Histone Deacetylase 6 (HDAC6) als therapeutisch doelwit in de axonale vorm van de ziekte van Charcot-Marie-Tooth

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system, affecting 1 in 2500 people. Progressive degeneration of the motor nerves leads to the development of motor problems such as muscle wasting and weakness, steppage gate and deformities of hand and feet. Also the sensory nerves are affected leading to reduced sensation of touch, pain and temperature. When only motor axons are affected, the disease is referred to as distal Hereditary Motor Neuropathy (distal HMN). Currently, no curative treatment exists for CMT or distal HMN patients. Previously, it was shown that pharmacologic inhibition of Histone Deacetylase 6 (HDAC6) is beneficial in a mouse model for the axonal form of CMT (CMT2), expressing mutations in small Heat Shock Protein B1 (HSPB1). HDAC6 is a major α-tubulin deacetylating enzyme and plays a role in the regulation of axonal transport. Defects in mitochondrial axonal transport are often associated with neurodegenerative disorders and peripheral neuropathies in particular. Specifically, we wanted to study if HDAC6 could serve as a potential target for the development of a therapeutic strategy. This research question was tackled in two different ways. CMT and distal HMN can be caused by mutations in more than 70 genes. Therefore, in a first step, we wanted to investigate whether pharmacological inhibition of HDAC6 has beneficial effects when motor and sensory axonal degeneration arises as a consequence of other genetic alterations in HSPB1 and in Glycyl-tRNA Synthetase (GARS). In a second step, we focused on the translation of selective HDAC6 inhibition into a clinical application. To investigate our first approach, we selected different mouse models for distal HMN or CMT2 in which we could study the effect of selective HDAC6 inhibition on the motor and sensory behavioral defects. First, we focused on other mutations in the gene encoding HSPB1 as a cause of distal HMN. Therefore, we used the previously developed and characterized mouse model for distal HMN, caused by the neuronal overexpression of human HSPB1P182L. Secondly, we extended our research to another animal model representative for CMT2. This mouse model was developed by inducing a mutation in the gene encoding GARS and became an interesting tool to study the pathogenesis of CMT2 as the motor and sensory deficits are caused by an endogenous mutation in Gars and thus no overexpression is needed to develop a phenotype. In both animal models for distal HMN and CMT2 we were able to demonstrate that the therapeutic potential of HDAC6 inhibition extends beyond mutant HSPB1-induced CMT2. We could show that inhibition of HDAC6 restores the motor and sensory problems on the behavioral level and also on the electrophysiological level. Moreover, these mouse models allowed us to study the presence of specific pathological deficits, such as alterations in the acetylation of α-tubulin and defects in the mitochondrial axonal transport, as possible hallmarks of peripheral nerve degeneration. Decreased acetylation of α-tubulin was present only in peripheral nerve tissue and in both the mutant HSPB1-induced distal HMN and in the mutant Gars-induced CMT2 mouse models. Also the disturbances in the axonal transport of mitochondria were observed in DRG neurons cultured from both disease models. Interestingly, these defects could be restored by selective inhibition of HDAC6. This indicates that decreased α-tubulin acetylation and mitochondrial transport defects are part of pathology common to peripheral nerve degeneration. Additionally, this can form the basis for the development of new therapeutic strategies for CMT. Lastly, to focus on the translation of HDAC6 inhibition into a clinical therapeutic strategy, we developed a compound screening based on the pathological findings of the mutant HSPB1-induced CMT2 mouse model. This screening method was used to develop and characterize HDAC6 inhibitors with improved pharmacokinetic properties suited for testing in clinical trials. In total, 70 chemical structures were tested in our screening method. The testing results of 35 compounds are summarized in this work. All the compounds were similar in their functional group, a hydroxamic acid, but different in their capping group which is responsible for HDAC6 surface recognition. The compound screening resulted in a selection of 3 molecules as potent and selective HDAC6 inhibitors that were able to restore the motor and sensory deficits of the mutant HSPB1-induced CMT2 mouse model. Interestingly, one of the inhibitors is already being tested in a phase IIb clinical trial for multiple myeloma. Thus, the positive results obtained in this screening will advance this compound faster into clinical trials for CMT patients. In summary, we were able to demonstrate that selective inhibition of HDAC6 is beneficial in several animal models of distal HMN and CMT2, further supporting the therapeutic potential of pharmacological inhibition of HDAC6 in specific subtypes of distal HMN and CMT patients. These data encourage further investigation of the effects of HDAC6 inhibition in other subforms of distal HMN and CMT, eventually aiming at the development of a curative treatment for distal HMN and CMT. Moreover, decreased acetylation of α-tubulin and defects in mitochondrial axonal transport are part of pathology common to mutant HSPB1-induced distal HMN/CMT2 and mutant GARS-induced CMT2. This indicates a more general mechanism for peripheral neuropathies involving decreased acetylation of α-tubulin and mitochondrial axonal transport defects. Finally, through our compound screening we were able to identify ACY-1215 with a good drug-like profile that acts as a potent and selective HDAC6 inhibitor for the treatment of CMT and distal HMN.TABLE OF CONTENTS v LIST OF ABBREVIATIONS viii SUMMARY xv SAMENVATTING xviii INTRODUCTION 1 1. Inherited peripheral neuropathies 1 1.1. Charcot-Marie-Tooth disease 4 1.2. Possible mechanisms underlying CMT 8 1.3. Therapeutic interventions to treat CMT 13 2. Acetylation of α-tubulin and HDAC6 in neurodegeneration 15 2.1. Neurons and their cytoskeletal architecture 15 2.2. The functionality of (de)acetylation of α-tubulin 17 2.3. Axonal transport involving acetylation of α-tubulin 18 2.4. Histone Deacetylase 6 (HDAC6) 19 2.5. Acetylation of α-tubulin and HDAC6 in human disorders 23 AIMS 25 MATERIALS AND METHODS 26 PART I HDAC6 as therapeutic target in distal HMN and axonal CMT 32 CHAPTER 1 HDAC6 inhibition restores motor deficits in a mouse model of distal HMN 32 1. Introduction 32 2. Results 33 3. Discussion 35 CHAPTER 2 HDAC6 is a therapeutic target in mutant Gars-induced axonal CMT 38 1. Introduction 38 2. Results 39 3. Discussion 43 CHAPTER 3 Pathological hallmarks of mutant Gars-induced CMT2 46 1. Introduction 46 2. Results 47 3. Discussion 50 CHAPTER 4 Role of HDAC6 in mutant Gars-induced CMT2 pathology 52 1. Introduction 52 2. Results 53 3. Discussion 56 PART II Screening for HDAC6 inhibitors with improved pharmacokinetic properties as a therapy for axonal Charcot-Marie-Tooth disease 58 General introduction 58 CHAPTER 5 Synthesis of benzothiophene-based hydroxamic acids as potent and selective HDAC6 inhibitors 61 1. Introduction 61 2. Results 62 3. Conclusions 64 CHAPTER 6 Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of potent and selective HDAC6 inhibitors 65 1. Introduction 65 2. Results 66 3. Conclusions 68 CHAPTER 7 Bicyclic-capped Histone Deacetylase 6 inhibitors with improved activity in a model of axonal CMT 69 1. Introduction 69 2. Results 70 3. Conclusions 76 CHAPTER 8 Development of improved HDAC6 inhibitors as pharmacological therapy for axonal CMT 77 1. Introduction 77 2. Results 78 3. Conclusions 85 GENERAL DISCUSSION AND FUTURE PERSPECTIVES 86 REFERENCES 94 CURRICULUM VITAE 115nrpages: 138status: publishe