Rad fragments

One of the selling points of FBLD is that it can find starting points against challenging targets such as protein-protein interactions. A major reason these targets are so tricky is that they often have large, flat interfaces with few pockets for small molecules to bind. An example is the interaction between the tumor suppressor BRCA2 and the recombinase RAD51, which is mediated in part by the phenyl ring of a phenylalanine residue – a very small moiety even by the standards of fragments. In a paper published recently in ChemBioChem, Marko Hyvönen and colleagues at the University of Cambridge describe how they’ve found fragments that bind to this site.

The researchers started with a microbial RAD51 homolog that had been humanized by mutagenesis; the human protein itself is unstable and difficult to work with. They performed a thermal screen with 1249 fragments. Thermal denaturation has been criticized for producing noisy data, and indeed, 96 fragments produced complex, uninterpretable results. However, the two best fragments both contained an indole core and were confirmed to bind by STD-NMR.

Competition experiments confirmed that these two fragments competed with a short peptide containing the critical phenylalanine, indicating that they bound at the desired spot. ITC revealed that they had dissociation constants around 2 mM. Their binding modes were also confirmed crystallographically.

The researchers then used one of these fragments as a probe in a round of STD-NMR experiments, in which they examined 42 fragments to see whether any of these could compete away the first fragment. This led to two new hits, both slightly more potent than the initial ones.

One of these new fragments was then used as a probe in another round of STD-NMR experiments with 120 fragments chosen as analogs or by in-silico screening. This led to four additional fragments, some of which had sub-millimolar affinities and good ligand efficiencies. All 6 of the new fragments from the two STD-NMR screens were characterized crystallographically and found to bind at the same site as the original indole fragments, though with some subtle differences that could be exploited for further elaboration.

This is a nice, thorough example of fragment discovery in academia. As the authors conclude:

Investment in a platform of orthogonal biophysical assays and screens is crucial for progression into a programme of medicinal chemistry. The elaboration of poorly validated hits not only has a high likelihood of failure, but without a variety of robust assays in place, the risk of being misled by badly behaving compounds increases.

Of course, these are still relatively weak fragments, but I’ve heard one of the authors speak at a conference in which he stated that they’ve been able to advance these to nanomolar leads with cell activity. Stay tuned!