Targeting HDAC6 with chemical modulators: catalytic inhibition and beyond

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Thesis

Reference

Targeting HDAC6 with chemical modulators: catalytic inhibition and beyond

DESCHAMPS, Nathalie

Abstract

Histone deacetylases (HDACs) are part of the epigenetic machinery, and, as such, are intricately involved in genes expression. HDAC6 is a unique isoform among the HDACs family through its enzymatic and non-enzymatic partners, but also through its structural organization.

It is involved in cell motility, immunomodulation, and in the misfolded proteins degradation in the aggresome pathway. Selectively targeting HDAC6 has a particular interest in onco-hematology, in combination with other oncodrugs. Using in vitro and in silico techniques (enzymatic study, protein-protein interaction, ELISA, MST, molecular docking, pharmacophore modeling and molecular dynamics simulations), this thesis aims at exploring the molecular requirements to reach HDAC6 selectivity, and demonstrates that relevant selectivity can be achieved without altering the catalytic activity.

DESCHAMPS, Nathalie. Targeting HDAC6 with chemical modulators: catalytic inhibition and beyond. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5168

URN : urn:nbn:ch:unige-1036276

DOI : 10.13097/archive-ouverte/unige:103627

Available at:

http://archive-ouverte.unige.ch/unige:103627

Disclaimer: layout of this document may differ from the published version.

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Section des Sciences Pharmaceutiques Professeur J.-L. Veuthey Dr. A. Nurisso

Dr. C. Simões-Pires

Targeting HDAC6 with Chemical Modulators: Catalytic Inhibition and Beyond

THÈSE

présentée à la Faculté des sciences de l’Université de Genève

pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques

par

Nathalie Deschamps de

Châteauroux (France)

Thèse N°5168

GENÈVE

Atelier d’impression ReproMail 2018

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TABLE OF CONTENTS

Figures and tables list i

Preface v

Scientific contributions vi

Acknowledgements viii

Résumé de la thèse x

Abbreviations xiii

Chapter 1. HDAC6, an exceptional enzyme among the histone deacetylase proteins family

1.1 Histone deacetylases: a family of intricate epigenetic enzymes……….. 2

1.2 Structural aspects of HDAC isoforms relevant to ligand binding and selective catalytic inhibition ………... 8

1.3 HDAC6, structure and biology in the context of drug discovery………. 18

1.4 Thesis aims and structure……….. 23

1.5 References………... 24

1.6 Appendix……….. 37

Chapter 2. HDAC inhibitors from diverse chemical libraries: looking for HDAC6 selectivity 2.1 Introduction……….. 41

2.2 Aurones as histone deacetylase inhibitors: identification of key features……… 43

2.3 Looking for HDAC6 selectivity: screening of diverse molecular libraries………….. 51

2.4 Discussion……… 56

2.5 Conclusion……… 60

2.6 Acknowledgements……….. 60

2.7 References……… 61

2.8 Methods……… 67

2.9 Appendix I……… 73

2.10 Appendix II………... 78

Chapter 3. Rational design of HDAC6-selective inhibitors through a pharmacophore approach 3.1 Introduction……….. 103

3.2 A rational approach for the identification of non-hydroxamate HDAC6-selective inhibitors………... 106

3.7 Methods……… 126

3.8 Appendix……….. 130

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Chapter 4. Accounting for HDAC6 selectivity: flexibility and solvent-mediated arguments

4.1 Introduction……….. 148

4.2 Unravelling the structural elements governing the selective inhibition of HDAC6 by capless compounds: a computational study………... 151

4.7 Methods……… 179

4.8 Appendix……….. 181

Chapter 5. Addressing the modulation of HDAC6 – ubiquitin interaction 5.1 Introduction……….. 199

5.2 Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin……….. 203

5.7 Methods……… 223

5.8 Appendix……….. 228

Chapter 6. Conclusion and perspectives 246

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i

FIGURES AND TABLES LIST Figures

 DNA packaging and selected examples of epigenetic writers, readers and erasers…….. 3

 HDACs phylogenetic classes, cellular localizations and catalytic mechanism……….... 5

 The HDAC catalytic inhibitor pharmacophoric scheme………... 6

 Transition between histone deacetylase 8 (HDAC8) conformations……… 12

 Transition between the open and closed conformational states of histone deacetylase 4 (HDAC4)………... 15

 HDAC6 functional domains……… 18

 A selection of HDAC6-selective catalytic inhibitors………... 20

 Comparison between the HDAC6 homology model and the crystallographic structure (CD2)………... 21

Chapter 2. HDAC inhibitors from diverse chemical libraries: looking for HDAC6 selectivity  The drug discovery process………. 41

 General synthetic route to chalcones 1-16 and aurones 17-32……… 45

 Procedure for the synthesis of hydroxylated aurones 33-36……… 45

 Validation of HDACi of compounds in living cells……… 48

 Compound 34 in complex with HDAC2 and HDAC6……… 49

 Scheme summarizing the screening campaign of one hundred aurone and oxadiazole compounds (ninety-one aurones and nine 1,3,4-oxadiazoles) on HDAC2 and HDAC6 52  121 in complex with HDAC2 and HDAC6……… 53

 Scheme summarizing the screening campaign of the three series of compounds on HDAC2 and HDAC6………... 55

 Docking of aurone 84 in the HDAC6 catalytic pocket: representation of the three clusters of docking solutions obtained with a RMSD cut-off of 1.8 Å………... 56

 Comparison between the HDAC6 homology model from Butler et al. with HDAC6 crystallographic structure………. 58

 Positive control and cytotoxicity of HDAC inhibitors in the BRET-based assay………. 73

 Cytotoxicity of inactive compounds in the BRET-based assay……… 74

 SAHA in complex with HDAC2 and HDAC6………. 74

 Compound 33 in complex with HDAC2 and HDAC6……….. 75

 Comparison between HDAC2 and HDAC6 pockets……… 77

 Chemical scaffolds of the compounds screened in the VS……… 78

 Compound 37 (indolyl-aurone) in complex with HDAC2 and HDAC6………... 78

 Aurones HDAC6 IC50 plots……….. 78

 Chalcones campaign……… 79

 General structure of the thiosemicarbazides series………... 79

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ii Chapter 3. Rational design of HDAC6-selective inhibitors through a pharmacophore approach

 Ligand-based and structure-based pharmacophores queries in FLAP……….. 103

 Prototypical pharmacophoric scheme for HDAC inhibition and the in-silico driven protocol adopted in this study………... 108

 Chemical structures of compounds selected for pharmacophore generation……… 109

 Pharmacophore model for selective HDAC6 inhibitors according to FLAPpharm…….. 110

 Chemical structure of the selected virtual screening hits from the SPECS database……. 114

 Relative α-tubulin acetylation in HeLa cells treated with AK-14 and Tubastatin A……. 115

 Western blot for Histone H4 acetylation levels in HeLa cells treated with AK-14 and Trichostatin A (TSA)………... 115

 ROC plots for pharmacophore-based and for ligand-based virtual screening on the HDAC ChEMBL subset………... 130

 The best-ranked docking pose of AK-14 into the catalytic site of HDAC6……….. 131

Chapter 4. Accounting for HDAC6 selectivity: flexibility and solvent-mediated arguments  Structure of the HDAC6 selective inhibitors Tubastatin A and the computational- based protocol adopted in this study……….. 152

 180 and 181 best-ranked docking poses into HDAC2, HDAC4, HDAC6 and HDAC8 catalytic sites……… 155

 Root Mean Square Fluctuation (RMSF) plots for HDAC8 complexes………. 159

 Zoom on HDAC8 fluctuations: comparison between 180- and 181- complexes……….. 160

 Radial distribution function analysis (Rdf) of the 180-HDAC2 complex………. 164

 Radial distribution function analysis (Rdf) of the 181-HDAC2 complex……… 164

 Radial distribution function analysis (Rdf) of the 181-HDAC8 complex……… 167

 The five loops characterizing the catalytic pocket of HDAC2, 4, 6, and 8……… 170

 180-HDAC2 equilibration plots………... 181

 181-HDAC2 equilibration plots……….. 181

 180-HDAC2 distances monitoring plots……….. 185

 181-HDAC2 distances monitoring plots………. 185

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Chapter 5. Addressing the modulation of HDAC6 – ubiquitin interaction

 Protein-protein interaction classes………... 200

 Ubiquitin, HDAC6 and the proteasome and aggresome pathways………... 201

 Overexpression of HDAC6-EGFP fusion proteins in HEK 293 cells and their interaction with recombinant mono-Ub measured by MST……….. 204

 Chemical structure of the 40 compounds selected through VS and screened using both the MST and the ELISA assays……… 207

 Microscale thermophoresis (MST) applied for the screening of HDAC6 ligands:

detection of binding on both catalytic and ZnF-UBP domains………. 211

 ELISA-based method for measuring the inhibition of the protein-protein interaction (PPI) between HDAC6 and Ub……… 213

 Ubiquitin binding pocket and its crystallographic configuration sampling……….. 215

 Calibration curve for recombinant GFP (rGFP) and MS-based catalytic activity of HDAC6-EGFP fusion proteins in lysates of transfected HEK 293 cells………... 228

 MST experiments with different batches of lysates diluted 2-, 4- and 8-fold indicated no significant competition between recombinant mono-ubiquitin and endogenous free ubiquitin or other Ub-like proteins………... 229

 MST binding experiments with HDAC6-EGFP fusion proteins in lysates of HEK 293 cells and His6-ubiquitin (His6-Ub)………... 230

 HDAC6-EGFP WT binds to mono-ubiquitin, K48-, and K63-linked polyubiquitin chains with similar affinities……… 231

 Mono-ubiquitin binds to EGFP-HDAC6 WT with 50-fold higher affinity than the ubiquitin-like proteins NEDD8 and ISG15……….. 233

 2D structures of biologically active compounds (197 and 211) selected from SBVS and positive/negative controls……… 235

 Binding of recombinant full length HDAC6 WT to mono-Ub labeled with NT-647 using NHS coupling chemistry……… 236

 HDAC6-ZnF-UBP Co-crystallized compounds structures……….. 236

 Summary of the SBVS results……….. 237

Tables

 Areas of flexibility detected in class I HDACs………. 13

 Areas of flexibility detected in class II and IV HDACs……… 16

 Epigenetic mechanisms and their respective involved protein families and domains…... 37

 HDAC6 crystallographic structures available on the Protein Data Bank (PDB)……….. 38

 HDAC6 partners and their demonstrated interacting domain(s)……….. 39

Chapter 2. HDAC inhibitors from diverse chemical libraries: looking for HDAC6 selectivity

 HDAC inhibition of selected aurones in a nuclear extract and towards various isoforms 47

 Activity profile of the seven best compounds from the aurones series………. 54

 Percentage viability and HDAC inhibition of compounds 2, 3 and 17-36……… 77

 Molecular docking and enzymatic results of 4,6-dihydroxylated aurones with the ring B replaced by an indole ring………. 80

 Molecular docking and enzymatic results of aurones bearing a sulfonyl group………… 85

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 Molecular docking and enzymatic results of 4,6-dihydroxylated aurones with a

hydroxylated ring B………. 85

 Molecular docking and enzymatic results of 4,6-dihydroxylated aurones bearing alkyloxy-substituents on the ring B……….. 87

 Molecular docking and enzymatic results of 4,6-dihydroxylated aurones bearing halogeno-substituents on the ring B………. 89

 Molecular docking and enzymatic results of 4,6-dihydroxylated aurones bearing other substituents on the ring B………. 91

 Molecular docking and enzymatic results of 6,7-dihydroxylated aurones………... 93

 Molecular docking and enzymatic results of other types of aurone……….. 94

 Molecular docking and enzymatic results of the 1,3,4-oxadiazole series………. 95

 Molecular docking and enzymatic results of the chalcones and derivatives compounds series……… 97

 Molecular docking and enzymatic results of the thiosemicarbazides series………. 99

 Molecular docking and enzymatic results of pyrimidinetriones………... 101

Chapter 3. Rational design of HDAC6-selective inhibitors through a pharmacophore approach  Enzymatic activity profile of the eight compounds selected from the virtual screening campaign……….. 113

 Cytotoxic properties of AK-14………. 116

 Number (N) of HDAC inhibitors (HDAC2, 4, 6, 8) retrieved from the ChEMBL search 131  The HDAC ChEMBL dataset……….. 132

 Isoform selectivity (ratio) for the compounds selected for HDAC6 pharmacophore building……… 136

 List of the 200 best-ranked compounds obtained with the pharmacophore based virtual screening approach……….. 136

 List of the 200 best-ranked compounds obtained with the ligand based virtual screening approach………... 137

 Forty compounds from the pharmacophore based and ligand-based virtual screening selected for in vitro testing………... 139

 Pocket volumes of HDAC2, 8, 4 and 6 calculated through Connolly’s surface using CASTp server……….. 145

Chapter 4. Accounting for HDAC6 selectivity: flexibility and solvent-mediated arguments  Summary of the molecular docking results for 180 and 181 conducted on HDAC isoform 2, 4, 6 and 8………. 154

 Details about the interactions between 180, 181 and HDAC2, HDAC4, HDAC6, and HDAC8 best-ranked docking poses………. 156

 The five loops surrounding the catalytic pocket of HDAC2, 4, 6 and 8……… 169

 HDAC proteins taken into account in this study. Structural details……….. 196

 Summary of the re-docking results for HDAC2, 4, 6, 8 complexes reported in the PDB 196  Average RMSD and distance values calculated from the MD trajectories of the eight complexes obtained by docking………... 197

 Average number of water molecules occupying the first and the second solvation shell calculated from the MD trajectories of the eight complexes obtained by docking……… 197

Chapter 5. Addressing the modulation of HDAC6 – ubiquitin interaction  Effect of selected hits on HDAC6 catalytic activity………. 245

 HDAC6-ZnF-UBP co-crystallized compounds screening using the ELISA-based assay 245

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v PREFACE

The Pharmacochemistry research group, directed by Pr. P.A. Carrupt, has long been dedicated to the development of original in vitro (Dr. G. Stezaert, 1998; Dr. G. Bouchart 2001; Dr. B. Bard, 2008, Y. Henchoz, 2009) and in silico (Dr. Zuaboni, 2008) tools and strategies to determine physico-chemical (lipophilicity, solubility, pKa) properties of new chemical entities (NCEs). Additionally, the permeation through the main human biological barriers (gastro-intestinal, blood-brain, and skin) of known drugs and chemicals, together with NCEs were predicted using parallel artificial membrane permeability assays (PAMPAs, Dr. M.E. Castella, 2005; Dr. G. Ottaviani, 2006; Dr. C. Le-Bourdonnec Passeleu, 2013; Dr. A. Bujard, 2015; Dr. C. Petit, 2016) and modeling (Dr. G. Ottaviani, 2006).

Lipophilicity is also a critical parameter in pharmacochemistry and drug development, and as such, has been another aspect investigated by the research group. A new descriptor was developed as a molecular interaction field: the molecular lipophilicity potential, MLP. Based on experimental LogP values, it enabled better description of lipophilicity in drug design projects (Dr. A. Galland, 2004; Dr.

A. Daina, 2006; Dr. N. Oberhauser, 2014) and in virtual screening approaches (Dr. A. Daina, 2006; Dr.

J. Bravo, 2009; Dr. N. Oberhauser, 2014).

Alongside the investigation of these important physico-chemical parameters, the Pharmacochemistry research group identified multi-target hits that inhibited enzymes involved in neurodegenerative disorders (monoamine oxidases, MAO, and acetylcholine esterase, AchE). In vitro (Dr. L. Novaroli, 2005; S. Di Givanni, 2006) and in silico (Dr. J. Bravo, 2009) strategies were successfully employed to discover natural and synthetic MAO or AchE inhibitors.

More recently, the research group has focused on the emerging field of epigenetics (Dr. L.

Ryckewaert, 2015, Dr. V. Zwick, 2016; Dr. L. Sacconnay 2016). The group has specialized on the histone deacetylase (HDACs) proteins family, investigating several classes (class I HDACs and class III HDACs, also called sirtuins) through computational and in vitro methods. Indeed this family of enzymes is involved in various diseases, including neurodegenerative disorders and cancer.

This manuscript reports the investigations on one specific HDAC isoform: the histone deacetylase 6. Using both in silico and in vitro techniques, it aims at getting insights into the molecular requirements to reach HDAC6 selectivity, at both the ligand and the protein structure levels, in the context of drug discovery.

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vi SCIENTIFIC CONTRIBUTIONS

Publications

 Aurones as histone deacetylases inhibitors: identification of key features, published in Bioorganic & Medicinal Chemistry Letters, 2014, 24, 5497-5501. Vincent Zwick^±, Alkiviadis- Orfefs Chatzivasileiou^±, Nathalie Deschamps^±, Marina Roussaki, Claudia A. Simões-Pires, Alessandra Nurisso, Iza Denis, Christophe Blanquart, Nadine Martinet, Pierre-Alain Carrupt, Anastasia Detsi, Muriel Cuendet.^±: equal contribution.

 How the flexibility of human histone deacetylases influences ligand binding - An overview, published in Drug Discovery Today, 2015, 20(6), 736-742. Nathalie Deschamps, Claudia A.

Simões-Pires, Pierre-Alain Carrupt, and Alessandra Nurisso.

 A rational approach for the identification of non-hydroxamate HDAC6-selective inhibitors, published in Scientific Reports, 2016. Laura Goracci^±, Nathalie Deschamps^±, Giuseppe Marco Randazzo, Charlotte Petit, Carolina Dos Santos Passos, Pierre-Alain Carrupt, Claudia Simões-Pires and Alessandra Nurisso. ^±: equal contribution.

 Unravelling the structural elements governing the selective inhibition of HDAC6 by capless compounds: a computational study, to be submitted. Nathalie Deschamps, Carolina Dos Santos Passos, Benjamin Grenier, Pierre-Alain Carrupt, Claudia Simões-Pires and Alessandra Nurisso.

 Methods for addressing the protein-protein interaction between histone deacetylase 6 and ubiquitin, submitted in Communications Biology 2017. Carolina dos S. Passos^±, Nathalie Deschamps^±, Yun Choi, Robert E. Cohen, Remo Perozzo, Stephen W. Michnick, Alessandra Nurisso and Claudia A. Simões-Pires. ^±: equal contribution.

Oral communications

 A rational approach for the identification of non-hydroxamate HDAC6-selective inhibitors. 24^th SCT Young Research Fellow Meeting, February 8-10^th 2017, Paris-Sud University, France.

 Addressing the HDAC6-Ubiquitin protein-protein interaction. PhD day, June 6^th 2017, Louis-Jeantet Fundation, Geneva, Switzerland.

Posters

 Aurones as histone deacetylases inhibitors: identification of key features. Vincent Zwick^±, Alkiviadis-Orfefs Chatzivasileiou^±, Nathalie Deschamps^±, Marina Roussaki, Claudia A.

Simões-Pires, Alessandra Nurisso, Iza Denis, Christophe Blanquart, Nadine Martinet, Pierre- Alain Carrupt, Anastasia Detsi, Muriel Cuendet. Swiss Pharma Day, August 2013, Bern, Switzerland.

 Capless compounds can inhibit HDAC6 selectively. Nathalie Deschamps, Carolina Dos Santos Passos, Pierre-Alain Carrupt, Claudia Simões-Pires and Alessandra Nurisso. 23^rd International Symposium on Medicinal Chemistry, EFMC-ISMC 2014, Lisbon, Portugal.

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 A rational approach for the identification of non-hydroxamate HDAC6-selective inhibitors. Laura Goracci^±, Nathalie Deschamps^±, Giuseppe Marco Randazzo, Charlotte Petit, Carolina Dos Santos Passos, Pierre-Alain Carrupt, Claudia Simões-Pires and Alessandra Nurisso. Journées Scientifiques du Médicament, June 2017, Grenoble, France.

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viii ACKNOWLEDGEMENTS

Ces cinq années de thèse ont été ponctuées de nombreuses rencontres, toutes ont enrichi cette expérience, que ce soit sur le plan humain ou sur le plan scientifique.

En tout premier lieu, je souhaite adresser ma plus profonde reconnaissance au Pr. Pierre-Alain Carrupt. En acceptant ma candidature spontanée, il m’a donné l’incroyable opportunité de faire cinq années de recherche passionnante, et de devenir, par la suite, docteure. Malgré son départ anticipé, je souhaite le remercier d’abord pour ses conseils scientifiques et ses encouragements, mais aussi pour les excellents moments partagés.

Ensuite, je souhaite remercier chaleureusement le Dr. Alessandra Nurisso. C’est grâce à elle que j’ai pu rejoindre le laboratoire de pharmacochimie ; elle est la première à avoir cru en moi. En me faisant partager sa recherche et ses compétences, elle m’a permis de mûrir en tant que scientifique.

J’adresse également ma profonde reconnaissance au Dr. Claudia Avello Simões-Pires. Toujours souriante, dynamique et disponible, elle a su me conseiller, me motiver et me soutenir à chaque nouveau projet. En devenant mes co-directrices, les Drs. Alessandra Nurisso et Claudia Avello Simões-Pires m’ont impliquée dans des projets passionnants en me poussant toujours vers l’excellence. Elles m’ont énormément apporté, autant scientifiquement qu’humainement. Je pense, en particulier, à toutes ces discussions informelles autour du café de 9h. Plus qu’une co-direction, nous avons formé une véritable équipe, et pour cela je souhaite leur dire un immense merci.

J’adresse au Pr. Jean-Luc Veuthey ma sincère reconnaissance. En acceptant de reprendre la direction de ma thèse après le départ du Pr. Carrupt, il m’aura permis de terminer ces cinq années de recherche dans les meilleures conditions. Je lui suis particulièrement reconnaissante de m’avoir intégrée à son groupe, mais aussi pour son écoute et ses conseils. J’ai été soutenue jusqu’au bout, et c’est aussi grâce à lui.

Parmi toutes les personnes m’ayant apporté leur soutien, je n’oublie pas Sylvia Passaquay-Rion.

Elle a toujours été présente, que ce soit grâce à son excellente gestion de mon dossier, que par son sourire et ses encouragements. Elle s’est toujours souciée de moi et m’a toujours apporté son aide avec bienveillance.

Je souhaite aussi remercier très chaleureusement l’équipe du Service égalité. Par l’attribution du Subside Tremplin 2017, ils m’ont permis de terminer ma thèse sereinement. Je remercie également la Pr. Frauke Müller pour son appui, toujours positif.

I would like to warmly thank all the jury members for being present and taking time to review my work: Pr. Ahcène Boumendjel, Dr. Yves Auberson and Pr. Patrycja Nowak-Sliwinska. I met each one of you either during my pharmacy studies, at the very beginning of my PhD thesis or later, and I have always appreciated our scientific discussions.

Je souhaite maintenant remercier tous les anciens membres du groupe Pharmacochimie pour tous les échanges et les bons moments passés ensemble : Dr. Alban Bujard, Dr. Stéphanie Romand, Dr.

Céline Le Bourdonnec-Passeleu, Christophe Francey, Fabrice Gillerat, Dr. Florine Lecerf-Schmidt, Dr.

Nils Oberhauser, Emilie Reginato, Dr. Sophie Martel, Virginie Beyeler, Laurent Starrenberger, Dr.

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ix Carolina Dos Santos Passos, Dr. Marco Randazzo, et Dr. Julietta Gradinaru. Mais j’ai une pensée toute particulière pour les Drs. Lucie Ryckewaert et Charlotte Petit. Lorsque je suis arrivée dans l’équipe, vous aviez déjà bien avancé vos thèses. Très vite, pourtant, vous m’avez intégrée et même adoptée.

Comment oublier ces longues heures passées ensemble au laboratoire, nos discussions scientifiques, culturelles ou complètement décalées autour d’un verre, nos motivations et démotivations, votre visite à la maternité, nos fou-rires. Vous étiez mon rayon de soleil au laboratoire, vous êtes devenues parmi mes plus proches amies. Je n’oublie pas le Dr. Lionel Saconnay avec qui j’ai partagé un bureau pendant plus d’une année. Nous nous sommes toujours entraidés sur les projets de modélisation moléculaire.

Même après avoir rejoint son nouveau poste, je lui suis très reconnaissante d’avoir continué à être un soutien.

Je n’oublierai pas non plus les moments passés avec la "team théâtre" avec qui j’ai eu quelques- uns de mes meilleurs fou-rires : Chiara Ambuehl, Dr. Stéphanie Romand, Dr. Lucie Ryckewaert, Dr.

Charlotte Petit, Dr. Florine Lecerf-Schmidt et Dr. Céline Passeleu – Le Bourdonnec. Mais aussi la "team running" commencée avec le Dr. Philippe Eugster, Dr. Leonardo Lauciello et Dr. Alessandra Monaco, puis avec Dr. Lucie Ryckewaert et Dr. Charlotte Petit, complétée enfin avec Aymeric Monteillier, Noémie Saraux, Léonie Pellissier et Antonio Azzollini.

Je pense maintenant aux membres de mon laboratoire d’adoption (Sciences analytiques) : Cédric Schelling, Christophe Francey, Jessica Ortelli, Emilie Reginato, Vida Sadat-Noorbakhsh, Aline Mutabazi, Arnaud Garcia, Nicolas Drouin, Yoric Gagnebin, Julian Pezzati, Vincent Desfontaine, Alexandre Goyon, Dr. Aline Dellicour, Dr. Marco Randazzo, Dr. Victor Gonzalez-Ruiz, Dr. Valentina D’Atri, Dr. Olivier Ciclet, Dr. Balazs Bobaly, Dr. Sabolcs Fekete, Dr. Julien Boccard, Dr. Julie Schappler, Dr. Davy Guillarme, Nicole Decrey et Pr. Serge Rudaz. Ils m’ont très vite intégrée au sein du groupe et je garderai beaucoup de souvenirs de discussions (non scientifiques) mémorables de la pause de midi !

Enfin, je tiens à remercier toute l’équipe d’encadrement des travaux pratiques où l’ambiance était toujours bon enfant : Dr. Davy Guillarme, Dr. Remo Perozzo, Pr. Leonardo Scapozza, Dr. Elisabeth Rivara-Menten, Elinam Gayi, Verena Santer, Dr. Stéphanie Romand, Dr. Charlotte Petit, Francesca Tessaro, Léonie Pellissier, Dr. Marco Randazzo, Micaela Freitas et Dr. Emmanuel Varesio. Avec un remerciement spécial au Dr. Sébastien Tardy pour son implication, et surtout pour l’arrivée du mégaphone qui nous aura valu des moments cocasses !

Plus personnellement maintenant, je souhaite dédier cette thèse à Marie et Dominique pour leur soutien indéfectible. Sans eux, rien de tout ceci n’aurait été possible. Je pense aussi aux petits et grands messages, aux soirées et aux week-ends off avec mes plus proches amis : Marion, Lydie, Boris, Isa, Hélo, Jérémie, Aurélie, Alex, Etienne, Sandy, Thomas et Alice.

Mais surtout, je dédie ces cinq années de travail à Ludovic et à Margot, qui, sans toujours le savoir, ont apporté leur contribution à chacun des chapitres de cette thèse.

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x RÉSUMÉ DE LA THÈSE

La recherche fondamentale et pharmaceutique dans le vaste domaine de l’épigénétique a explosé ces dix dernières années. Au début de l’année 2017, quarante-six molécules étaient en phase d’essais cliniques, et huit autres avaient déjà été approuvées par les autorités compétentes, témoignant, ainsi, du dynamisme de ce secteur de recherche. L’immense majorité de ces candidats médicaments ont une visée anticancéreuse. En effet, l’étymologie du mot épigénétique signifie autour et au-dessus de la génétique, c’est-à-dire les processus qui régulent l’accès et l’expression des gènes sans modifier leur séquence nucléotidique. Les mécanismes épigénétiques font intervenir plusieurs types de domaine protéique et plusieurs familles d’enzyme. Parmi ces dernières, la famille des histone déacétylases (HDACs) fait l’objet d’un intérêt particulier. Les HDACs catalysent, entre autres, la déacétylation de certaines lysines spécifiques sur les histones, des protéines globulaires permettant l’organisation compacte de l’ADN en chromatine. Par ailleurs, les localisations tissulaire et cellulaire de ces enzymes sont variées : les isoformes de la classe I (HDACs 1-3, et 8) sont très majoritairement nucléaires, les isoformes des classes IIa et IV (HDACs 4, 5, 7, 9, et 11, respectivement) naviguent entre le cytosol et le noyau, alors que les isoformes de la classe IIb (HDACs 6 et 10) sont principalement cytosoliques. Cependant, cette famille enzymatique a bien d’autres substrats et partenaires, donnant lieu à des rôles dans la maturation neuronale, l’immunité, le cycle cellulaire en général, ou bien encore dans l’infection par certains virus.

Du fait de cette diversité, les HDACs sont impliquées dans divers processus pathologiques. Le plus documenté étant l’oncologie, et plus particulièrement les cancers hématologiques.

Il existe, à l’heure actuelle, quatre médicaments autorisés ayant pour cible les HDACs pour le traitement de seconde ou troisième intention des lymphomes cutanés et du myélome multiple (vorinostat, Zolinza® - Merck, 2006 ; romidepsin, Istodax® - Celgène, 2009 ; belinostat, Beleodaq® - Spectrum Pharmaceuticals ; et panobinostat, Farydak® - Novartis, 2015). Ces médicaments sont autorisés aux Etats Unis d’Amérique, mais font l’objet d’un statut de médicament orphelin ou d’autorisation temporaire d’utilisation en Europe, sauf pour le Farydak® dont le niveau SMR (service médical rendu) est jugé modéré. Quoi qu’il en soit, les quatre principes actifs de ces médicaments suivent tous le même schéma structural. Ils sont composés, sur le plan chimique, de trois segments : le premier comporte un groupe fonctionnel capable de chélater le zinc catalytique du site actif des HDACs (localisé au fond du site actif), le second est un segment de connexion, souvent hydrophobe, pour occuper le site actif en forme de tunnel, et enfin le troisième segment est un motif de taille et de composition variable que l’on appelle le groupe « chapeau », car il occupe la surface du site actif. Ce dernier segment est jugé responsable de la sélectivité entre les isoformes de la famille HDACs. Bien que présentant une avancée pour les patients, via une re-sensibilisation des cellules tumorales au chimiothérapie conventionnelles, ils montrent des faiblesses en termes de pharmacocinétique et d’effets indésirables (fatigue profonde, nausées et vomissements dose-limitants, toxicités cardiaque et hématopoïétique). Par ailleurs, ces inhibiteurs ciblent la famille des HDACs sans discrimination, et leurs effets indésirables ont été récemment attribués à l’inhibition des isoformes de la classe I. Aussi est-il critique de développer de nouvelles molécules plus sélectives. Le défi proposé est grand car les sites actifs de ces enzymes présentent un haut niveau d’homologie.

Cependant, l’isoforme 6 fait figure d’exception. Sa structure (1215 acides aminés, deux domaines catalytiques en tandem, et un domaine en doigt de zinc), sa localisation cellulaire, et ses partenaires (aussi bien enzymatiques que non-enzymatiques), font d’elle une protéine unique au sein de la famille des déacétylases. HDAC6 a, entre autres protéines associées, la chaperone HSP90, l’α-

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xi tubuline et la cortactine pour substrats, et l’ubiquitine comme partenaire d’interaction protéine-protéine.

HDAC6 joue donc un rôle majeur dans la dégradation des protéines mal repliées (voies du protéasome et de l’aggrésome), dans la mobilité cellulaire, et dans la régulation de certains processus immunitaires (lymphocytes T régulateurs). Du fait de son profil exceptionnel, tant sur le plan structural que sur le plan biologique, l’intérêt de l’inhibition sélective d’HDAC6 a peu à peu émergé. En 2011, le premier essai clinique (de phase I et IIa) avec un inhibiteur catalytique sélectif, ACY-1215, était lancé, avec pour objectif son étude en combinaison avec un inhibiteur du protéasome chez des patients atteints de myélome multiple, réfractaire ou en rechute. La structure d’ACY-1215 suit le schéma classique pour l’inhibition des HDACs, son groupe chélatant étant, comme pour les inhibiteurs déjà sur le marché, un acide hydroxamique. Cette stratégie de combinaison exploite la position centrale d’HDAC6 dans l’aggrésome, une voie de secours pour la dégradation des agrégats protéiques toxiques largement stimulée par les cellules tumorales lorsque la voie classique du protéasome est altérée.

Malgré la grande attractivité d’HDAC6, beaucoup d’inconnues demeurent concernant les éléments structuraux permettant d’obtenir une sélectivité catalytique, tout comme les mécanismes moléculaires intervenant pour l’inhibition de l’aggrésome. De plus, ces questions soulèvent la possibilité d’un lien dynamique entre les différents domaines fonctionnels d’HDAC6, et donc d’une relation potentielle entre inhibition catalytique et interaction protéine-protéine.

Cette thèse a donc pour but principal d’apporter certaines pièces manquantes au puzzle HDAC6 dans le contexte de la découverte de nouveaux modulateurs sélectifs. L’isoforme 6 étant une protéine multi-domaine, ce manuscrit est organisé en fonction de ces domaines. Les chapitres 2 à 4 traitent du domaine catalytique 2 (CD2). Dans ces chapitres, des inhibiteurs catalytiques sélectifs ont été identifiés et rationnalisés par, i) une approche basée sur la structure du CD2 (criblage virtuel utilisant un modèle par homologie et un criblage enzymatique – chapitre 2), et par ii) une approche basée sur le ligand (modélisation d’un pharmacophore utilisé comme support pour un criblage virtuel, validé par un criblage enzymatique des meilleures molécules – chapitre 3). Les chapitres 2 et 3 ont montré que le respect du schéma classique d’inhibition catalytique des HDACs n’est pas discriminant concernant HDAC6. Le groupe chélatant peut être remplacé par un bioisostère (comme un hydrazide), et le groupe chapeau peut être supprimé. Quant au segment de connexion, une insaturation de type sp² en position α du groupe chélatant s’est avérée une excellente alternative. Ces deux chapitres ont également mis en lumière l’aspect déterminant de la bonne appréhension du zinc catalytique pour l’identification correcte de composés. Ils ont aussi laissé présupposer d’une certaine flexibilité du CD2 vis-à-vis du ligand et une possible influence du solvant. Le chapitre 4 a répondu à ces questions en utilisant une simulation de dynamique moléculaire à l’aide des nouvelles données cristallographiques mises à disposition en 2016.

Ce domaine catalytique est apparu significativement flexible : deux boucles bordant le site actif ont montré des capacités d’accommodation du ligand, qu’il suive ou non le schéma structural classique. De plus, lors de cette simulation, HDAC6 a été la seule isoforme à ne pas héberger de molécule d’eau au sein de son site actif. Finalement, les deux domaines catalytiques, CD1 et CD2, ainsi que le domaine en doigt de zinc de liaison à l’ubiquitine (ZnF-UBP) ont été conjointement étudiés dans le dernier chapitre (chapitre 5). Le but était d’obtenir des éléments de réponse au mécanisme moléculaire de l’inhibition de l’aggrésome par l’inhibition catalytique d’HDAC6. Une influence directe des CD1 et 2 sur la formation du complexe HDAC6-ubiquitine a été démontrée par des techniques de thermophorèse et d’ELISA dont le schéma a été spécialement modifié. Un criblage virtuel, suivi d’un criblage in vitro par ces deux techniques, ont montré qu’il était également possible d’empêcher la formation du complexe HDAC6- ubiquitine sans altérer l’activité catalytique.

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xii Ce dernier chapitre ouvre le débat sur comment moduler HDAC6 : par voie catalytique, en sachant que cela risque d’impacter tous les processus biologiques dans lesquels cette enzyme est impliquée (y compris les processus faisant intervenir des interactions protéine-protéine), ou bien en développant uniquement des modulateurs de l’interaction HDAC6-ubiquitine ?

Ce dernier type de modulateur pourrait être d’une grande efficacité tout faisant l’économie d’un grand nombre d’effets indésirables, car, à la fin, le bénéfice pour le patient prime toujours.

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xiii ABBREVIATIONS

Chapter 1

ATP: adenosine triphosphate

BRCA1: breast cancer susceptibility gene 1 BRCT: BRCA1 C-terminal

CD: catalytic domain

CNS: central nervous system

CTLA4: cytotoxic T lymphocyte antigen 4 DMB: dynein motor binding

DNA: deoxyribonucleic acid

FDA: Food and Drug Administration GPU: graphics processing unit

HATs: histone acetyltransferases

HDACs: histone deacetylases

HDLP: histone deacetylase-like protein

HMTs: histone methyltransferases

HSP90: heat shock protein 90 IC50: concentration for 50% inhibition IP4: inositol tetraphosphate

KDMs : lysine demethylases

MBT: malignant brain tumor MD: molecular dynamics

MOE: Molecular Operating Environment N-CoR: nuclear receptor co-repressor NES: nuclear export signal

NLS: nuclear localization signal

NMR: nuclear magnetic reasonance

PDB: Protein Data Bank PD1: programmed cell death PHD: plant homeodomain

PRMTs: protein arginine methyltransferases

PWWP: proline-tryptophan-tryptophan-proline domain

RMSD: root mean square deviation

SAHA: suberoylanilide hydroxamic acid

SE14: Ser-Glu rich domain SIRT: sirtuin

SMRT: silencing mediator for retinoid and thyroid- hormone receptors

SUMO: small ubiquitin-like modifier Tregs: regulatory T-cells

Ub: ubiquitin

WD40: repeated tryptophan and aspartic acid motifs over 40-60 residues

ZBG: zinc-binding group

ZF-CW: zinc-finger conserved cysteine and tryptophan

ZnF-UBP: ubiquitin-binding domain

Chapter 2

BRET: bioluminescence resonance energy transfer DMSO: dimethylsulfoxide

DNA: deoxyribonucleic acid FA: fluorescence artefact

FDA: Food and Drug Administration FWHM: full width at half maximum HATs: histone acetyltransferases HDAC: histone deacetylase

HDACi: histone deacetylase inhibitor HESI: heated electrospray ionization HTS: high throughput screening IC50: concentration for 50% inhibition MMPs: matrix metlloproteases NA: no activity

ND: not determined NDA: new drug application PCR: polymerase chain reaction PDB: Protein Data Bank

RMSD: root mean square deviation RNA: ribonucleic acid

SAHA: suberoylanilide hydroxamic acid SD: standard deviation

SEM: standard error of the mean SIRT: sirtuin

TLC: thin layer chromatography TMS: tetramethylsilane

TNFα: tumor necrosis factor α TSA: trichostatin A

UHPLC: ultra high performance liquid chromatography

VS: virtual screening ZBG: zinc-binding group

Chapter 3

AUC: area under the curve BBB: blood-brain barrier

FDA: Food and Drug Administration

FLAP: Fingerprints for Ligands And Proteins software

HA: hydroxamic acid

HBA: hydrogen bond acceptor HBD: hydrogen bond donor HDACs: histone deacetylases HSP90: heat shock protein 90 IC50: concentration for 50% inhibition

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xiv IUPAC: International Union of Pure and Applied

Chemistry

LBVS: ligand-based virtual screening MIFs: molecular interaction fields MW: molecular weight

ND: not determined

PAMPA: passive artificial membrane permeability assay

PBVS: pharmacophore-based virtual screening Pe: effective passive permeability value

PIFs: pharmacophoric interaction fields PLD: phospholipidosis

ROC: receiver operating characteristic SAR: structure-activity relationship SD: standard deviation

SI: selectivity index SIRT: sirtuin TSA: trichostatin A VS: virtual screening ZBG: zinc-binding group

Chapter 4

AMBER: Assisted Model Building with Energy Refinement

CD: catalytic domain DNA: deoxyribonucleic acid

FDA: Food and Drug Administration GIST: grid inhomogeneous solvation theory GPU: graphics processing unit

HDAC: histone deacetylase

HIV: human immunodeficiency virus IC50: concentration for 50% inhibition IST: inhomogeneous solvation theory MD: molecular dynamics

MOE: Molecular Operating Environment NMR: nuclear magnetic resonance NPT: isothermal-isobaric

NVT: isothermal-isovolumic PDB: Protein Data Bank PME: particle mesh Ewald Rdf: radial distribution function RMSD: root mean square deviation RMSF: root mean square fluctuation SIRT: sirtuin

VMD: Visual Molecular Dynamics ZBG: zinc-binding group

Chapter 5

ATP: adenosine triphosophate

CD: catalytic domain

CFTR: cystic fibrosis transmembrane conductance regulator

EGFP: enhanced green fluorescent protein ELISA: enzyme linked immunosorbent assays FP: fluorescence polarization

FRET: Förster resonance energy transfer HDAC: histone deacetylase

MOE: Molecular Operating Environment MST: microscale thermophoresis NMR: nuclear magnetic resonance PDB: Protein Data Bank

PPI: protein-protein interaction SEM: standard error of the mean SBVS: structure-based virtual screening SPR: surface plasmon resonance T-jump: temperature jump Ub: ubiquitin

UBP: ubiquitin-binding domain WT: wild type

ZnF-UBP: zinc finger ubiquitin-binding domain

Chapter 6

CD: catalytic domain HDAC: histone deacetylase HSP90: heat shock protein 90 MD: molecular dynamics PPI: protein-protein interaction Ub: ubiquitin

ZBG: zinc-binding group

ZnF-UBP: zinc finger ubiquitin-binding domain

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Chapter 1. HDAC6, an exceptional enzyme among the

histone deacetylase proteins family

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2

1.1 Histone deacetylases: a family of intricate epigenetic enzymes Epigenetics: past and current definition

One of the first use of the word “epigenetic” was the illustration of the genes influence on cell fate decision through an “epigenetic landscape”. Indeed, Waddington, working on the fruit fly developmental biology, linked epigenesis, a theory of embryology that describes specialized tissue developing from non-specialized states, and genetics to coin the concept of an epigenetic landscape ^1-5. Nanney, who worked with a bacterial model in which he assumed that epigenetic mechanisms were related to cellular memory, reconsidered Waddington’s theory in 1958 ⁶. This idea was further developed by Riggs and Holliday ⁷, who described DNA methylation as a potential transcriptional regulatory mechanism that could be maintained by cell division, insisting on the cellular memory mechanism previously assumed by Nanney. ^2,⁸. It is noteworthy that, by defining DNA methylation as an epigenetic modification, the term has evolved from a cellular development mechanism (involved in what is investigated in the current field of developmental biology) into a molecular mechanism of gene regulation ⁹. Although some debate exists about its rigorous definition ², epigenetics can be translated from its direct Greek etymology meaning the inheritance of variation (-genetics) above, and beyond (epi-) changes in the DNA sequencing ¹⁰. Its expanded definition currently includes not only DNA methylation, but also the molecular mechanisms involved in chromatin remodeling, such as histone modifications ^11,¹².

The mediation of the DNA accessibility through its folding/unfolding around the nucleosomes (the fundamental subunits of chromatin), and how nucleosomes are positioned along the DNA material is at the heart of the epigenetic regulation of gene expression (Fig. 1) ¹³. The molecular mechanisms involved in this process are ruled by protein families that, either catalyze reversible covalent modifications (methylation, acetylation, phosphorylation, ubiquitination, or SUMOylation, among others), also called the “writers” and the “erasers”, or that “read” these marks on DNA (e.g. cytosine methylation) and on histones (e.g. histone acetylation) (Fig.1). As a matter of clarification in the context of the present thesis, “epigenetic enzymes” are considered as those proteins catalyzing the reactions involved in the selective addition or removal of chemical marks on DNA and chromatin, thereby altering chromatin and DNA accessibility towards the transcriptional machinery. Accordingly, “epigenetic drugs” refer to drugs used to modulate the activity of epigenetic enzymes.

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3

Figure 1. DNA packaging and selected examples of epigenetic writers, readers and erasers. The DNA material is not free within the nucleus, but highly packaged through the chromatin fiber by wrapping around nucleosome cores. One nucleosome unit is composed of 147 base pairs of DNA which are wrapped around an octamer of histone proteins that contains 2 copies of H2A, H2B, H3 and H4 histone proteins. Histones are globular proteins comprising an N-terminal domain that is the place for post-translational modifications (lysine methylation or acetylation, arginine methylation being the most abundant marks). Epigenetic proteins which add these marks are called “writers”

(HATs: histone acetyl-transferases; HMTs: histone methyltransferases; PRMTs: protein arginine methyltransferases), those which remove these marks are the “erasers” (HDACs: histone deacetylases; KDMs: lysine demethylases) whereas the proteins that recognize and bind such modifications are the “readers”. This picture has been built according to Arrowsmith et al.¹¹, Falkenberg et al.¹⁴, Azad et al.¹³, Schmauss 2017 ¹⁵, and Luo et al.¹⁶, and using Servier Medical Art, under the Common Creative license 3.0. Note: only a selection of epigenetic enzymes, mainly histones modifiers, are given for scheme clarity and because this thesis focuses on HDAC6. A more complete list of epigenetic enzymes is provided in section 1.6, table S1.

Epigenetic state in disease and epigenetic drugs

The epigenetic state of a cell, and its corresponding phenotype, evolves along with the differentiation processes and the development stage of the organ and the organism it belongs to. Because epigenetic changes are believed to allow cell plasticity in response to the environment (toxins, stress, diet, etc.), they have also been considered as a target for dysregulation associated to diverse diseases such as cancer. The first link between epigenetic processes and human disease was established in cancer cells showing less 5-methylcytosine than the corresponding normal cells ¹⁷. Increased DNA methylation was observed in the promoters of tumor suppressor genes that appeared to be silenced ¹⁸. Epigenetic aberrations, originating from loss or gain of function of epigenetic regulatory proteins, significantly take part in the onset and progression of human cancers.

Since then, there has been a broad enthusiasm around the manipulation of epigenetic enzymes in an attempt to reverse altered processes for cellular reprogramming, as demonstrated by the huge body of data found when entering the key words “epigenetics” and “drug discovery” in search engines with a gradually increase these last 4 years (551 articles on PubMed with 76, 105, 111 and 104 items found from 2014 to 2017, respectively, https://www.ncbi.nlm.nih.gov/pubmed, accessed on October, 25^th 2017; 119,000 research articles found on Google Scholar, https://scholar.google.fr/, accessed on

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4

October, 25^th 2017). More than fifty small molecules targeting one of the epigenetic protein families (Fig. 1, Table S1) were under clinical trials in 2017 ¹⁹. Among them, two families are heavily targeted for hematological malignancies treatment: the histone deacetylases having sixteen compounds under clinical trials and four drugs approved (vorinostat - Zolinza®, Merck, 2006 ²⁰; romidepsin - Istodax®, Celgene, 2009 ²¹; belinostat - Beleodaq®, Spectrum Pharmaceuticals, 2014 ²²; panobinostat - Farydaq®, Novartis, 2015 ²³) by the Food and Drug Administration (FDA), and the DNA methylases with three candidates under clinical trials and two FDA-approved drugs (azacitidine - Vidaza®, Celgene, 2004 ²⁴; decitabine - Dacogen®, MGI Pharma, 2006 ²⁵).

The histone deacetylase (HDAC) proteins family

One of the most abundant post-translational modifications in epigenetics, and one of the most studied (after DNA methylation), is histone acetylation ¹¹. This histone mark is balanced by two protein families: the histone acetyl-transferases (HAT, Fig.1) that add acetyl groups to lysine residues on histone amino-terminal regions, and the histone deacetylases that remove these groups. These 2 counterparts were first viewed as direct modulators of the transcription through the chromatin compacting/de- compacting effects driven by the neutralization of the lysine basic charge that the acetyl group produces.

However, this was a simplistic picture, and it’s now widely recognized that a more sophisticated interplay between several epigenetic mechanisms and marks is involved to regulate gene transcription

14, 26, 27. While DNA methylation state can be in some cases inherited and maintained through mitosis and meiosis (contributing to cell memory mechanisms described in the definition of epigenetic regulation), the relevance of histone modifications to cellular memory is uncertain ².

HDACs’ name originally stems from their discovery in the mid-1990s, being the identified enzymes responsible for histone deacetylation and a contributor to gene regulation ^28-30. Actually, it is nowadays more justified to call them lysine deacetylases as this family of enzymes has many more substrates, other than histones, such as transcriptional factors (p53, Rb among more than sixty others), cytoskeleton proteins (α-tubulin, actin, cortactin) and a number of proteins implicated in cell signaling, apoptosis, DNA repair and replication, chaperones, and viral infection ³¹. The HDAC family comprises eleven + seven members classified in subgroups according to phylogenetics, homology to yeast and co- factors (Fig. 2) ^11,³². Class I HDACs (1-3, 8, homologous to yeast Rpd3-like proteins ^33, ³⁴), class II HDACs (IIa: 4, 5, 7, and 9, IIb: 6 and 10, homologous to Hda1-like proteins ³⁴) and class IV HDAC (11 is the sole member) contain zinc-dependent catalytic domain(s), whereas the class III HDACs, also called sirtuins (Sirt1-7) are a structurally and mechanistically unrelated group of NAD⁺-dependent hydrolases. Class I, II and IV hydrolyze the amide bond to release acetate from their substrates through a histidine tandem, one being a single general acid – base catalyst, and the other one remaining protonated along the catalytic cycle to be employed as an electrostatic catalyst. The process is assisted by a water molecule that is bound to the metal to be deprotonated by the neighboring histidine residue , hence activating it for a nucleophilic attack on the substrate carbonyl (Fig. 2) ³⁵.

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5

Figure 2. HDACs phylogenetic classes, cellular localizations, and catalytic mechanism. (A) Phylogenetic tree of the HDAC family, reproduced from Bradner et al.³² and from Arrowsmith et al.¹¹. (B) Simplified view of HDACs cellular localization ³¹. HDACs 1-3 are nuclear, whereas HDAC8, class IIa and class IV HDACs are able to shuttle between the cytoplasm and the nucleus.

Conversely, class IIb HDACs are mainly found in the cytoplasm. The figure was built according to Roche and Bertrand 2016 ³¹ and using Servier Medical Art, under the Creative Common License 3.0.

(C) Catalytic scheme of HDACs. The residues numbering corresponds to HDAC8 isoform, as this new mechanism was proposed by Gantt et al. ³⁵. His143 has a dual role: it serves both as a general base to catch a proton from the water molecule that, in turn, attacks the substrate carbonyl. The tetrahedral oxyanion is stabilized by the neighboring Tyr306 and the zinc ion. Finally the substrate catches a proton from His143 (which, this time, plays the role of a general acid) to release the acetate byproduct. Scheme reproduced and adapted from Gantt et al. ³⁵.

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6

HDAC enzymes share a high degree of homology (sequence identity > 50% within class I isoforms, 22% of sequence similarity between HDAC1 and HDAC6, 33% of sequence similarity between HDAC6 and HDAC11), particularly when considering their catalytic site where only punctual differences characterize them ^36-38. Because their cellular localizations and substrates are diverse (Fig.

2), HDAC enzymes have been shown to be involved in diverse diseases. Indeed, this family is implicated in multiple biomolecular pathways and cell signaling at multiple levels. HDACs have roles in brain function regulation and neurological development ¹⁵. As an example of their high degree of intricacy, HDAC1 and 2 are important for synaptic transmission in immature neurons, whereas only the altered expression of HDAC2 takes part in pathogenesis in mature neurons ^39-41. Additionally, HDACs play a role in both inflammation and viral infection processes, in which class I HDACs (1-3) are in part responsible for the regulation of the innate immunity (cytokines production regulation), whereas class IIa isoforms are hypothesized to be central for adaptive immunity (influences on T-cell functions).

Overlaps between the two immune responses were also reported ^42-45. What originally triggered the development of HDACs inhibitors was their large role in cancer onset and progression ¹⁴. HDACs function and/or expression is aberrant in a broad variety of cancers, and is often correlated with poor prognosis ⁴⁶. However, similarly to the other physio-pathological fields in which HDACs are involved, there are paradoxes at the molecular level. The function of HDACs 1-3 remains unclear: although their inhibition demonstrated efficacy in the clinic, they might also have a tumour-suppressor role ¹⁴.

Figure 3. The HDAC catalytic inhibitor pharmacophoric scheme. The four FDA-approved compounds are also reported.

Nevertheless, sufficient efficacy profiles in cutaneous T-cell lymphoma, relapsed or refractory peripheral T-cell lymphoma, and multiple myeloma were reached, leading to FDA approval of four HDAC catalytic inhibitors (Fig. 3) ^{47, 48}. Many others are under clinical trials, still for oncology purposes

19. These catalytic inhibitors all follow the same pharmacophoric scheme which is composed of three regions: a zinc-binding group (ZBG, usually a hydroxamic acid) to chelate the catalytic zinc ion located at the bottom of the active site, a surface recognition domain (also called the cap group) that occupies the pocket entrance (or channel rim), and a hydrophobic linker to connect the two and that fits into the tunnel-like catalytic pocket (Fig. 3).

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7

The FDA-approved compounds are either non-selective (Vorinostat, Belinostat, and Panobinostat) or class I selective (Romidepsin).While effective, they are subject to strong side effects, mainly attributed to class I HDACs catalytic inhibition (cardiac and hematopoietic toxicities, dose - limiting diarrhea and vomiting) ^13, ^49-52. Moreover, the hydroxamic acid ZBG is subject to rapid hydrolysis, hence weakening the pharmacokinetic profile of these drugs ^53, ⁵⁴. Although HDAC inhibitors represent a hope for patients suffering from leukemia, they are still poorly successful in solid tumours. Research efforts are thus ongoing to build the new generation of HDAC inhibitors. One of the possibilities to do so is reaching isoform selectivity. HDAC enzymes do not operate alone but are very often embedded in multiple subunits complexes. And promising compounds that are found active on one isolated purified isoform are not necessarily active when confronted to the corresponding HDAC- containing complex ⁵⁵. Thus, unravelling the HDACs levels of complexity starts with in-depth structural knowledge on each isoform, especially regarding their respective flexible behavior. This requires selective molecular probes to study the first molecular level of HDACs, being their catalytic domain.

Indeed, selectively targeting one isoform begins with structurally knowing its adversaries. For this purpose, in the next section, information from class I and IIa HDACs, co-crystallized with various compounds, were gathered and analyzed altogether to highlight the catalytic domains plasticity regarding the isoform and the ligand properties.

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1.2 Structural aspects of HDAC isoforms relevant to ligand binding and selective catalytic inhibition

This section is adapted from the article “How the flexibility of human histone deacetylases influences ligand binding - An overview”, published in Drug Discovery Today 2015, 20(6), 736-742.

Nathalie Deschamps¹, Claudia A. Simões-Pires¹, Pierre-Alain Carrupt¹, and Alessandra Nurisso^1,^⌂ .

⌂ Corresponding author.

Affiliation

1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne Quai Ernest - Ansermet, 30, CH- 1211, Geneva 4, Switzerland.

Contribution

Article designing, article writing, article proofreading.