Last year Practical Fragments highlighted substrate activity screening (SAS) as a means for identifying enzyme inhibitors. The idea is to make libraries consisting of potential substrates, modified to reveal interactions: for example, amides that release a fluorescent reporter group when cleaved by a protease. These libraries are then screened against a protein of interest, and any new substrates identified can be transformed into inhibitors by replacing the cleavable bond with some sort of warhead. At the end of that post, we asked why more people weren’t using SAS. In a new paper in ChemBioChem, Pieter Van der Veken and colleagues at the
have partially answered that question,
and provided a solution. University of Antwerp
The researchers were interested in the oncology target urokinase (uPA), a trypsin-like protease. They built potential substrates from an amino-methylcoumarin designed to fluoresce when cleaved. They used this to assemble a library of 137 molecules, each consisting of the amino-methylcoumarin coupled to a variable fragment. Of these, about 50 contained positively charged moieties likely to interact with the large S1 pocket of uPA, which has a predilection for cationic species. (The rest were diverse molecules.) The library was then screened against the enzyme to look for substrates, but the researchers ran into several difficulties.
First, since all the substrates are poor, the researchers had to use quite a bit of enzyme (about 2.5 micrograms per well) to get a good signal. Second, for the same reason, they had to run the assay for a long time (6 hours). Third, and somewhat unexpectedly, it turns out that SAS is susceptible to an interesting artifact: low levels of contaminating enzymes can cleave substrates, giving false positives. Indeed, the researchers found that commercial uPA isolated from human urine produced a number of hits that did not repeat with recombinant (and presumably purer) enzyme and could not be competed by addition of a potent uPA inhibitor.
Despite these challenges, the researchers identified 11 hits. However, notably absent were some of the fragments known to have affinity for the S1 pocket, such as several guanidines. This is not surprising: for a molecule to be processed as a substrate it needs to be able to fit in the S1 pocket as well as to position the cleavable bond near the catalytic machinery – subtle changes in geometry will prevent processing. This got the researchers thinking about alternative ways to use their library.
The approach they came up with, “modified substrate activity screening” (MSAS), starts by first looking for inhibitors rather than substrates, since a poor substrate can behave as an inhibitor. The idea is to incubate library members with the enzyme and a single potent substrate. This allowed the researchers to reduce the enzyme concentration by 10-fold and run a much shorter assay (10 minutes). It also reduces the risk that contaminating enzymes will be responsible for activity, though of course inhibition assays are susceptible to all sorts of other artifacts.
When the researchers applied MSAS to uPA, not only did they rediscover the 11 hits they had identified as substrates previously, they also identified 17 additional molecules, including all the guanidine-containing fragments.
The researchers propose a flowchart for MSAS in which compounds are first screened for inhibition. These hits are then followed up using SAS to determine whether some of these inhibitors are substrates too. Any substrates thus identified can be readily transformed into inhibitors by adding an appropriate warhead. Inhibitors identified in the first step that aren’t substrates can also be useful to provide structure-activity relationships and new fragments to take forward.
Of course, one could argue that if you are doing inhibition assays, there is no point in going to all the trouble of making custom libraries for MSAS. That said, if you’ve already got the substrate-based libraries, doing an initial inhibition screen is probably a good idea.