg.de/diss/z2021/0095/cover.png
2020-11-06
2021
Philipps-Universität Marburg
ths
Prof. Dr.
Klebe
Gerhard
Klebe, Gerhard (Prof. Dr.)
Pharmazeutische Chemie
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
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
https://doi.org/10.17192/z2021.0095
urn:nbn:de:hebis:04-z2021-00954
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