ReviewKeynoteSmall molecule drug discovery for Huntington's Disease
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.
References (101)
Transcriptional signatures in Huntington's disease
Prog. Neurobiol.
(2007)Tissue transglutaminase: a novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases
Eur. J. Pharmacol.
(2008)The therapeutic role of creatine in Huntington's disease
Pharmacol. Ther.
(2005)- et al.
Neuroprotection for Huntington's disease: ready, set, slow
Neurotherapeutics
(2008) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo
Cell
(1997)A microtiter plate assay for polyglutamine aggregate extension
Anal. Biochem.
(2001)Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation
BMC Neurosci.
(2005)Inhibition of polyglutamine protein aggregation and cell death by novel peptides identified by phage display screening
J. Biol. Chem.
(2000)- et al.
Gene transfer methods for CNS organotypic cultures: a comparison of three nonviral methods
Mol. Ther.
(2001) - et al.
A single-chain Fv intrabody provides functional protection against the effects of mutant protein in an organotypic slice culture model of Huntington's disease
Brain Res. Mol. Brain Res.
(2004)
Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles
Neurobiol. Dis.
A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor
Neuron
Time-lapse analysis of aggregate formation in an inducible PC12 cell model of Huntington's disease reveals time-dependent aggregate formation that transiently delays cell death
Brain Res. Bull.
High throughput quantification of mutant huntingtin aggregates
J. Neurosci. Methods
Discovery of a novel small-molecule targeting selective clearance of mutant huntingtin fragments
J. Biomol. Screen.
p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease
Neuron
RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells
J. Biol. Chem.
Sp1 is up-regulated in cellular and transgenic models of Huntington disease, and its reduction is neuroprotective
J. Biol. Chem.
Decreased cAMP response element-mediated transcription: an early event in exon 1 and full-length cell models of Huntington's disease that contributes to polyglutamine pathogenesis
J. Biol. Chem.
Drug targeting of dysregulated transcription in Huntington's disease
Prog. Neurobiol.
Progressive and selective striatal degeneration in primary neuronal cultures using lentiviral vector coding for a mutant huntingtin fragment
Neurobiol. Dis.
A cell-based screen for drugs to treat Huntington's disease
Neurobiol. Dis.
Compounds blocking mutant huntingtin toxicity identified using a Huntington's disease neuronal cell model
Neurobiol. Dis.
Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons
Neuron
Intrastriatal injections of quinolinic acid or kainic acid: differential patterns of cell survival and the effects of data analysis on outcome
Exp. Neurol.
Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice
Cell
Phenotypic abnormalities in the YAC128 mouse model of Huntington disease are penetrant on multiple genetic backgrounds and modulated by strain
Neurobiol. Dis.
Environmental, pharmacological, and genetic modulation of the HD phenotype in transgenic mice
Exp. Neurol.
A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration
Neuron
Huntington's disease: progress and potential in the field
Expert Opin. Investig. Drugs
Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain
Science
Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders
Nature
Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death
Nature
Bioenergetics in Huntington's disease
Ann. N.Y. Acad. Sci.
Proton magnetic resonance spectroscopy in Huntington's disease: evidence in favour of the glutamate excitotoxic theory
Mov. Disord.
Decreased expression of striatal signaling genes in a mouse model of Huntington's disease
Hum. Mol. Genet.
Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease
Hum. Mol. Genet.
Mutant huntingtin's effects on striatal gene expression in mice recapitulate changes observed in human Huntington's disease brain and do not differ with mutant huntingtin length or wild-type huntingtin dosage
Hum. Mol. Genet.
Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease
Science
Interaction of Huntington disease protein with transcriptional activator Sp1
Mol. Cell Biol.
The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription
Proc. Natl. Acad. Sci. U. S. A.
Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity
Science
Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease
Hum. Mol. Genet.
Modulation of nucleosome dynamics in Huntington's disease
Hum. Mol. Genet.
Global changes to the ubiquitin system in Huntington's disease
Nature
Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy
Hum. Mol. Genet.
Apoptosis and caspases in neurodegenerative diseases
N. Engl. J. Med.
RNA interference improves motor and neuropathological abnormalities in a Huntington's disease mouse model
Proc. Natl. Acad. Sci. U. S. A.
Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity
Proc. Natl. Acad. Sci. U. S. A.
Coenzyme Q10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects
Proc. Natl. Acad. Sci. U. S. A.
Cited by (36)
Recombinant Adeno Associated Viral (AAV) vector type 9 delivery of Ex1-Q138-mutant huntingtin in the rat striatum as a short-time model for in vivo studies in drug discovery
2016, Neurobiology of DiseaseCitation Excerpt :It is crucial from a drug discovery prospective to create animal models that, in addition to presenting symptoms typical of striatal neurodegeneration, also recapitulate the genetic and molecular mechanisms underlying the degenerative processes of the human pathology. To that end, the combination of knowledge of the genetic basis of the disease and the emergence of transgenic and gene transfer technologies has allowed the creation of animal models of HD (from the invertebrate C. Elegans to primates) that recapitulate the genetic defect found in humans and are able to reproduce phenotypes reminiscent of HD such as nuclear huntingtin inclusions, neurodegeneration, motor deficits and cognitive impairment (Menalled, 2005, Ramaswamy et al., 2007; Fecke et al., 2009). In most cases, these models are based on mutant huntingtin bearing ca. 100 or more glutamines (e.g. R6/2, BACHD, YAC128 and Knock-In mice, with ca. 130, 97, 128 and 111 polyglutamine repeats respectively; Ferrante, 2009; Menalled and Brunner, 2014).
Towards small molecules as therapies for alzheimer's disease and other neurodegenerative disorders
2014, Drug Design and Discovery in Alzheimer's DiseaseA phenotypic screening assay for modulators of huntingtin-induced transcriptional dysregulation
2013, Journal of Biomolecular ScreeningTargeting mutant huntingtin for the development of disease-modifying therapy
2012, Drug Discovery TodayCitation Excerpt :Understanding the genetics of HD has facilitated disease diagnosis and also enabled the generation of numerous HD model systems including in vitro assays, cell- and brain-tissue-based models, as well as a range of transgenic and knockin animals (for review, see Ref. [8]). Several excellent overviews of the molecular pathomechanisms implicated in HD and approaches toward innovative HD therapeutics have been published (e.g. [8–11]). However, despite nearly two decades of research on mHTT mechanisms, only the vesicular monoamine transporter inhibitor tetrabenazine (Xenazine®) is currently approved for pharmacotherapy of HD acute motor symptoms [12].
Pluripotent Stem Cells Models for Huntington's Disease: Prospects and Challenges
2012, Journal of Genetics and GenomicsCitation Excerpt :There has been very little progress in the discovery of treatments that slow disease progression. There is a particular interest in the discovery of small molecule compounds that inhibit the toxic effects of mutant HTT and reduce neurodegeneration (Fecke et al., 2009; Morse et al., 2011). Stem cell models for HD have the potential as power tools for high-throughput screening of compounds and small molecules that may mediate HD toxicity.
- 1
Current address: GenKyoTex, c/o Eclosion, 14, Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland.