Fragment merging revisited: CYP121

Last year we highlighted a paper from Chris Abell and colleagues at the University of Cambridge in which they applied FBLD to CYP121, a potential anti-tuberculosis target. Several fragments with different binding modes were identified, and while some could be successfully merged to produce higher affinity binders, others couldn’t. In a new paper in ChemMedChem, the researchers take a closer look at why some of their initial attempts at fragment merging failed, and figure out how to succeed.

In the original paper, fragment 1 was particularly interesting for two reasons. First, crystallography revealed that it did not make direct interactions with the enzyme’s heme iron, as do most inhibitors of CYPs, suggesting that higher specificity might be achievable. Moreover, the co-crystal structure revealed that fragment 1 could bind in two nearly overlapping orientations, practically begging to be merged. Unfortunately, the resulting merged compound 4 actually bound worse than the initial fragment.

Computational modeling suggested that a primary reason for this disappointing result is the steric clash between two hydrogen atoms on the two phenyl rings of compound 4. These are forced into an unfavorable configuration when the molecule binds the protein. To fix this, the researchers sought to introduce a new interaction with the protein that would allow the molecule to relax into a lower energy conformation, alleviating the steric clash. This led to compound 5, with a satisfying increase in affinity. But lest folks become too cocky, an attempt to pick up an additional hydrogen bond to the protein (compound 8) actually led to a decrease in affinity despite the presence of the designed hydrogen bond, as assessed by crystallography. More successfully, building into a cavity led to the most potent compound 9. (Geeky aside: the aminopyrazole versions of fragment 1 had similar affinities as fragment 1, suggesting that the aminopyrazole moiety per se only gives a boost in potency in the context of the merged molecule.)

High-resolution co-crystal structures were solved for several of the molecules; the figure below uses color-coded carbons to show the overlay of the two different binding modes of fragment 1 (green), compound 4 (cyan) and compound 5 (magenta). What’s striking is how closely all the molecules superimpose, despite their very different affinities.

This is a nice case study in fragment merging that emphasizes just how difficult the strategy can be, even when it looks like it should be easy. And while Practical Fragments has not always looked kindly on computational methods, this is a beautiful example of how modeling can be used to understand why things that look good on paper don’t work, as well as how to fix them.