Who's Regulating the Regulators?

In two recent ASAP papers in J. Med Chem. Chung et al. from GSK in Stevenage report on their recent efforts in discovering bromodomain inhibitors. Bromodomains are part of the "hot" target class largely lumped together in epigenetic targets. Bromodomains are the sole reader for Acetyl-lysine (AcK) and are thus important components of maintaining the "histone code". These domains are small (~110 aa) and have a common fold of four anti-parallel helices with the peptide recognition site in loops at one end of the helices. There are at least 56 bromodomains encoded in 42 proteins in the human genome.

The GSK group assembled a library that mimicked AcK: compounds had hydrogen bonding functionality and a small alkyl group. 1376 compounds were tested in a fluorescence anisotropy assay. Of these, 132 (~10%) showed >30% displacement of the fluorogenic ligand. After all actives were fully titrated, compounds were soaked into apo crystals. 40 structures were then analyzed by X-ray. The figure below shows how the native peptide is bound, analysis of the crystals showed that the H-bond interaction with the bridging water dominates that with N156, but both influence ligand positioning.
Below the structure of one bromodomain is shown, the yellow spheres are AcK sites. The other figure shows the preferred binding surface and low energy waters involved in AcK mimetic binding. These interacting residues tend to be conserved among bromodomains. The authors conclude the first paper by stating that their fragments are unlikely to discriminate between bromodomains. But, the real question is: is it possible to create compounds from these fragments which can?

And the answer is....[I did say there were two papers] maybe.

People have found compounds which work against bromodomains before (Cpds 1 and 2).
As can be seen they are relatively potent (nM) and decently ligand efficient. Cpd 3 and 4 are fragments found in the screen from GSK. Cpd 3 had ~30 % activity against two bromodomains. The authors admit it was far from the best fragment, but "it was small, efficient, novel, and chemically attractive." Who can argue with that.

Following up with this cpd, they found this compound binds in a manner consistent with its selection: one methyl of 3 mimics the terminal methyl of AcK, the second one overlaps the e-CH2 of AcK, and isoxazole N and O mimic the carbonyl. At 2A resolution, they couldn't differentiate which heteroatom was where, so they made 4 to prove their placement: it was.

As a start to the hit expansion, they used a 3D pharmacophore model to search for commerically avaailable analogs: analog by catalog. The found a series of phenyl-isoxazole with meta-sulfonamides on the phenyl ring. Compound 5a is ~100x more potent than the parent fragment and is very efficient (0.39). The xtal structure of 5a shows that it binds exactly as expected. SAR around this compound showed an increase in affinity, but with the expected loss of LE. This series also suffered from poor solubility. They could not add functionality to the sulfonamide (its pocket is rather hydrophobic), but instead where able to increase solubility through para-phenols (this points towards the solvent). They were able to increase solubility via this route, while keeping potency, but losing LE. This series also had cellular activity.
















This work shows that not all protein-protein interactions are flat and featureless. This work also shows that you can target PPI without having to change the rules: no need to relax the Lipinski Rules. These exciting new results show that the hot new targets in drug discovery play by the same rules as the same old targets.