Stabilizing protein-protein interactions: part 3 (fragment linking)

Stabilizing protein-protein interactions is becoming increasingly popular, and not just for PROTACs. Nearly three years ago we highlighted the use of crystallographic screening to find fragments that could stabilize interactions between the adapter protein 14-3-3δ and peptides derived from p53, a prominent cancer target. After noting how much work lay ahead, we ended the post with, “expect a part 3!” This has now been published (open access) in Angew. Chem. by Adam Renslo, Luc Brunsveld, Michelle Arkin, Christian Ottmann, and collaborators at UCSF and Eindhoven University of Technology.

 

In addition to crystallographic fragment screening, the researchers had previously performed a disulfide Tethering screen on the 14-3-3δ protein, which we described here. The fragments from the two screens bound next to one another, so the researchers decided to link them. They started by solving the crystal structure of compound 1 disulfide-bonded to 14-3-3δ in the presence of fragments from the crystallographic screen as well as a peptide derived from estrogen receptor alpha (ERα, another anti-cancer target). These co-structures guided the synthesis of new linked molecules, and these were soaked into crystals of 14-3-3δ and the ERα peptide. Compound 6 gave strong electron density and overlayed nicely on the initial fragments.

 

 

To determine whether the linked molecule could stabilize the 14-3-3δ/ERα complex, the researchers developed a fluorescence anisotropy assay with a dye-labeled peptide from ERα. Some of the linked molecules produced an increase in anisotropy, suggesting stabilization of the 14-3-3δ/ERα complex, but when the researchers ran the important control of repeating the experiment in the absence of 14-3-3δ they found that several molecules still increased anisotropy, which could be due to aggregation. (Adam published a nice early paper on aggregation and is thus particularly attuned to the dangers.)

 

Fortunately, some of the molecules passed this control, and with a robust crystallography system the researchers were able to use structure-based design to improve them, ultimately arriving at compound 24, which increased the affinity of the 14-3-3δ/ERα complex by 25-fold. It was also quite specific towards ERα, and did not increase the affinity of nine peptides from from other proteins for 14-3-3δ. The researchers attribute this selectivity to the fact that most other peptides would sterically clash with compound 24. (Not reported was the peptide from p53, which would be interesting.)

 

This is a nice paper on several levels. In addition to selectively stabilizing a therapeutically relevant protein-protein interaction, this is a rare example of starting with a covalent fragment and developing a non-covalent binder. (For another, see here.) Also, this is a good example of fragment linking, which is often challenging.

 

There is still a long way to go. The most potent molecules all contain amidine moieties, whose high polarity is a liability for cell permeability, let alone oral bioavailability. Moreover, the affinity of compound 24 is still quite weak, with a low ligand efficiency.

 

That said, with a wealth of structural and biological understanding I am optimistic further progress can be made, perhaps by rebuilding the covalent linkage to the protein, as was the case of sotorasib or this more recent paper from the UCSF team. I look forward to part 4!