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.
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