University of Groningen
Fragment-based Discovery Aiming at a Novel Modulation of Malate Dehydrogenase and
Beyond
Reyes Romero, Atilio
DOI:10.33612/diss.150386440
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Publication date: 2021
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Reyes Romero, A. (2021). Fragment-based Discovery Aiming at a Novel Modulation of Malate Dehydrogenase and Beyond. University of Groningen. https://doi.org/10.33612/diss.150386440
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Fragment-based Discovery Aiming
at a Novel Modulation of
Malate Dehydrogenase and Beyond
Atilio Reyes Romero
The research presented in this PhD thesis was performed in the group of Drug Design within the Groningen Research Institute of Pharmacy at the University of Groningen, The Netherlands. The research was financially supported by the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014 – 2020) under the Marie Skłodowska – Curie Grant “AEGIS” (Accelerated Early staGe Drug Discovery, Agreement No. 675555).
Printing of this thesis was financially supported by the University Library and the Graduate School of Science, Faculty of Mathematics and Natural Sciences, University of Groningen, The Netherlands.
Layout: Legatron Electronic Publishing Cover design: Atilio Reyes Romero
Cover picture: ‘Piazza d’Italia’ (ca. 1950 – 1951) – Oil on canvas, 40x50 cm., by Giorgio de Chirico (1888 – 1978), private collection.
Printing: Ipskamp Printing
© Copyright 2020, Atilio Reyes Romero. All rights reserved. No part of this thesis may be reproduced in any form or by any means without prior permission of the author
Fragment-based Discovery Aiming
at a Novel Modulation of Malate
Dehydrogenase and Beyond
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. C. Wijmenga
and in accordance with the decision by the College of Deans. This thesis will be defended in public on
Monday 11 January 2021 at 9.00 hours
by
Atilio Reyes Romero
born on 29 May 1988 in Isola della Scala, ItalySupervisors
Prof. A.S.S. Dömling Prof. T.A. Holak
Co-supervisor
Dr. M.R. Groves
Assessment Committee
Prof. A. Salvati Prof. C.J. Camacho Prof. A.M. Dolga
TABLE OF CONTENTS
Chapter 1: General Introduction and Scope of the Thesis 1
Oligomeric interfaces 3
Macrocycles 4
Covalent inhibitors 5
Rerefences 9
Chapter 2: A Fragment-Based Approach Identifies an Allosteric Pocket that 13 impacts Malate Dehydrogenase Activity
Abstract 14 Introduction 15 Materials and Methods 17
Recombinant protein expression and purification 17
NMR experiments 18
Microscale thermophoresis 19
Thermal shift assay 19
Crystallization, X-ray data collection and processing 19
Activity assay and data analysis 20
Enzymatic assay and kinetics experiment of 4-phenylthiazol-2-amine 21
Screening of 4-(3,4-difluorophenyl) thiazol-2-amine derivatives 21
Statistical analysis 21
Computational analysis 22
Small-angle X-ray scattering experiments 22
Dynamic light scattering 22
Enzymatic assay and kinetics experiment of 4-phenylthiazol-2-amine 24
Screening of 4-(3,4-difluorophenyl) thiazol-2-amine derivatives 24
Statistical analysis 24
Computational analysis 25
Small-angle x-ray scattering experiments 25
Dynamic light scattering 25
Results 26
Initial identification and orthogonal validation of the fragment hit 26
4-(3,4-difluorophenyl) thiazol-2-amine binds at the oligomeric interface 26
Structural rearrangements related to 4-(3,4-difluorophenyl) thiazol-2-amine binding 29
6.7. 4-(3,4-difluorophenyl) thiazol-2-amine derivative screening 32 Thermal destabilization related to 4-(3,4-difluorophenyl) thiazol-2-amine derivatives 33 Validation of the allosteric nature of the pocket with 4-phenylthiazol-2-amine as 33 a model molecule
Small Angle X-ray Scattering 35
Docking of 4-(3,4-difluorophenyl) thiazol-2-amine in other MDHs 37
Discussion 38 Acknowledgment 40 References 41 Supporting Information 45
Chapter 3: Benchmark of Generic Shapes for Macrocycles 59
Abstract 60 Introduction 61
Materials and methods 63
Dataset 63
Preparation of the input structures 64
Software tested and parametrization 64
Comparison parameters 66
Statistical analysis 69
Results 69
Exhaustiveness 69 Accuracy 69
Diversity and sampling efficiency 72
Speed 74
Study cases 75
Intramolecular interactions 80
User-defined energy threshold for improved accuracy and diversity 81
Discussion 82 Conclusion 87 Supporting Information 90
Statistical analysis p-values 90
User-defined energy threshold for improved accuracy and diversity 101
Chapter 4: Discovery of Covalent Phosphatase Inhibitors Across Six 108 Synthetic Mass Scales
Abstract 109
Acknowledgement 120
References 121
Supporting Information 124
Protein expression and purification 124
Activity assay 124
Mass spectrometry analysis 125
Covalent docking 126
Docking scores and binding poses to Cys121 128
References 131 Chapter 5: Gliptin repurposing for COVID-19 134
Abstract 135 Introduction 136 Materials and Methods 138 Results and discussion 138 References 142 Supporting Information 144
Virtual screening methodologies 145
Consensus score 146
Vildagliptin as study case 148
Comparison against the non-covalent inhibitor of PLpro of SARS-CoV 150
References 150 Chapter 6: Summary and Future Perspectives 154
References 159
Appendix 164 Scientific contributions 165 List of publications 165 List of posters 166 List of presentations 167 Secondments 167 Chapter contributions 168 Acknowledgments 169
