Our group was recently part of a study that looked at the role of a specific enzyme, NEU4, in the survival of glioblastoma cells (a type of brain cancer.) I wanted to summarize some of the elements of the work here. Our group’s contribution was the provision of an essential reagent, which was used to test the hypothesis that this enzyme could be targeted with a small molecule drug. The bulk of the work was done by a group at the University of Milan, led by Profs. Venerando and Tringali.
First – what is NEU4? NEU4 is one of four different enzymes in humans known as a neuraminidases. You may know that neuraminidase enzymes are important in the pathology of influenza. The virus needs this enzyme to escape and reinfect host cells, and drugs like oseltamivir and zanamivir target the viral form of it. But human cells have their own use for NEU enzymes, and these four enzymes are ones that we are still learning a lot about. Neuraminidases are named for their substrate, neuraminic acid (also known as sialic acid – and the enzymes are alternatively called sialidases.) Neuraminic acids are carbohydrate residues that tend to be on the ends of complex glycoproteins. In fact, many pathogens (like influenza) use these groups as a handle to enter host cells. But in general, our cells are making glycoproteins (and other glycoconjugates) that contain neuraminic acid, and the NEU enzymes are there to remove those residues. Exactly what molecules are involved is still an area that is being studied. There are four human isoenzymes: NEU1, NEU2, NEU3, and NEU4; development of inhibitors for these enzymes is a relatively new area.
So why do we care about NEU4 specifically? It turns out that Prof. Tringali and coworkers had previously shown that the enzyme was over expressed in some glioblastoma cancers. Based on that, they had initiated studies to understand what the enzyme was doing. It was during those studies (summarized in the title paper) that my group approached them with the offer of an inhibitor that we had just found which we thought could target the enzyme specifically.
The story of that inhibitor is a bit interesting, and is a lesson in how research doesn’t always go as planned. We first made the compound as part of a study we published in 2010. At that time, we only had the assay for one isoenzyme, NEU3. When we tested this compound (and a few others) we found that they had moderate activity for NEU3, but they left a lot of room for improvement. A few years later, we had developed a panel of all four isoenzymes. We’ve used that panel to identify selective inhibitors for several of the isoenzymes already. However, the most notable were some compounds we reported in 2013 which were remarkably potent against just one of the isoenzymes: NEU4. Some of these were the same compounds we had tested back in 2010, and while they still had weaker activity for NEU3, they were very powerful inhibitors of NEU4 alone. The best of these, compound 6, was a 30 nM (nanomolar, or 10^-9 molar) inhibitor. Most importantly, compound 6 was selective, it was 500-times more active against NEU4 than the next best isoenzyme target.
Of course our hope in finding these compounds was that you could use them to determine the role of specific NEU enzymes. The glioblastoma example reported by the group in Milan was a perfect fit for our hypothesis: malignant cells that had unusually high levels of NEU4. In principle, our compound would turn off the action of that enzyme.
The overexpression of NEU4 in these cells resulted in some changes that could be detected, first the cells had increased growth (proliferation) and expressed specific stem cell markers. When NEU4 was ablated genetically, both of these reversed: cell growth slowed and the markers went away. Most exciting for us, when the cells were treated with compound 6, they found the same observations: cell proliferation was reduced and the markers returned to normal levels. These results provide the first evidence that targeting of specific human NEU isoenzymes could be a strategy for an anticancer therapeutic.
Of course there is still a lot of work to do before this is something that could be used for therapy. First and foremost, although our compound was very active in vitro, the cells were not as cooperative. Very high concentrations of the inhibitor were needed to see the effects observed in glioblastoma cells. This is most likely due to the compound just not penetrating into the cells, and might be something we could improve chemically. Another potential concern would be that we don’t know what this compound might do in other tissues – i.e. what could its side effects be? Although NEU4 is upregulated in these cancer cells, it probably does something in normal cells. Inhibition by compound 6 (or a related compound) could lead to other undesired side effects. Our hope is that by targeting just one isoenzyme, negative effects in normal cells will be minimal as the other isoenzymes could still compensate and minimize side effects – but that all remains to be tested.
Our group is continuing to work on identifying selective inhibitors for the other human NEU isoenzymes, and hopefully we will be able to use these in the future to look at the role of the enzymes in health and disease.