Fragments vs BRPFs: A chemical probe

Bromodomains, a type of functional module within proteins, recognize acetylated lysine residues in other proteins to act as epigenetic “readers.” Humans have 61 of them spread across 46 different proteins, and figuring out what recognizes what and in which context has been a major undertaking for the past several years. Fragment-based approaches have proven very successful: Practical Fragments devoted the entire month of last July to bromodomains. In a new paper in J. Med. Chem., a large group of academic and industry investigators led by Paul Fish describe their successful efforts to find a chemical probe for the four members of the BRPF family.

The story actually starts with a fragment screen against a different bromodomain, PCAF, which we discussed last year. Compound 5b was a hit against that target, but bromodomain fanciers will recognize this as a privileged pharmacophore against the target class, and it turned out to be even more potent against BRPF1.

Crystallography confirmed that compound 5b bound in the acetyl-lysine pocket as expected, and also revealed a potential vector for growing, as exemplified by compound 6. Introducing a methyl group led to a 10-fold boost in affinity for compound 7, which as the researchers point out is near the limit of what you can expect for hydrophobic interactions. Further growing ultimately led to NI-42, with low nanomolar potency.

Of course, potency is one thing, but when you’re dealing with dozens of related proteins you really need specificity to understand the biology. Happily, NI-42 was quite selective for members of the BRPF family. Importantly, it has only 4.5 µM activity against BRD4, which can dominate cell phenoytpes when inhibited. The selectivity is actually remarkable given the close structural similarity of NI-42 to PFI-1, which hits BRD4 and which Teddy wrote about here. The researchers suggest that the difference between the N-H in PFI-1 and the N-methyl in NI-42 drives the selectivity – so one could argue that the probe actually makes use of two magic methyls.

NI-42 also showed good cell permeability and target engagement in cells, adequate solubility, decent pharmacokinetics, and respectable oral bioavailability in mice. Although a screen of 211 cancer cell lines did not reveal stunning activity, cell models for other diseases are being evaluated. Also, the researchers have generated an inactive but closely related control simply by replacing both methyl groups with ethyl groups.

This is a lovely fragment optimization story. It is also a useful reminder that, as with phosphodiesterases and kinases, a nonselective fragment can ultimately yield a selective chemical probe.