If you go to clinicaltrial.gov, one of the only websites for up-to-date clinical trial information, and search for Niemann-Pick Type C (NP-C), you will notice a small set of trials in various stages of development. Some of the trials are focused on better understanding disease progression, including finding disease biomarkers. Others are to test novel therapies such as small molecules, stem cell replacement or enzyme replacement. Small-molecule therapeutics to treat Niemann-Pick Type C and other rare genetic diseases are of personal interest since that is the space we are exploring at Perlstein Lab.
Patients with NP-C suffer from seizures, difficulty walking, slurred speech, and trouble moving eyes up and down. As the disease progresses, learning disabilities develop and dementia can occur and eventually require a feeding tube. Currently there is no approved therapy in the United States to treat NP-C. The first drug studied in an NP-C patient population was Miglustat, which is used to treat another rare genetic disease, Gaucher disease (GD1). Miglustat is the first drug approved to treat the neurological complication in NP-C patients, however it has only been approved in the European Union. Based on a study led by MC Patterson et. al., horizontal saccadic eye movement (HSEM), an endpoint measurement of disease progression, improved in Miglustat treated patients. Additionally, in patients older than 12 years, there was an improvement in the ability to swallow and hear clearly. Based on further studies disease progression was delayed, but not in all patients. The FDA is requesting more information on the drug prior to approval in the US.
Two other drugs currently in clinical trial are cyclodextrin and vorinostat. Cyclodextrin is widely used in the food and drug industry for various purposes, including as a preservative, but in the case of NP-C, is used to bind cholesterol and potentially free the cell of cholesterol accumulation. We will cover the cyclodextrin story in a future blog entry.
Vorinostat (aka SAHA) is a small molecule shown to reverse the effects of NP-C based on yeast and mammalian assays and further mouse studies, and is currently in clinical trial for NP-C. Vorinostat was first discovered as a potential drug candidate by Munkacsi, et al. The idea was to determine what proteins play a role in the NP-C disease phenotype, and would it be possible to find a drug to regulate those interacting pathways? They went about this by using yeast as a model organism. Yeast is an excellent choice (yes, good old beer brewing and bread baking yeast) because they are small, grow extremely rapidly, are easy to manipulate genetically, have similar cellular processes to those in mammalian cells and have homologous genes to many that are associated with human disease. Therefore yeast is widely used as a model organism to study human genetic disease. Check out YeastMine on SGD if you are interested in learning more.
Further justification for using yeast to study NP-C as Munkacsi et al. did, is the conservation between the yeast and mammalian NPC proteins. Niemann-pick type C is primarily caused by mutations in the NPC1 gene as mentioned in previous blog entries. The yeast ortholog to this human protein complements function in cell culture, meaning that NP-C mammalian cells can be supplemented with the yeast protein variant, and the disease phenotype can be reversed. The yeast NPC1 ortholog amino acid sequence is 35% similar to the human NPC1 protein, as shown in a partial protein alignment in the figure below from Berger et al. 2005, with the human NPC1 sequence on top and the yeast ortholog, Ncr1p, on the bottom:
Therefore, studying this disease in yeast will allow for a better understanding of the disease biology as well as lead to potential therapies.
Munkacsi et al. deleted the yeast NCR1 gene to make the ncr1Δ yeast strain and crossed it with a yeast deletion collection of other genes to create double ncr1Δ and ORF (open reading frame) knockouts. This was done via synthetic genetic array analysis, as shown in the figure below. They found that individual deletion of 13 genes inhibits the growth of the ncr1Δ mutant in an oxygen-deprived environment. Knowing the cellular phenotype of NP-C is the accumulation of lipids/toxic metabolites in cellular compartments, making them unavailable for other processes, it is possible that the interacting genes found in the study are involved in lipid transport or compensate for the accumulation of toxic metabolites.
The researchers took note that both eaf1 and yaf9, which upon deletion completely inhibited growth of the ncr1Δ strain, encode for proteins that are a part of histone acetyltransferase (HAT) complex. HATs are responsible for acetylating histone proteins. Then they made a small leap of genetic reasoning. What they found is that if you inhibit histone deacetylases (HDACs), enzymes that perform the opposite function of HATs, the NP-C disease phenotype is reversed in mammalian cells.
Filipin , a fluorescent stain, binds to cholesterol and is used widely in research and diagnostically to look at cholesterol accumulation in patient cells. If cholesterol accumulates, as in diseased cells, more intense fluorescence is observed. Above are images we took of patient cells containing the most common NP-C mutation, I1061T. On the left are wildtype dermal fibroblasts stained with filipin, and on the right are the NP-C patient fibroblasts stained with filipin. The difference in phenotype is striking.
Diseased cells accumulate cholesterol as shown above and as also seen below in the leftmost fluorescence microscopy images of the NPC-26 and NPC-J4 patient cell lines from Munkacsi et al. Cells that are not diseased or are rescued from the disease phenotype will show a clearance/usage of cholesterol by the cell. SAHA (aka Vorinostat), which is inhibiting the HDACs, allows for clearance of cholesterol, resulting in a decrease in filipin staining (the right microscopy image). However, vorinostat is not a specific HDAC inhibitor and will alter the regulation of many other genes.
Subsequently, Norbert Wiech founded Lysomics LLC, to assist in bringing NP-C treatments to patients, based on the work out of Olaf West’s and Paul Helquist’s laboratories at Notre Dame. They further showed that HDAC inhibitors reverse the NP-C cholesterol accumulation phenotype in mammalian cells. Vorinostat’s usage in patients was expedited by the Orphan Drug Act, and is in phase I/II clinical trial to assess the safety, tolerability and efficacy in NP-C patients.
At Perlstein Lab, our goal is to use model organisms such as yeast, fly and worm to find novel therapeutics for NP-C and other rare diseases via a high throughput approach. In the case of worm and fly, we are using a whole body approach such that the therapeutics we find might not be directly targeting the mutated protein causing the disease but other pathways contributing to the disease state with the added benefit of being able to monitor full life cycle development, not just a single cell. The drugs currently in clinical trial will hopefully prove to be effective, however, our goal is to discover novel therapeutics that are more efficacious and of higher specificity.
Feature image modified from wallpapersafari.com