Review
Keynote
Small molecule drug discovery for Huntington's Disease

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Huntington's Disease (HD) is a rare neurodegenerative disease caused by mutation of the huntingtin gene that results in a protein with an expanded stretch of glutamine repeats (polyQ). Knowledge of validated targets is in its infancy, and thus, traditional target-based drug discovery strategies are of limited use. Alternative approaches are needed, and early attempts were aimed at identifying molecules that inhibited the aggregation of polyQ huntingtin fragments. More recently, phenotypic assays were used to find molecules able to reverse some of the pathogenic mechanisms of HD. Such discovery strategies have an impact on the configuration of screening cascades for effective translation of drug candidates toward clinical trials.

Introduction

Huntington's Disease (HD) is a fatal, progressively degenerative brain disorder for which only symptomatic treatments but no efficacious anti-neurodegenerative therapy exists at present [1]. The disease is caused by an expansion of glutamine repeats (polyQ, >35 glutamine residues) at the N-terminal part of a large protein called huntingtin (Htt), which is ubiquitously expressed. Mutant Htt is cleaved by proteolytic enzymes, which results in the release of N-terminal fragments containing the expanded polyQ sequence. These fragments are able to aggregate with themselves and other proteins, and form large nuclear and cytoplasmatic inclusions [2]. There are conflicting reports on whether these large inclusions mediate subsequent cell death [3] or if they are rather cytoprotective, being the result of a mechanism by which the cell protects itself against the production of soluble toxic monomers or oligomers [4].

In any case, the production of these polyQ-containing fragments has several pathophysiological consequences for the affected neurons. They suffer from mitochondrial dysfunction, resulting in reduced ATP levels, decreased Ca2+ uptake and oxidative stress [5]. Mitochondrial impairment leads to excitotoxicity, that is, hypersensitivity to excitatory amino acids, in particular glutamate [6]. Several genes from key signaling pathways, such as the ones induced by cAMP and retinoic acid, were found to be downregulated in different rodent HD models [7], with short N-terminal fragments of Htt showing a much stronger effect than the full-length recombinant protein [8]. The changes in gene expression patterns between these rodent models and human HD post-mortem tissue are comparable [9], underlining the idea that altered transcription is a key mechanism in HD pathogenesis [10]. The polyQ fragments enter the nucleus and mediate transcriptional deregulation by sequestration of transcription factors 11, 12, 13, 14, 15 and histone acetyltransferases [16]. Constitutive production of mutated Htt and aggregate formation overcomes the ability of cells to degrade these proteins by the proteasome [17] and autophagy [18] pathways. All these pathogenic mechanisms eventually lead to apoptotic or necrotic cell death [19] (Fig. 1) even if transgenic animal models for HD can show the full symptoms of the disease before any cell death is measurable.

Mutated Htt protein cannot be regarded as a tractable drug target for small molecules itself, mainly owing to a lack of functional activity, unsolved structure, no known and relevant binding sites for small molecules and ubiquitous expression in many cell types. However, alternative approaches using RNAi [20] or intrabodies [21] have shown recent promise in either preventing the production of mutated Htt or the associated toxicity. Companies and academic groups have, therefore, looked for ‘downstream’ enzymatic targets that might be involved in the pathophysiology of HD. Examples include the use of transglutaminase inhibitors to interfere with aggregation of polyQ fragments [22], the application of creatine [23] and ubiquinone [24] to restore the activity of the mitochondrial electron transport chain, histone deacetyltransferase (HDAC) inhibitors for reversal of transcriptional repression 25, 26, 27, and caspase inhibitors to prevent neuronal apoptosis and proteolysis of Htt [28].

In addition, the pathogenic mechanisms themselves (e.g. mutant Htt aggregation and proteolysis, proteasome and autophagy activation, mitochondrial dysfunction and oxidative stress, excitotoxicity, transcriptional deregulation and apoptosis) have been utilized as assay readouts for the identification of novel, potent and HD-specific small molecules for drug discovery. Here we will describe the design and outcome of these phenotypic primary screening assays (Table 1), and also look at some of the innovative technologies and models that are being used to validate hit compounds from these initial screens. Although information on the ultimate fate of the hits is sometimes difficult to obtain, several efficacious compounds in animal models are described and are currently in clinical development 29, 30.

Section snippets

In vitro aggregation assays

As aggregation of polyQ fragments is an early consequence of mutated Htt expression, this mechanism has been targeted by many different approaches, often with the aim of identifying inhibitors of the aggregation process. It could be demonstrated that stable amyloid-like aggregates were formed as soon as a glutathione S-transferase (GST) tag of an E. coli fusion protein, containing 51 glutamines and corresponding to exon 1 of the Htt gene, was removed proteolytically [31]. In this assay the GST

Yeast assays

Besides assays that were based on the use of mammalian cells, aggregation of Htt has also been studied in simpler yeast systems. These systems have the advantage of being less vulnerable to polyQ-mediated toxicity, while at the same time being readily amenable to genetic analysis for elucidating the participation of other cellular factors in the aggregation process. In one study for instance, the N-terminal region of Htt with repeats from 25 up to 103 glutamines was fused with GFP and expressed

Htt clearance assays

An alternative approach to the inhibition of Htt aggregation is the search for compounds that are able to increase the cellular degradation of soluble or aggregated forms of the mutated protein. Using a Q74 extended and EGFP-tagged exon 1 fragment of the HD gene in both transiently transfected COS cells, and with a stable inducible, PC12 cell line, researchers found accumulation of the polyQ-containing fragments when cells were treated with different inhibitors of the autophagy–lysosome pathway

Assays measuring transcriptional dysregulation

Apart from protein aggregation and impaired clearance mechanisms, one other important factor in the pathology of HD is an overall change in gene transcription. For instance, it could be shown that mutated Htt sequesters the cAMP response element-binding protein (CREB) co-activator, CREB-binding protein (CBP) through direct polyglutamine interactions, which then leads to decreased CREB-mediated transcription [14]. Reporter gene assays in PC12 cells transfected with inducible polyQ exon 1 coupled

Cell death assays

As polyQ expression ultimately leads to cell death, this downstream consequence is also being used as an assay readout for the identification of neuroprotective compounds. Caspase-3 activation as a mediator of apoptotic cell death was measured in one screen assessing the effects of a truncated version of the androgen receptor with a 112 glutamine stretch [65], a model system that could be used for other polyQ diseases. The construct was transiently transfected in 293/HEK cells, which resulted

Non-rodent in vivo model systems of HD

Many genetic models of the disease have been generated in model organisms, such as the nematode worm Caenorhabditis elegans (C. elegans) [72], the fruit fly Drosophila melanogaster 73, 74 and the zebrafish (Danio rerio) [75].

C. elegans is an organism commonly used in developmental biology. When expanded polyQ is expressed in sensory neurons, signs of neuropathology and neuronal dysfunction occur. Among other effects, time-dependent protein inclusions at random locations in the cytoplasm can be

Rodent in vivo model systems of HD

Evaluating active compounds in rodent models of the disease is usually the last part of a screening cascade for proof-of-concept studies before testing compounds in clinical trials. As opposed to other neurodegenerative diseases, many animal models exist (both pharmacologically and genetically induced) that reproduce at least some of the characteristics of HD. However, despite the availability of several alternatives, there are as yet no data demonstrating correlations between effects in a

Primate models

The close physiological, neurological, and genetic similarities between humans and primates would make a monkey model very useful for better understanding of human HD. Recently, the important development of a non-human primate model with rhesus macaques was reported [99]. The model expresses the exon1 of the human Htt gene with 84 glutamine repeats. The severity of observed phenotypes depends on the expression level of mutant protein in the different monkey clones; only one Macaques clone

Conclusions

Since it is too early to define the predictive value of the described phenotypic or mechanism-based assays – measured in terms of identified compounds with real disease-relevance – it is important to characterize active compounds in a broad panel of secondary assays in order to provide further evidence for efficacy before investing in long and expensive in vivo studies with vertebrate animal models. In addition, as the molecular targets in such screening assays are not known, target

Disclosure statement

The authors declare no competing financial interests.

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