Stabilizing protein-protein interactions: part 2

Stabilizing – rather than disrupting – protein-protein interactions is increasingly popular, particularly in the context of PROTACs and molecular glues. Last year we highlighted research in which covalent Tethering was used to identify fragments that could stabilize a protein-protein interaction, but only when the fragments were disulfide-bonded to one of the proteins. In a new (open access) J. Med. Chem. paper some of the same researchers, including Christian Ottmann and a large group of collaborators at Eindhoven University of Technology, University of Lille, AstraZeneca, and UCSF, have extended the approach to non-covalent fragments.

 

As before the researchers were interested in the adapter protein 14-3-3; the various isoforms of this “hub” protein each interact with hundreds of other proteins, many of which are implicated in disease. Natural products such as fusicoccin A stabilize interactions between 14-3-3 and some protein partners, suggesting ligandability, but the 13 chiral centers make analoging somewhat daunting. Two 14-3-3 partners implicated in oncology include the transcriptional coactivator TAZ and the tumor suppressor p53. Stabilizing the interactions of either of these proteins with 14-3-3 could have anti-cancer effects.

 

To find fragments that could stabilize these interactions, the researchers first grew crystals of 14-3-3σ in complex with peptides derived from either TAZ or p53. These crystals were then soaked in 100 pools of five fragments, each at 10 mM. Electron density was seen for several fragments; all fell into one of two scaffolds. Interestingly, both scaffolds contained an amidine moiety that forms a salt bridge with a glutamic acid side chain in 14-3-3σ.

 

One fragment, AZ-003, made interactions both with 14-3-3σ as well as with the TAZ-derived peptide. The peptide itself actually changed conformation so that a carbonyl oxygen could form a hydrogen-bond to the fragment, and three additional peptide residues could be resolved in the electron density that were not seen in the absence of fragment.

 

In the case of the other complex, none of the fragments interacted directly with the p53-derived peptide, though they bound nearby. Fragment growing and crystallography revealed that some of these larger molecules made water-mediated interactions to the peptide and were specific for p53 over TAZ, demonstrating that selectivity can be achieved. In the case of one molecule, AZ-008, crystallography was unsuccessful but modeling and protein-observed NMR experiments suggested direct interactions with the p53-derived peptide. Fluorescence polarization and SPR experiments revealed that 1 mM AZ-008 improved the affinity of 14-3-3σ for the peptide by an unambiguous but modest two-fold.

 

As with the previous paper, there is still much to do, and it will likely not be easy. The Supporting Information contains more than 30 crystal structures of small molecules bound to 14-3-3, both covalently and non, hinting at the effort required. However, the fact that both AstraZeneca and Novartis are working on 14-3-3 proteins – along with at least one startup – bodes well. Expect a part 3!