University of Groningen Expanding the toolbox of protein-templated reactions for early drug discovery Unver, Muhammet

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University of Groningen

Expanding the toolbox of protein-templated reactions for early drug discovery

Unver, Muhammet

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Publication date: 2017

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Unver, M. (2017). Expanding the toolbox of protein-templated reactions for early drug discovery. University of Groningen.

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Expanding the toolbox of protein-templated

reactions for early drug discovery

Muhammet Yağz Ünver

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The work described in this thesis was carried out at the Stratingh Institute for Chemistry, University of Groningen, The Netherlands.

This work was financially supported by the University of Groningen and the Ministry of Education, Culture and Science (Gravitation program 024.001.035, Research Center for Functional Molecular Systems).

Printed by: Proefschriftmaken Cover design: Kaja Sitkowska

ISBN

978-94-034-0026-6 (printed version) 978-94-034-0029-7 (digital version)

Expanding the toolbox of

protein-templated reactions for early drug

discovery

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with the decision by the College of Deans.

This thesis will be defended in public on Monday 25 September 2017 at 16.15 hours

by

Muhammet Yağz Ünver

born on 30 November 1987 in Sarayönü, Turkey

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The work described in this thesis was carried out at the Stratingh Institute for Chemistry, University of Groningen, The Netherlands.

This work was financially supported by the University of Groningen and the Ministry of Education, Culture and Science (Gravitation program 024.001.035, Research Center for Functional Molecular Systems).

Printed by: Proefschriftmaken Cover design: Kaja Sitkowska

ISBN

978-94-034-0026-6 (printed version) 978-94-034-0029-7 (digital version)

Expanding the toolbox of

protein-templated reactions for early drug

discovery

PhD thesis

to obtain the degree of PhD at the University of Groningen

on the authority of the Rector Magnificus Prof. E. Sterken

and in accordance with the decision by the College of Deans.

This thesis will be defended in public on Monday 25 September 2017 at 16.15 hours

by

Muhammet Yağz Ünver

born on 30 November 1987 in Sarayönü, Turkey

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Supervisors Prof. A. K. H. Hirsch Prof. B. L. Feringa Assessment committee Prof. A. J. Minnaard Prof. G. Roelfes Prof. J. Rademann

To my dear family

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Supervisors Prof. A. K. H. Hirsch Prof. B. L. Feringa Assessment committee Prof. A. J. Minnaard Prof. G. Roelfes Prof. J. Rademann

To my dear family

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Table of Contents

Chapter 1. Protein-templated hit identification strategies in drug discovery

1.1 Drug discovery and development process 2

1.2 Fragment-based drug design 3

1.3 Target-guided synthesis 5

1.3.1 Dynamic combinatorial chemistry (DCC) 5 1.3.2 Kinetic target-guided synthesis (KTGS) 6

1.3.2.1 Reactions used 7

1.3.2.2 Practical aspects 9

1.3.2.3 Therapeutic scope 12

1.4 Aspartic proteases 22

1.5 Protein-protein interactions 23

1.6 Outline of this thesis 25

1.7 References 27

Chapter 2. Fragment-based drug design facilitated by protein-templated click chemistry: fragment-linking and -optimization of inhibitors of the aspartic protease endothiapepsin

2.1 Introduction 32

2.2 Results and discussion 34

2.2.1 Fragment-based drug design 34

2.2.2 Synthesis of building blocks (azides and alkynes) 35

2.2.3 Generation of the library 36

2.2.4 Synthesis of triazoles identified 39

2.2.5 Biochemical evaluation 41

2.2.6 Discussion 42

2.3 Conclusions 44

2.4 Experimental section 44

2.4.1 Fluorescence-based inhibition assay 44

2.4.2 Modeling and docking 45

2.4.3 PTCC experiments 45

2.4.4 General experimental details 47

2.4.5 Synthesis of azides, alkynes and triazoles 48

2.5 Contributions from co-authors 51

2.6 References 52

Chapter 3. In situ Ugi four-component reaction for the protein-templated identification of inhibitors of endothiapepsin

3.1 Introduction 56

3.2.1 Design of the inhibitor with Ugi-4CR scaffold 57

3.2.3 In situ Ugi reaction 59

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Table of Contents

Chapter 1. Protein-templated hit identification strategies in drug discovery

1.1 Drug discovery and development process 2

1.2 Fragment-based drug design 3

1.3 Target-guided synthesis 5

1.3.1 Dynamic combinatorial chemistry (DCC) 5 1.3.2 Kinetic target-guided synthesis (KTGS) 6

1.3.2.1 Reactions used 7

1.3.2.2 Practical aspects 9

1.3.2.3 Therapeutic scope 12

1.4 Aspartic proteases 22

1.5 Protein-protein interactions 23

1.6 Outline of this thesis 25

1.7 References 27

Chapter 2. Fragment-based drug design facilitated by protein-templated click chemistry: fragment-linking and -optimization of inhibitors of the aspartic protease endothiapepsin

2.1 Introduction 32

2.2.1 Fragment-based drug design 34

2.2.2 Synthesis of building blocks (azides and alkynes) 35

2.2.4 Synthesis of triazoles identified 39

2.2.6 Discussion 42

2.3 Conclusions 44

2.4.3 PTCC experiments 45

2.4.5 Synthesis of azides, alkynes and triazoles 48

2.6 References 52

Chapter 3. In situ Ugi four-component reaction for the protein-templated identification of inhibitors of endothiapepsin

3.1 Introduction 56

3.2.1 Design of the inhibitor with Ugi-4CR scaffold 57

3.2.3 In situ Ugi reaction 59

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3.2.5 Synthesis and biochemical evaluation of the inhibitors 65

3.2.6 Docking results 68

3.3 Conclusions 69

3.4.3 Experimental procedures 70

3.4.5 Synthesis of Ugi products 73

3.6 References 79

Chapter 4. Protein-templated reductive amination for the identification of inhibitors of protein-protein interactions

4.1 Introduction 82

4.2.1 Design of the inhibitor 84

4.2.3 Synthesis of the core scaffold 3 85 4.2.4 Protein-templated reductive amination 86 4.2.5 Synthesis and biochemical evaluation of the inhibitors 88

4.3 Conclusions 91

4.4.1 Modeling 92

4.4.2 Protein purification procedure 92

4.4.3 FP assay 93

4.4.4 1_H-15_{N heteronuclear single quantum coherence (HSQC) NMR experiment 93}

4.6 References 102

Chapter 5. Protein-templated esterification reaction for the inhibitors of aspartic protease endothiapepsin

5.1 Introduction 106

5.2.1 Design of the ester inhibitor 107 5.2.2 Synthesis and biochemical evaluation of the designed inhibitor 108

5.2.3 Optimization studies 109

5.2.4 Generation of the library and the library reaction 110 5.2.5 Synthesis and biochemical evaluation of the library 111

5.2.6 Discussion 112

5.3 Conclusions 113

5.4.5 General procedure for Steglich esterification /de-protection reaction 114

5.6 References 117

Chapter 6. Design and synthesis of bioisosteres of acylhydrazones as stable inhibitors of the aspartic protease endothiapepsin

6.2.1 Design of the bioisosteres 121

6.2.2 Synthesis of the bioisosteres 122

6.2.4 Crystallographic studies 124

6.3 Conclusions 126

6.4.3 Crystallization, data collection and processing 127

6.6 References 132

Summary and outlook 133

Samenvatting 137

Acknowledgment 143

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3.2.5 Synthesis and biochemical evaluation of the inhibitors 65

3.2.6 Docking results 68

3.3 Conclusions 69

3.4.5 Synthesis of Ugi products 73

3.6 References 79

Chapter 4. Protein-templated reductive amination for the identification of inhibitors of protein-protein interactions

4.1 Introduction 82

4.2.1 Design of the inhibitor 84

4.2.3 Synthesis of the core scaffold 3 85 4.2.4 Protein-templated reductive amination 86 4.2.5 Synthesis and biochemical evaluation of the inhibitors 88

4.3 Conclusions 91

4.4.1 Modeling 92

4.4.2 Protein purification procedure 92

4.4.3 FP assay 93

4.4.4 1_H-15_{N heteronuclear single quantum coherence (HSQC) NMR experiment 93}

4.6 References 102

Chapter 5. Protein-templated esterification reaction for the inhibitors of aspartic protease endothiapepsin

5.2.1 Design of the ester inhibitor 107 5.2.2 Synthesis and biochemical evaluation of the designed inhibitor 108

5.2.3 Optimization studies 109

5.2.4 Generation of the library and the library reaction 110 5.2.5 Synthesis and biochemical evaluation of the library 111

5.2.6 Discussion 112

5.3 Conclusions 113

5.4.5 General procedure for Steglich esterification /de-protection reaction 114

5.6 References 117

Chapter 6. Design and synthesis of bioisosteres of acylhydrazones as stable inhibitors of the aspartic protease endothiapepsin

6.2.1 Design of the bioisosteres 121

6.2.2 Synthesis of the bioisosteres 122

6.2.4 Crystallographic studies 124

6.3 Conclusions 126

6.4.3 Crystallization, data collection and processing 127

6.6 References 132

Summary and outlook 133

Samenvatting 137

Acknowledgment 143

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Protein-templated hit-identification strategies in drug discovery

Today’s drug discovery mainly relies on the synthesis of large numbers of compounds and testing with a variety of targets. Target-guided-synthesis (TGS) has emerged as a powerful alternative technique to current drug-discovery methods, which can accelerate the hit-identification process, and thus this long-term trajectory. In this chapter, we firstly discuss the drug-discovery process and fragment-based drug design (FBDD) briefly. Secondly, we introduce TGS in a broad context and discuss kinetic target-guided synthesis (KTGS) in more detail.

M. Y. Unver, A.K.H. Hirsch, in preparation