Merging two of the same fragments for FABP4

The fatty acid binding proteins (FABPs) are a family of 10 proteins that – as their name suggests – shuttle fatty acids around cells. FABP4 has been implicated in a host of diseases, from atherosclerosis to nonalcoholic steatohepatitis. A recent paper in J. Med. Chem. by Yechun Xu and collaborators mostly at Shanghai Institute of Materia Medica describes how a fragment led to a compound with in vivo efficacy. It is a lesson in both recognizing and capitalizing on the fact that fragments often have multiple binding modes.

The researchers screened just 500 fragments, each at 1 mM, looking for displacement of a fluorescent ligand. Two hits were identified, of which compound 1 was by far the most potent. The researchers characterized the binding mode using crystallography, which itself was challenging because the protein co-purified with bound fatty acids. They had to denature the protein, strip fatty acids, and then refold it to obtain the apo form. When they were finally able to determine the crystal structure, they were surprised to find that compound 1 adopted three different binding modes under two different conditions (pH 6.5 and 7.5). These experimental results were supported by molecular dynamics calculations.

It is not uncommon for fragments to assume different binding modes. Indeed, the 7-azaindole fragment that led to vemurafenib, pexidartinib, and other clinical compounds has been found to bind in multiple orientations. In this case, the researchers recognized that the three binding modes put the two phenyl rings in three positions, suggesting that grafting a third phenyl ring onto compound 1 could improve affinity. This proved successful, and the resulting compound 3 had an affinity more than two orders of magnitude better as assessed both in the displacement assay and by isothermal titration calorimetry. Crystallography revealed that the molecule bound as expected.

Further structure-based design ultimately led to compound 17, with low nanomolar affinity. This molecule is also active in a cellular assay and has surprisingly good pharmacokinetic properties in mice. Given these encouraging results, the researchers tested whether the molecule could protect mice from multiorgan damage promoted by inflammatory lipopolysaccharides. The results were positive.

Unfortunately, compound 17 does show low micromolar activity against FABP3, whose inhibition would likely cause cardiac toxicity. Still, this is a nice example of fragment “self-merging”. Although merging two different fragments is common, merging a fragment onto itself is relatively rare, and – as shown here – not necessarily easy. It is an approach worth keeping in mind the next time you encounter a fragment with multiple binding orientations.