Novartis is a company that produces many pharmaceutical drugs. They also actively research how to find new ones. In 2016, they published a paper on their discovery of a molecule that inhibits a phosphatase enzyme implicated in many cancers. We would very much like to have drugs that inhibit the phosphatases associated with many human diseases, but we aren’t there yet. We have evidence that this type of drug would eliminate cancer, alleviate Alzheimer’s disease, and even prevent diabetes and obesity, but the drugs themselves don’t exist. But in this study, Novartis discovered an inhibitor that may well act as a future pharmaceutical.
First, the researchers looked at cancer cell lines. A cancer cell is one that keeps surviving, keeps dividing, and never stops. There are pathways associated with this malfunction, and pathways associated with normal cell growth. Novartis scientists noted the association of a phosphatase known as SHP2 with the malfunctioning pathways, but they know that correlation does not imply causation.
They decided to test the hypothesis that SHP2 leads to a cell growth pathway gone cancerous. To do that, they introduced short hairpin RNA molecules (shRNA) into cancerous cell lines. Now, shRNA is a double stranded RNA molecule that is designed to match a gene of interest. The central dogma of biology tells us that DNA codes for RNA codes for proteins; that means that prior the being an enzyme, a SHP2 gene had to be copied from the DNA of the cell, transported by RNA into the cytoplasm, and read by RNA into a protein.
You can design shRNA to bind to the RNA copy of a gene. When an enzyme in the cell finds shRNA, it cuts it into a single-stranded RNA molecule that identifies and binds the gene of interest – then cuts it in half! The broken RNA can no longer code for that protein, effectively silencing the gene. Thus, the researchers took cells that depended on cancerous pathways to survive and introduced shRNA that silences SHP2. Sure enough, these cells died. The cancer stopped, at least in a petri dish!
That establishes it: SHP2 is needed to make the cellular pathways go awry. How, then, can we find inhibitors? In their case, they took 100,000 compounds and screened them against SHP2. Of those 100,000 diverse molecules, 900 of them inhibited SHP2 (at a rather low threshold of 30% or more reduced enzymatic activity). That’s just under 1% of the library proving itself useful. Welcome to the world of research.
Now, molecules can inhibit enzymes in different ways. There are active inhibitors, for example, and allosteric inhibitors. Active inhibitors bind to the active site of the enzyme, blocking activity. Allosteric inhibitors bind elsewhere on the enzyme, changing its shape so that it is no longer active.
When it comes to phosphatases like SHP2, the active site is highly conserved. That means that, among all 107 phosphatases in the human genome, the differences between each active site is pretty small. For phosphatases that are close cousins (the tumor suppressor SHP1 is very similar to tumor inducing SHP2), the active sites may be virtually indistinguishable for an inhibitor. The last thing we want is a drug that goes around blindly inhibiting human enzymes! Of these 900 SHP2 inhibitors, we need to check if they’re specific to SHP2.
To address this problem, Novartis screened their inhibitors against the catalytic domain of SHP2. That limited the screen to a small fraction of the SHP2 enzyme – the part that contains the active site. If an inhibitor reduced activity of these catalytic domains, they decided that the odds were too great for it to be an active inhibitor, and they threw such samples out. What was left is something that binds and inhibits the full-length SHP2 only. That increases the odds that it’s an allosteric inhibitor.
How many molecules do you think made it through this screen? Six. A grand total of six molecules have now been selected as potential drug targets that inhibit SHP2. They picked the strongest inhibitor from that list, and optimized the sequence further. The result was (rather unromantically) named SHP099, and it not only a very effective inhibitor of SHP2; screening it against 20+ other phosphatases demonstrated no detectable inhibition. That’s a powerful selective inhibitor, and an exciting new candidate for anticancer drug development.