The researchers were interested in CYP121, which is unique to Mtb and important for its viability. They used a thermal-shift assay to screen 665 commercial fragments at 5 mM, of which 66 increased the melting point by at least 0.8 ˚C. 56 of these were further characterized by STD and WaterLOGSY NMR, and 26 showed interactions with the protein and could also be competed with a known substrate. Eight of the most soluble of these were then soaked into crystals of CYP121, leading to four high-resolution structures, three of which are shown here. Isothermal titration calorimetry was used to determine dissociation constants.
Interestingly, two of these fragments (1 and 2 in blue and red, respectively) coordinate to the heme iron, while the other two do not. Fragment 4 (green in figure above) showed two binding modes in the crystal structure, leading the researchers to make molecules that merge both binding modes. Although the resulting molecules bind in a similar fashion as compound 4 as judged crystallographically, they show at best marginal improvements in affinity and sizable losses in ligand efficiency. Quantum mechanical calculations suggested that this lack of improvement was due to conformational strain within the molecules.
Happily, merging compounds 1 and 2 was much more successful, leading to compound 14, which maintained ligand efficiency and improved affinity. A crystal structure revealed that, as designed, compound 14 binds in a very similar manner as the initial fragments. The molecule was also selective for CYP121 over a different CYP from Mtb as well as several human CYPs.
This is a nice paper not only because it reports a successful example of fragment merging on a new class of targets, but because it also describes several approaches that didn’t work. Fragment merging and fragment linking probably fail more often than they succeed, and this report really digs into the SAR and addresses why merging can be so challenging.
Of course, what would be really cool would be to link compound 14 with compound 4 (ie, link all of the fragments in the top figure), and the paper ends with the statement that this is currently ongoing. It will be fun to see the results.