Linking lessons on EGFR

From the earliest days of FBLD, fragment linking has been an aspirational goal. Snapping together two low affinity binders to get an additive or even superadditive boost in affinity is conceptually appealing but, as our poll showed, not always easy. In a new open-access J. Med. Chem. paper, Hung Nguyen, David Heppner, and collaborators at The State University of New York at Buffalo and elsewhere propose a general workflow.

 

Epidermal growth factor receptor (EGFR) is a prominent cancer target with multiple approved drugs. It has been studied for decades and thus makes an excellent model system; we wrote about some of Heppner’s work on this protein a couple years ago. As with most kinases, many known ligands bind in the so-called hinge-binding region where the adenine of ATP normally sits. But some ligands have been identified binding to an adjacent pocket, and in a 2024 open-access Comm. Chem. paper the Heppner group reported linking ligands at these two sites.

 

The new paper systematically expands on this work, diving deep into the SAR of compounds binding to the oncogenic L858R/T279M EGFR mutant. Specifically, the researchers vary connecting points as well as the length and composition of the linker.

 

 

As expected, the point of connection can make the difference between improved activity and dead compounds. Linker length matters too, as shown for compounds 4, 5, and 6, where the “Goldilocks” two-carbon linker (compound 5) binds orders of magnitude more tightly than the one- or three-carbon linker. Remarkably, compounds 4 and 6 are less active than one of the initial fragments! Linker composition is also important, but perhaps less than one might expect: the amide-containing linker in compound 8 is only marginally more potent than compounds 9 and 10.

 

The researchers characterize several of their linked molecules both crystallographically and using molecular dynamics. They argue that “maximal flexibility is likely the most important design criterion for linking molecules that occupy adjacent pockets.” That is consistent with this post from 2012 but doesn’t quite fit with the fact that the amide-containing compound 8 is better than either of the (presumably more flexible) compounds 9 and 10.

 

The paper is actually more of a fragment-merging than a fragment linking story: the central phenyl ring is common to both starting compounds, so it is not really possible to assess the extent of additivity. Also, activity is based on inhibition rather than thermodynamic measurements, so it is impossible to say whether the improved affinities of the linked molecules are due to entropic, enthalpic, or some combination of factors.

 

Still, this is a nice contribution to the literature of fragment merging/linking, and the general guidelines are sound: assess multiple connection points and linker lengths before refining the linker itself.