New hope for Huntington’s disease drug discovery

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Maelle D.  
By Maelle D.    time to read 4 min

Huntington’s disease is a rare hereditary degenerative brain disorder that was described for the first time by George Huntington in 1872. Symptoms include disturbance in motor (movement), behavioral (mood, anxiety, apathy), and cognitive (such as understanding) systems, generally appearing in the middle of adult life. There is currently no therapy to effectively treat the disease, even though there are treatments that can reduce symptoms and improve the quality of life of those affected.

The origin of Huntington’s disease: Huntingtin (HTT) protein

Huntington’s disease (HD) is a progressive and fatal neurodegenerative genetic disorder in which autosomal-dominant mutation is present on either of an individual’s two copies of the Huntingtin (HTT) gene coding for the Huntingtin protein located on chromosome 4 (Ref 1). The HTT gene contains a repeat of CAG codon coding for glutamine. If the repeat contains 40 or more repeats, it will lead to the development of Huntington’s disease within a person’s normal lifetime. This mutation results in the production of an altered protein leading to dysfunction and neuronal death in the brain’s striatum region.

Molecular consequences of huntingtin mutation on Huntington’s disease
Representation of the molecular genesis of Huntington’s disease.

Model of mHTT-LC3 Linker compound mechanism of action

Several drug discovery strategies have been investigated to treat HD. Recently, a group of scientists used the cellular autophagy mechanism as a way to counter mHTT aggregation leading to neuronal degeneration (Ref 2). Autophagy is a physiological cellular process that enables the degradation of misfolded or aggregated proteins and damaged organelles (Ref 3). Li et al., identified four small molecule compounds that link both the mHTT and LC3 (not wtHTT) triggering the selective decrease of mutant HTT levels.

Model of huntingtin reduction using the cellular autophagy mechanism
Model showing how mHTT-LC3 linker compounds may induce mutated HTT degradation. This diagram illustrates the concept of lowering target protein levels using autophagosome-tethering compounds.

During autophagy, autophagosomes surround cytoplasmic components, including cytosolic proteins and organelles. In parallel, LC3 protein is recruited to autophagosome membranes. Autophagosomes fuse with lysosomes to form autolysosomes and intra-autophagosome components are degraded by lysosomal hydrolases. In the article by Li et al., each identified linker successfully associated the mutated version of Huntington protein specifically to the LC3 protein, leading to a decrease in mHTT cellular levels.

Lowering of mHTT in Huntington’s disease patient cells due to mHTT-LC3 linker compounds

In the study, the effects of the compounds were assessed in primary cells from patients affected with HD using HTRF 2. HTRF technology consists of a signal that is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other (Ref 4). The HTRF assay used in this study was comprised of specific antibodies: 2B7 that recognizes HTT protein in Nterm; 2166 targeting the region at around amino acid 44; and MW1 that binds the polyQ epitope. The antibody pairs 2B7/2166 and 2B7/MW1 allow the specific assessment of wt and mutated HTT respectively (Figure 3.). Using HTRF technology, researchers showed that lowered mHTT protein levels were due to autophagy induction through the action of the four compounds in fibroblasts from Huntington’s disease patients and induced pluripotent cell-derived neurons (iPS cells). Interestingly, the four compounds did not have any effect on wtHTT levels in fibroblast from healthy donors or from patients with Parkinson’s disease. These first results showed the specificity of linker action to mHTT proteins in primary cells from patients affected by HD.

HTRF assay principle for mutated Huntingtin level measurement
Diagram of the HTRF method used to determine mHTT and HTT cellular levels (Ref 5 and Ref 6).

Furthermore, researchers wanted to confirm that the lowered mHTT was related predominantly to activation of the autophagy pathway. To that end, the effect of several compounds were studied in ATG5 knockdown fibroblasts from a patient with HD. ATG5 is a key autophagy gene required for autophagosome formation (Ref 7). The decrease of mHTT levels upon treatment with the different linker compounds was fully wiped out in absence of ATG5. Several autophagy inhibitors were also tested, such as NH4CL and chloroquine, leading to an absence of the mHTT lowering effect in the presence of linker compounds. Together, these results confirmed the implication of autophagy in the reduction of mHTT levels. Additionally, researchers sought to determine the mechanism of action of the identified compounds. Two of the four identified compounds (10O5 and 8F20) are already known as being inhibitors of c-Raf and KSP (Ref 8 and Ref 9). To investigate the role of these linkers, the effects of several known c-Raf and KSP inhibitors were studied. Among all the c-Raf or KSP inhibitors tested, none were able to reduce HTT levels. These results show that c-Raf and KSP have no influence in reducing HTT levels.

Li et al., identified four specific mHTT-LC3 linker compounds that can significantly decrease mHTT levels at nanomolar concentrations in HD cellular models, while showing no effect with respect to wtHTT. Modulation of the autophagy pathway may therefore represent a new paradigm for treating Huntington’s disease. In this article, the selected active compounds were tested mostly on Huntington’s disease models, but promising data suggests that their therapeutic effect can be extended to treat other poly-Q diseases.

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