Fragments as enzyme activators

About half of all approved drugs are small molecules that inhibit enzymes. This makes sense intuitively: an enzyme is like a complicated little machine, and there are lots of ways you can wreck a machine. But there are times when you might want to activate an enzyme, and this is conceptually more difficult. In a new paper in Angew. Chem. Int. Ed., Rod Hubbard and colleagues at the University of York show how fragments can help.

The researchers were interested in the enzyme O-GlcNAc hydrolase (OGA), which removes N-acetylglucosamine from proteins. They were looking for inhibitors of a bacterial version of this enzyme, so they screened it against 100 fragments using three ligand-observed NMR methods (STD NMR, PO-WaterLOGSY, and T_1ρ-filter). This resulted in a very high hit rate: 22 fragments showed binding in all three assays, and 18 of these were competitive with a known substrate-like inhibitor, PUGNAc. Some of these were also active in an enzymatic assay.

More interestingly, the four hits that were not competitive with PUGNAc actually appeared to bind more strongly to the protein in the presence of that inhibitor. In this case, the inhibitor can be considered a stand-in for the natural substrate, and the results suggested that the fragments might enhance binding of the enzyme to its substrate. Indeed, when these fragments were tested in the enzymatic assay, one of them (compound 2) actually activated the enzyme, with an “AC₅₀” value of 3.5 mM.

The activator was further characterized by several orthogonal methods. NMR revealed that, in the presence of PUGNAc, compound 2 bound with a dissociation constant of 3.1 mM, very close to its AC₅₀ value. In the absence of PUGNAc, the dissociation constant was too weak to be determined. Isothermal titration calorimetry experiments revealed that compound 2 increased the affinity of the enzyme for PUGNAc by more than three-fold, while Michaelis-Menten kinetics revealed that, at a concentration of 8 mM, compound 2 nearly doubled the k_cat/K_M. A crystal structure of both compound 2 and PUGNAc bound to the enzyme revealed that the two molecules bind near one another in what appears to be a catalytically active conformation of the protein.

Next, the researchers took an “SAR by catalog” approach to find more potent molecules, leading to compound 4, with an AC₅₀ value roughly ten-fold better than compound 2. Detailed enzymatic characterization revealed that the molecule acts as a “nonessential reversible activator,” and improves substrate binding roughly 7-fold while improving k_cat by a factor of 1.7.

Interestingly, differential scanning fluorimetry (DSF) revealed that all the activators destabilize the enzyme, while PUGNAc stabilizes the enzyme. This is yet more evidence that compounds can decrease the melting temperature of a protein while still binding specifically.

This is an academic study in the best sense of the phrase. The immediate utility of these molecules is tenuous, as they do not work with human OGA (which has some important active site differences), though there may be industrial applications. The more important finding of this rigorous paper is that discovering enzyme activators might be easier than expected. There aren’t that many reported, but this may just be because people tend not to look for them: if you find an activator of an enzyme you are trying to shut down you probably won’t pursue it. That said, a few companies have been founded on enzyme activators. Perhaps fragments can help discover more.