Case study: PCSK9 cholesterol-lowing drugs

Here, we will use the development of pcsk9 cholesterol lowering drug as an example. Now the pcsk9 cholesterol lowering drug was approved for, by the FDA for treating atherosclerosis related disease. Let’s look at what is atherosclerosis. It is the accumulation of cholesterol in the vessels which form a cholesterol plaque that blocks the blood flow. If it happens in the coronary artery, it will cause the death of heart muscle. Without immediate treatment, the patients will die within minutes. Therefore, the cholesterol lowering drugs prevent formation of atherosclerosis.

There are several parameters you need to know in atherosclerosis. First, cholesterol. As we mentioned earlier, high plasma cholesterol levels are associated with atherosclerosis. Second, LDL cholesterol. It is also known as “bad cholesterol” because it can carry cholesterol from livers to the artery. At high levels, it leads the plaque accumulating on your artery walls and induce heart attack or stroke. Third, LDL receptors. The LDL receptors bind LDL particles and complex are taken up by hepatocytes. This figure shows the fate of LDL particles and LDL receptors. LDL receptors are synthesized in endoplasmic reticulum which will be exported to cell surface through Golgi apparatus.

Circulating LDL particles binds LDL receptors on the surface of hepatocytes and is taken up by the hepatocytes by endocytosis. The internalized LDL particle/LDL receptor complex are in the endosome which will fuse with an organelle called lysosome. Then, LDL particles will be separated from the LDL receptors. Importantly, the LDL receptor containing membrane is recycled to cell surface to bind another LDL particle. The internalized LDL particles are degraded in the lysosome. Free amino acid and cholesterol are released in cytoplasm. Therefore, preventing LDL receptors from degradation helps the intake of cholesterol into liver and decreases the plasma cholesterol levels.

The association of PCSK9 mutations and atherosclerosis was first discovered in 2003 in the

paper: Natural Genetics. They studied on the patients of autosomal dominant hypercholesterolemia and later on more and more PCSK9 mutations were discovered. The mutations can be divided into two groups. Gain-of-function mutations which are associated with high cholesterol levels. Loss-of-function mutations which are associated with low cholesterol levels. This figure shows some of the PCSK9 mutations that has been reported previously. Each mutant is labeled as amino acid changed in specific position in the polypeptide chain of PCSK9. Some of them are associated with higher LDL cholesterol levels and some are associated with lower LDL cholesterol levels.

Therefore, the next step is to determine whether the mutated PCSK9 influence the LDL receptor amounts and LDL uptake in hepatocytes. The scientists introduce the mutant genes into hepatocytes and find, indeed, gain of function mutations decrease the LDL receptors and the LDL uptake. In contrast, loss-of-function mutations increase the LDL receptors and increase LDL uptake.

This approach can also apply to animals such as pig. In the paper published in 2013 Science Translational Medicine, the gain-of-function mutations transgenic pig mimic familiar hypercholesterolemia. These patients are normal diets still have high levels of bad cholesterol and routinely develop atherosclerosis.

The second approach is to determine whether RNAi mediated PCSK9 silencing influence LDL receptor amounts, LDL uptake and LDL cholesterol in vitro and in vivo. The paper published in the journal, PNAS 2009, demonstrating the RNAi approach significantly decreases PCSK9 amount and plasma cholesterol level in rodents and in non-human primates.

Through this approach, we know the PCSK9 is a secreted enzyme that bind to the LDL receptor and the LDL particle complex in hepatocytes. After PCSK9 bind to the complex, it change the fate of LDL receptors. It directs the LDL receptor to lysosome for degradation which inhibit the recycle of LDL receptors to cell surface. Therefore, PCSK9 decreases the amount of LDL receptors on the cell surface and increases circulating cholesterol levels.

Finally, the scientist design monoclonal antibody to disrupt the binding of PCSK9 and LDL receptors which can restore the LDL receptors recycling to the cell surface and decreases the cholesterol levels. The strategy is effective in mice and non-human primates as shown in the paper of PNAS published in 2009. And finally there are two anti-PCSK9 antibody will approve by the FDA as anti-cholesterol drug in 2015.