Histone Deacetylase 6 (HDAC6) as therapeutic target in axonal Charcot-Marie-Tooth disease

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

Charcot-Marie-Tooth Disease and Other Peripheral Neuropathies

Peripheral nerves connect the central nervous system with peripheral tissues in the body and are therefore crucial for all living animals to communicate with the environment. Due to the length of their axons, peripheral neurons are extremely vulnerable to insults. Inherited peripheral neuropathies comprise a large group of disorders characterized by progressive loss of axons or myelin that affect motor, sensory and/or autonomic nerves. Charcot-Marie-Tooth disease is the most common form of these inherited peripheral neuropathies. Peripheral nerves can also be damaged by a wide variety of stressors such as inflammation, infection, trauma, systemic disease, toxins/drugs and metabolic disturbances giving rise to several clinical subtypes of the disease. These disorders are referred to as acquired peripheral neuropathies. Ongoing research is focused on unraveling the pathogenic mechanisms underlying these debilitating diseases in order to find possible therapeutic strategies. So far, no drug therapy has been proven effective and patients have to rely on symptomatic treatments that are largely insufficient. Although there is no existing cure for peripheral neuropathies to date, some encouraging advances have been made which are also discussed in this chapter.edition: 1ststatus: publishe

The role of histone deacetylase 6 (HDAC6) in neurodegeneration

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Primary Active Ca2+ Transport Systems in Health and Disease

Calcium ions (Ca2+) are prominent cell signaling effectors that regulate a wide variety of cellular processes. Among the different players in Ca2+ homeostasis, primary active Ca2+ transporters are responsible for keeping low basal Ca2+ levels in the cytosol while establishing steep Ca2+ gradients across intracellular membranes or the plasma membrane. This review summarizes our current knowledge on the three types of primary active Ca2+-ATPases: the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps, the secretory pathway Ca2+- ATPase (SPCA) isoforms, and the plasma membrane Ca2+-ATPase (PMCA) Ca2+-transporters. We first discuss the Ca2+ transport mechanism of SERCA1a, which serves as a reference to describe the Ca2+ transport of other Ca2+ pumps. We further highlight the common and unique features of each isoform and review their structure-function relationship, expression pattern, regulatory mechanisms, and specific physiological roles. Finally, we discuss the increasing genetic and in vivo evidence that links the dysfunction of specific Ca2+-ATPase isoforms to a broad range of human pathologies, and highlight emerging therapeutic strategies that target Ca2+ pumps.status: publishe

Defective axonal transport: A common pathological mechanism in inherited and acquired peripheral neuropathies

Peripheral neuropathies are characterized by a progressive and length-dependent loss of peripheral nerve function. This can be caused either by genetic defects, classified as 'inherited peripheral neuropathies', or they can be acquired throughout life. In that case, the disease is caused by various insults such as toxins and mechanical injuries, or it can arise secondary to medical conditions such as metabolic disorders, nutritional deficiencies, inflammation and infections. Peripheral neuropathies are not only very heterogeneous in etiology, but also in their pathology and clinical presentation. A commonality amongst all peripheral neuropathies is that no pharmacological disease-modifying therapies currently exist that can reverse or cure these diseases. Moreover, the length-dependent nature of the disease, affecting the longest nerves at the most distal sites, suggests an important role for disturbances in axonal transport, directly or indirectly linked to alterations in the cytoskeleton. In this review, we will give a systematic overview of the main arguments for the involvement of axonal transport defects in both inherited and acquired peripheral neuropathies. In addition, we will discuss the possible therapeutic strategies that can potentially counteract these disturbances, as this particular pathway might be a promising strategy to find a cure. Since counteracting axonal transport defects could limit the axonal degeneration and could be a driving force for neuronal regeneration, the benefits might be twofold.status: publishe

Development of improved HDAC6 inhibitors as pharmacological therapy for axonal Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, with an estimated prevalence of 1 in 2500. The degeneration of motor and sensory nerve axons leads to motor and sensory symptoms that progress over time and have an important impact on the daily life of these patients. Currently, there is no curative treatment available. Recently, we identified histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, as a potential therapeutic target in axonal CMT (CMT2). Pharmacological inhibition of the deacetylating function of HDAC6 reversed the motor and sensory deficits in a mouse model for mutant "small heat shock protein B1" (HSPB1)-induced CMT2 at the behavioral and electrophysiological level. In order to translate this potential therapeutic strategy into a clinical application, small drug-like molecules that are potent and selective HDAC6 inhibitors are essential. To screen for these, we developed a method that consisted of 3 distinct phases and that was based on the pathological findings in the mutant HSPB1-induced CMT2 mouse model. Three different inhibitors (ACY-738, ACY-775, and ACY-1215) were tested and demonstrated to be both potent and selective HDAC6 inhibitors. Moreover, these inhibitors increased the innervation of the neuromuscular junctions in the gastrocnemius muscle and improved the motor and sensory nerve conduction, confirming that HDAC6 inhibition is a potential therapeutic strategy in CMT2. Furthermore, ACY-1215 is an interesting lead molecule as it is currently tested in clinical trials for cancer. Taken together, these results may speed up the translation of pharmacological inhibition of HDAC6 into a therapy against CMT2.status: publishe

Synthesis and SAR assessment of novel Tubathian analogs in the pursuit of potent and selective HDAC6 inhibitors

The synthesis of novel isoform-selective HDAC inhibitors is considered to be an important, emerging field in medicinal chemistry. In this paper, the preparation and assessment of thirteen selective HDAC6 inhibitors is disclosed, elaborating on a previously developed thiaheterocyclic Tubathian series. All compounds were evaluated in vitro for their ability to inhibit HDAC6, and a selection of five potent compounds was further screened toward all HDAC isoforms (HDAC1-11). The capability of these Tubathian analogs to inhibit alpha-tubulin deacetylation was assessed as well, and ADME/Tox data were collected. This thorough SAR evaluation revealed that the oxidized, para-substituted hydroxamic acids can be recognized as valuable lead structures in the pursuit of novel potent and selective HDAC6 inhibitors

Synthesis of potent and selective HDAC6 inhibitors bearing a cyclohexane- or cycloheptane-annulated 1,5-benzothiazepine scaffold

Selective inhibitors of histone deacetylase 6 (HDAC6) are an emerging class of pharmaceuticals due to the involvement of HDAC6 in different pathways related to neurodegenerative diseases, cancer, and immunology. Herein, the synthesis of ten new benzohydroxamic acids, constructed by employing the tetrahydrobenzothiazepine core as a privileged pharmacophoric unit, is described. This is the first report on the synthesis and isolation of octahydrodibenzothiazepines and octahydro-6H-benzocycloheptathiazepines, which were then used to develop a new class of HDAC6 inhibitors. Evaluations of their HDAC-inhibiting activity resulted in the identification of cis-N-(4-hydroxycarbamoyl-benzyl)-1,2,3,4,4a, 5,11,11a-octahydrodibenzo[b, e] [1,4]thiazepine-10,10-dioxide and cis-N-(4-hydroxycarbamoyl-benzyl)-7-trifluoromethyl-1,2,3,4,4a, 5,11,11a-octahydrodibenzo[b,e][1,4]thiazepine-10,10-dioxide as highly potent and selective HDAC6 inhibitors with activity in the low nanomolar range, which also show excellent selectivity on the enzymatic and cellular levels. Furthermore, four promising inhibitors were subjected to an Ames fluctuation assay, which revealed no mutagenic effects associated with these structures

Bicyclic-Capped Histone Deacetylase 6 Inhibitors with Improved Activity in a Model of Axonal Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth (CMT) disease is a disorder of the peripheral nervous system where progressive degeneration of motor and sensory nerves leads to motor problems and sensory loss and for which no pharmacological treatment is available. Recently, it has been shown in a model for the axonal form of CMT that histone deacetylase 6 (HDAC6) can serve as a target for the development of a pharmacological therapy. Therefore, we aimed at developing new selective and activity-specific HDAC6 inhibitors with improved biochemical properties. By utilizing a bicyclic cap as the structural scaffold from which to build upon, we developed several analogues that showed improved potency compared to tubastatin A while maintaining excellent selectivity compared to HDAC1. Further screening in N2a cells examining both the acetylation of α-tubulin and histones narrowed down the library of compounds to three potent and selective HDAC6 inhibitors. In mutant HSPB1-expressing DRG neurons, serving as an in vitro model for CMT2, these inhibitors were able to restore the mitochondrial axonal transport deficits. Combining structure-based development of HDAC6 inhibitors, screening in N2a cells and in a neuronal model for CMT2F, and preliminary ADMET and pharmacokinetic profiles, resulted in the selection of compound 23d that possesses improved biochemical, functional, and druglike properties compared to tubastatin A.status: publishe

Synthesis of benzothiophene-based hydroxamic acids as potent and selective HDAC6 inhibitors

A small library of 3-[(4-hydroxycarbamoylphenyl)aminomethyl]-benzothiophenes was prepared and assessed as a novel class of HDAC6 inhibitors, leading to the identification of three representatives as potent and selective HDAC6 inhibitors. Further tests with regard to inflammatory responses indicated that HDAC6 inhibition can be uncoupled from transcriptional inhibition at the level of activated NF-kappa B, AP-1, and GR