Regular readers will be aware that there are lots of ways to find fragments, but one approach we haven’t covered yet is substrate activity screening, or SAS. A new paper in J. Med. Chem. by Jon Ellman and coworkers at Yale uses this technique to find inhibitors of striatal-enriched protein tyrosine phosphatase (STEP), which is implicated in cognitive decline in a variety of diseases.
Many enzymes can accept a wide range of substrates, and these are often fragment-sized. The basic idea behind SAS is that, since substrates (by definition) bind to a target, finding new substrates gets you new binders, and for some target classes it is straightforward to transform substrates into inhibitors. Of course, you could screen for inhibitors from the start, but the nice thing about looking for substrates is that you are far less likely to encounter artifacts. This is because artifacts normally muck up assays; it’s harder to envision a spurious substrate.
Phosphatases clip phosphates from their substrates. Protein tyrosine phosphatases (PTPs), for example, dephosphorylate tyrosine residues in proteins; they essentially perform the opposite reaction of protein tyrosine kinases. Like kinases, though, finding selective inhibitors can be challenging. The researchers started by building a small library of 140 phosphorylated fragments (previously described here) and looking for those that were particularly good substrates. One of the best for STEP was substrate 8, which looks quite different from phosphotyrosine.
Replacing the substrate phosphate group with a bioisostere (difluoromethylphosphonic acid) that could not be hydrolyzed by the enzyme gave compound 12, which had an inhibition constant (Ki) similar to the Michaelis constant (Km) of substrate 8. Subsequent optimization led to compound 12s, with a low micromolar Ki and at least 18-fold selectivity against four other PTPs.
Unfortunately, the highly acidic phosphate bioisosteres in these molecules limit membrane permeability: although compound 12s inhibits STEP activity in rat neuronal cell cultures, it is not permeable in a model of the blood-brain barrier. Perhaps some of the less polar phosphate bioisosteres discovered in a previous virtual screen could help.
SAS is an interesting method, and I’m curious as to why more people aren’t using it. Of course, it does require generating bespoke libraries of fragment substrates, but once you have these they are useful for many members of a target class. What do you think?