Despite the fact that the second FDA-approved fragment-derived drug targets a protein-protein interaction (PPI), these types of targets have a well-earned reputation for being difficult. Most researchers try to disrupt PPIs. An alternative is to stabilize PPIs. This is not as crazy as it sounds: rapamycin, tafamidis, and PROTACs all stabilize PPIs. In a paper just published in J. Am. Chem. Soc., Michelle Arkin, Christian Ottmann, and collaborators at UCSF, Eindhoven University of Technology, Novartis, and the University of Duisburg-Essen bring fragments to bear on the problem.
The researchers were interested in the protein 14-3-3δ, a “hub” protein that binds to more than 300 other proteins (not all at the same time). One of these is estrogen receptor α (ERα): binding prevents the transcription factor from dimerizing and binding to DNA. The natural product fusicoccin A (FC-A) binds at the interface of 14-3-3δ and ERα and stabilizes that interaction, thereby inhibiting the growth of breast cancer cells. Because FC-A is a structurally complex natural product, the researchers sought fragments that would have a similar effect. They used Tethering, in which reversible disulfide bond formation stabilizes a protein-ligand complex, allowing its identification (see here and here). Specifically, fragments that bind near a cysteine residue are resistant to reduction, and the extent of binding can be detected by mass spectrometry.
The 14-3-3δ protein conveniently contains a cysteine residue in the vicinity of the ERα binding groove; the researchers used this native protein and also created two additional mutant proteins in which the native C38 cysteine was removed and new cysteine residues were introduced nearby. These three proteins were then screened against a library of 1600 disulfide-containing fragments under mildly reducing conditions in the presence or absence of a phosphopeptide derived from ERα. Most of the hits against the native protein were weak, but several hits against the N42C mutant were both resistant to reduction and also bound preferentially to the 14-3-3δ/ERα peptide complex compared to 14-3-3δ alone. Thus, ERα could enhance the binding of fragments to 14-3-3δ.
Next, the researchers used a fluorescently labeled peptide derived from ERα to show that one fragment could improve the apparent dissociation constant for the peptide and 14-3-3δ about 40-fold, from 1.3 µM 32 nM. Crystallography revealed that the cooperative fragments bound at the PPI interface, as expected given the location of the cysteine residues. The cooperative fragments placed a phenyl group in close proximity to a valine residue from the ERα peptide.
The researchers then examined the selectivity of one of their stabilizing fragments for other 14-3-3δ client proteins. In the case of a phosphopeptide derived from TASK3, which has a similar sequence to that of the ERα peptide, the fragment also showed cooperative binding. However, two peptides from other client proteins competed with the fragment for binding, and crystal structures revealed that the binding modes would be incompatible.
This is a nice illustration of site-directed fragment discovery to identify fragments that can modulate protein function in a more sophisticated manner than simple inhibition. One of the nice features of Tethering is that – like crystallography – it is able to identify extraordinarily weak binders. Unfortunately, this sometimes makes the hits challenging to advance: NMR experiments do show binding between a non-disulfide-containing derivative of one of the fragments and the 14-3-3δ/ERα peptide complex, but at high concentrations. It will be interesting to see whether this can be built into a potent non-covalent binder, and/or whether other types of covalent modifiers will be able to produce useful chemical probes for this target.