Through extensive experiments in cell and animal models of spinal muscular atrophy (SMA), investigators have demonstrated the possible usefulness of two protease inhibitors — compounds that stop certain enzymes from degrading proteins — to treat SMA, a study reports.
Researchers reported that these two protease inhibitors can increase the levels of survival motor neuron (SMN) protein in cells.
The study, “Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology,” was published in the journal Life Science Alliance.
SMA is caused by decreased levels of functional SMN protein. Specifically, SMA patients usually lack correctly working copies of the SMN1 gene. There is also an SMN2 gene, though it tends to create a version of the protein that is less stable, so it can’t normally compensate for the lack of SMN1.
Targeting the SMN2 gene to make more protein, or a more stable version of the protein, is one of the therapeutic avenues currently being explored.
In this study, the researchers needed a way to easily measure the amount of SMN made from the SMN2 gene. Using a gene-editing technique called CRISPR, the researchers generated a cell line that had the code for a fluorescent protein inserted into the SMN2 gene. The details of this model are somewhat complicated, but on a basic level, the researchers could treat cells with a chemical and then see if the cells fluoresced more brightly, which would indicate they had more SMN protein.
“Because we specifically targeted the SMN2 gene in human cells, an important advantage of this approach is that the SMN2 reporter has all the regulatory elements and thus will be effective in screening for therapeutic agents,” the investigators said.
After doing preliminary tests to make sure the model worked, they tested a number of chemicals to see if any had a particular effect. The most noteworthy was Z-FA-FMK, which markedly increased SMN protein levels in these cells.
Having identified this compound, the researchers tested it in a number of different models to see if it would have the same effect of making more SMN protein, and whether this would provide any benefit.
Testing the compound in skin cells, or fibroblasts, from SMA patients, they found that Z-FA-FMK increased the SMN levels in these cells as well.
They then used induced pluripotent stem cells from an SMA patient. Because stem cells can grow into other types of cells with the right chemical stimulation, the researchers grew them into neurons — the kind of cell predominantly affected in SMA. In these neurons, Z-FA-FMK increased the amount of SMN protein; it also improved the movement of organelles such as mitochondria within the cells, which is impaired in SMA.
Further experimentation in cells suggested that Z-FA-FMK probably works by inhibiting proteases — proteins that are responsible for degrading other proteins as part of the cell’s recycling machinery. Specifically, Z-FA-FMK probably targets CAPN1, CAPN7, CTSB, and CTSL proteases. This, in turn, helps prevent the degradation of the unstable form of SMN protein produced by the SMN2 gene.
Finally, the researchers tested Z-FA-FMK in a mouse model of SMA. As expected, the treatment increased the amount of SMN protein in the brain and spinal cord. Treated mice also tended to live a bit longer, though the difference wasn’t large enough to be statistically significant, meaning this trend might just be chance. Further studies will be needed to be sure.
Additionally, the investigators tested another compound, E64d, which works similarly to Z-FA-FMK, but can cross the blood-brain barrier more easily. The blood-brain barrier is a semipermeable membrane that protects the brain from the external environment, and is a major barrier for the efficient delivery of certain therapeutics that need to reach the brain and central nervous system.
E64d had similar effects and significantly extended the lifespan of mice, suggesting that it and other protease inhibitors might be viable candidates for treating SMA. But more research is still needed before these compounds are used in humans.
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