From covalent to noncovalent 14-3-3 modulator, unintentionally

The seven members of the 14-3-3 family act as “hub proteins” that bind to other proteins, bringing them together or affecting their subcellular localization. Some of the client proteins, such as estrogen receptor alpha (ERα), are associated with diseases such as cancer, and stabilizing the interaction with 14-3-3 could thus be useful therapeutically. The idea is that a small “molecular glue” could bind at the interface between 14-3-3 and a client protein, enhancing the interaction. We’ve written here, here, and here about successful examples, both covalent and noncovalent. An open-access paper in ACS Med. Chem. Lett. earlier this year by Richard Doveston and colleagues at University of Leiscester starts with a similar strategy, but ends up somewhere else entirely.

 

The researchers had previously found that WR-1065, the active metabolite of the approved drug amifostine, can covalently bind to C38 of 14-3-3σ. (This is the same cysteine residue that had been targeted by Michelle Arkin and coworkers in 2019 using tethering.) Disulfide bond formation between WR-1065 and 14-3-3σ enhances the affinity of ERα for 14-3-3σ by a modest 2.8-fold.

 

In the new paper, the Doveston group made a small set of analogs in which the thiol was replaced by other warheads. Compound 7, containing an acrylamide, improved the affinity of ERα for 14-3-3σ by nearly two orders of magnitude, from 206 to 2.8 nM. But surprisingly, mass spectrometry revealed no covalent modification. Indeed, compound 7 was also active at low micromolar concentrations against the C38A mutant form of 14-3-3σ, while WR-1065 was inactive.

An examination of the structure-activity relationships (SAR) revealed that the acrylamide was essential; reducing the double bond abolished activity, while replacing the amide nitrogen with an oxygen made it significantly less active. Removing the primary amine or replacing it with a hydroxyl or carboxylic acid was also not tolerated.

 

If compound 7 does not bind covalently to C38, how does it work? Surprisingly, the molecule does not even bind at the interface between 14-3-3σ and ERα. Indeed, it shows additivity with a well-characterized molecular glue (fusicoccin A) known to bind at the interface. Where exactly compound 7 does bind is uncertain. A series of biophysical experiments suggests that it may stabilize the dimeric form of 14-3-3σ, though the mechanism remains to be determined.

 

This paper is a useful reminder that, as the authors conclude, “even rational design approaches can lead to unexpected outcomes.” Sometimes a warhead does not behave as one.