Protein flexibility can be both an opportunity and a barrier – quite literally, when a solid wall of protein seems to block opportunities for fragment growing. But like secret doorways, protein domains can yawn open to expose tunnels and cavities. An example of this was published earlier this year in Bioorg. Med. Chem. Lett. by Stuart Francis and collaborators at the CRUK Beatson Institute.
The researchers were interested in fascin 1, which increases the invasiveness of multiple cancers by helping pack filamentous actin into bundles important for cell migration. The team began their search for an inhibitor by performing a surface plasmon resonance (SPR) screen of 1050 fragments, generating an impressive 53 hits. Although a number of these were reported to bind to multiple sites on the protein, only one is discussed.
Compound 1 binds between two domains of the protein in a pocket that does not exist in unbound fascin. However, the fact that the pocket completely envelopes the fragment “hampered attempts to develop the series.” Fortunately, the researchers were following the patent literature, and when they characterized compound 2 (not a fragment, and reported by a different group), they discovered that while it binds in the same pocket as compound 1, additional conformational changes occur to accommodate the larger molecule.
Next, the researchers looked for analogs of compound 2 and also performed a virtual screen against the enlarged pocket. Of 110 commercial compounds tested, three gave dissociation constants better than 100 µM, including compound 3. The researchers recognized that compound 3 lacks the halogens found on both the original fragment and compound 2, and by adding these they were able to improve the affinity more than ten-fold. Further optimization ultimately led to BDP-13176, with mid-nanomolar affinity by SPR and ITC as well as activity in a functional assay. Although the molecule has reasonable solubility and stability against liver microsomes, it has low permeability and high efflux.
This is a nice structure-based design story, and while the fragment did provide some information about the binding site, one could argue that the real breakthrough came with determining the binding mode of compound 2. Indeed, without this information, it would have been all too easy to assume that the pocket was not ligandable. This is an important reminder that crystal structures usually only reveal one form of a protein. The system is also a good test case for modelers who want to see how their algorithms perform against a dynamic protein. Breakthroughs are often unexpected, and it is always worth making a few compounds that don’t look like they’ll fit.