The following two summaries have been kindly provided by Alison Stevenson, Research Officer – Ataxia UK

SUMMARY OF FINAL REPORT

Time dependence and dose response of HDAC inhibitors of the pimelic diamide family on the chromatin structure of the frataxin gene

Principal researchers: Elisabetta Soragni & Joel Gottesfeld, the Scripps Research Institute, California (August 2008 - August 2009)

Background and aims

This project is aimed at the development of HDAC inhibitors as an effective treatment for Friedreich’s ataxia (FRDA). FRDA is caused by the frataxin gene, which encodes the essential mitochondrial protein frataxin, being abnormally ‘switched off’, or repressed.

We recently identified a novel class of histone deacetylase (HDAC) inhibitors that relieve repression of the frataxin gene in white blood (lymphoid) cells derived from people with FRDA.

A library of HDAC inhibitors, all based on the structure of the commercially available HDACi BML-210, was developed at Repligen corporation. These small molecules were tested for pharmacokinetic, cell permeability, cytotoxicity and other properties. The most active HDAC inhibitors were identified and tested for cytotoxicity and ability to increase frataxin gene expression. From this initial screening a group of 7 lead compounds was selected at Repligen Corporation for further analysis (compounds 44, 106, 109, 123, 136, 526, 531). One of these molecules, compound 106, is able to increase frataxin transcription in a mouse model of FRDA.

To gain further understanding of the effects of our HDAC inhibitors, we analyzed the dose and time of exposure necessary to restore function of the frataxin gene and the duration of this effect. The experiments were carried out in cells; in a FRDA lymphblastoid cell line and in FRDA primary lymphocytes. We also investigated the cellular target(s) and specificity of effective HDAC inhibitors.

Results

The HDAC inhibitors were analyzed for their ability to overcome repression of the frataxin gene in primary lymphocytes taken from people with FRDA. The levels of frataxin mRNA and the acetylation state of the gene were measured as indicators of its function (FRDA-causing mutations in the frataxin gene are associated with a low level of acetylation whereas higher acetylation levels are associated with a functional gene).

Of the compounds tested, 106 and 109 achieved the highest increase in frataxin mRNA and this was seen at low doses; 5µM for both molecules. As compound 109 was consistently better than 106 at increasing frataxin transcription, it was used in further experiments to determine the timing of induction, the duration of effect and the minimum exposure time required to achieve frataxin activation.

Primary lymphocytes from an FRDA donor were treated with 5 and 10 µM 109 for 5 to 46 hours and frataxin mRNA levels were measured as an indicator of the function of the gene. Maximum induction of frataxin mRNA was seen after 46 hours of treatment with 10 µM 109, and 10 hours of treatment with 5 µM 109 was sufficient to cause a two-fold increase in frataxin mRNA. An increase in the acetylation of the frataxin gene was seen after 12 hours of treatment.

In terms of the duration of this response, frataxin mRNA was still detectable at therapeutically significant levels 12 hours after the inhibitor had been removed from the media. Four hours of treatment was sufficient to induce a two-fold increase of frataxin mRNA levels.

In summary, the minimum effective dose of compound 109 for the treatment of FRDA primary lymphocytes is 5µM. The results show that a 10 hour treatment at this dose is necessary to achieve a two-fold increase in frataxin transcription; such an increase is considered therapeutically significant for most people with FRDA. To achieve such activation in 10-12 hours, the inhibitor needs to be present for a minimum of 4 hours, and after removal of the inhibitor the effect lasts for 12 hours.

To investigate the cellular targets of effective HDAC inhibitors, two compounds with different HDAC specificity were investigated; compounds 3 and 106. Both compounds were found to inhibit two types of class I HDACs; HDAC1 and HDAC3. However, compound 3 was more selective for HDAC1 enzyme whilst compound 106 was more selective for HDAC3 enzyme. Only compound 106 was able to induce a therapeutically significant increase of frataxin expression. These results show that HDAC3 has an important role in frataxin repression and targeting this enzyme is crucial to restore expression of the mutant frataxin gene.

Lay Summary of results

The different HDAC inhibitor compounds were analyzed for their ability to ‘switch on’ the frataxin gene, in cells taken from people with FRDA. This was done by measuring the ‘message’ from the frataxin gene (frataxin mRNA), and assessing the acetylation state of the gene (which is known to decrease when the gene is mutated).

Of the compounds tested, 106 and 109 were the best at switching the frataxin gene back on, although 109 was more effective. Therefore, compound 109 was used in further experiments to determine what dose and timing for delivery of the drug is necessary to achieve maximum activation of the frataxin gene. This was done by taking cells from a person with FRDA and treating them with different concentrations compound 109 for different lengths of time.

The results showed that the minimum effective dose of compound 109 is 5µM and that a 10 hour treatment at this dose is necessary to achieve a two-fold increase in frataxin mRNA. Such an increase is considered therapeutically significant for most people with FRDA and the effects of the inhibitor lasted for over 12 hours.

There are different types, or classes, of HDAC enzymes and the specific targets of HDAC inhibitors that are effective at switching on the frataxin gene were investigated. To do this, two HDAC inhibitor compounds targeting different HDAC enzymes were examined; compounds 3 and 106. Both compounds were found to inhibit two types of HDAC enzyme; HDAC1 and HDAC3. However, compound 3 was more selective for HDAC1 enzyme and compound 106 was more selective for HDAC3 enzyme. Only compound 106 was able to induce a significant increase of frataxin expression, showing that improved therapeutics can be obtained in the future by targeting specifically this enzyme.

Benefits to people with ataxia arisen/likely to arise from this research

Results from these experiments support the idea that development of HDAC inhibitors of the pimelic diamide family with HDAC3 specificity can lead to new improved therapeutics for Friedreich’s ataxia. Specifically, the results are an important contribution to the establishment of a dose regimen for clinical trials that will be performed by Repligen Corporation in the future.

SUMMARY OF FINAL REPORT

Development of histone deacetylase inhibitors as treatment for Friedreich’s ataxia

Principal researchers: Elisabetta Soragni & Joel Gottesfeld, the Scripps Research Institute, California (July 2009 – July 2010)

Background and aims

Friedreich’s ataxia (FRDA) is caused by the frataxin gene, which encodes the essential mitochondrial protein frataxin, being abnormally ‘switched off’. This is due to a mutation in the gene that consists of an increased number of ‘GAA•TTC’ repeats in the DNA; this is called a triplet-repeat expansion. The mutation changes the structure of the DNA and this leads to ‘switching off,’ or repression, of the gene. HDAC inhibitors (HDACi) work by switching the frataxin gene back on and this project was aimed at developing HDACi as an effective treatment for FRDA.

Specifically, the aims of the project were 1) to analyse the effect of HDACi on the frataxin gene “environment” (chromatin composition) and expression in white blood cells (lymphocytes) taken from people with FRDA, 2) to screen new inhibitors that specifically inhibit class 1 HDAC enzymes and 3) look at the ability of HDACi to turn the frataxin gene back on in neuronal Friedreich’s ataxia cells.

Lay summary of Results

1) Analysing the effects of HDACi on the frataxin protein expression in lymphocytes taken from people with FRDA

To help the development of HDACi for FRDA, a suitable dose regimen needs to be established for clinical trials. To this end, we took white blood cells (lymphocytes) from people with FRDA, treated them with HDACi and analyzed the timing of frataxin protein expression in the cells.

We have previously demonstrated that a 10-hour treatment with our lead molecule HDACi 109 is necessary to achieve a therapeutically significant increase in frataxin expression. An increase in frataxin protein is first detectable 24 hours after the start of the treatment, and continues for up to 72 hours. After the treatment finishes, therapeutically significant levels of frataxin protein are still detectable for 24 hours, indicating that the frataxin protein is very stable. These results show that HDACi 109 has a long lasting effect, making it an optimal candidate for clinical trials.

2) Screening new inhibitors that specifically inhibit class 1 HDAC enzymes

We wanted to develop new HDACi that were more effective and showed better penetration of the brain. Using a method called ‘click’ chemistry which involves joining different parts of different molecules together to get the most effective compound, a new group of HDACi was synthesized in our laboratory. Very encouraging preliminary results were obtained with two of these compounds; click1 and click2.

When these compounds were compared to our lead compound, HDACi 109,for their ability to increase expression of the frataxin gene in cells from people with FRDA, click1 was found to be as effective, or more effective than compound 109. In blood samples from people with FRDA that did not respond to treatment with compound 109, Click2 was able to increase frataxin considerably (by a factor of 2.5). Moreover, click1 displayed excellent penetration of the blood-brain barrier in rats, meaning that it is readily available to the brain. Taken together, the above results show that although further characterization is necessary, the click compounds could be new improved therapeutics for FRDA.

3) Looking at the ability of HDACi to turn the frataxin gene back on in neuronal Friedreich’s ataxia

Research into Friedreich’s ataxia lacks an appropriate cellular model for studying the condition and screening potential therapeutic compounds. The currently available cell models differ greatly from the cell types primarily affected in FRDA, ie heart cells and brain cells (neurons). They also rely on the availability of people with FRDA to obtain blood donations and skin biopsies from which to source the cells. Very recently it has been shown that skin cells can be reprogrammed into stem cell-like cells, called induced pluripotent stem (iPS) cells, and these can be developed (differentiated) into different cell types, eg neurons and heart cells.

Following a previously published protocol, we obtained FRDA iPS cells. Our FRDA iPS cells exhibit the same abnormalities as FRDA cells ie switching off of the frataxin gene, and other features of FRDA. Hence these cells could be used to study the development of FRDA and possibly also to screen for new potential therapeutics.

To establish whether the FRDA iPS cells could be used to screen for new therapeutics, we wished to determine whether treatment with HDACi could restore frataxin gene expression. Encouragingly, treatment with compound 109 improved the expression of the frataxin gene and increased frataxin protein levels in the cells.

Obtaining neuronal cells from our iPS cells is the more challenging goal. Using a protocol previously published, we were able to generate neuronal-type cells from the iPS cells. Many different types of neuronal cells exist and it is likely that our population of cells is a mixture of these. Therefore our next effort will focus on improving the development (differentiation) of these cells and devising a method to select or separate the different types of neuronal cells from their neighbouring cells. After this, we will use the neuronal cells to study the mechanism o FRDA and test our HDACi.