20 July 2016

Fragments deliver a chemical probe for Family VIII bromodomains

Today’s post continues the theme of July as bromodomain month at Practical Fragments. The 61 human bromodomains (found in 46 proteins – some proteins have more than one) have been divided into eight families based on their sequences. Family VIII contains ten members, some of which are involved in keeping stem cells from differentiating. Two papers describe chemical probes that target some or most members of this family.

The first paper, which actually came out last year in Science Advances, is from a multinational group including Thomas Günther (Universität Freiburg), Stefan Knapp and Susanne Müller (both University of Oxford) and collaborators at Pfizer. The researchers started by screening libraries of acetyl lysine mimetics that had yielded inhibitors against other bromodomains. These came up empty; even promiscuous bromodomain inhibitors failed to hit Family VIII members. As is so often the case, when all else fails, the researchers turned to fragments. A thermal shift assay revealed that salicylic acid – the polypharmacological metabolite of aspirin – binds to the bromodomain PB1(5). Isothermal titration calorimetry (ITC) confirmed this result, providing a dissociation constant of 250 µM.

The researchers were also able to obtain a crystal structure of PB1(5) bound to salicylic acid in the acetyl lysine binding site common to all bromodomains, with the carbonyl making the usual hydrogen bond with a conserved asparagine. But whereas most other bromodomain binders make a water-mediated bridge to a conserved tyrosine, the phenol makes a direct hydrogen bond. The benzene ring also binds deeper in the pocket, displacing four highly conserved water molecules.

The subsequent medicinal chemistry optimization of this fragment is described in a paper published earlier this year in J. Med. Chem. by Dafydd Owen and colleagues at Pfizer, along with collaborators at the University of Oxford, DiscoveRx, Eurofins, the University of Massachusetts Worcester, and Johann Wolfgang Goethe University. Testing commercial and proprietary analogs of salicylic acid quickly revealed that uncharged enamides such as compound 2 were more effective at stabilizing PB1(5) against thermal denaturation than salicylic acid, and crystallography confirmed a similar binding mode.

Two rounds of library synthesis were conducted, first with 130 amines and then with 320 amines, with physicochemical properties of target compounds chosen in advance such that cLogP would range between 1 and 4. Seven family VIII bromodomains were screened in parallel, and compounds were identified with differing specificities. Some of the compounds were unstable in water, but introducing steric hindrance around the amine improved stability and led to compounds such as PFI-3. This is potent against the family VIII bromodomains PB1(5), SMARCA2A, and SMARCA4 and did not hit at least 40 other bromodomains tested. A related compound is active against more of the family VIII bromodomains while still maintaining good selectivity against other bromodomains.

Both of these probes are able to bind to family VIII bromodomains in cells and were used to explore the proteins’ biological roles. A variety of cellular phenotypic assays showed minimal changes, and the compounds do not appear to be toxic. They did attenuate myocyte or adipocyte differentiation, while PFI-3 caused embryonic stem cells to differentiate. One gets the impression that the researchers were hoping for more profound effects, but that’s why you make chemical probes in the first place. Whether or not these compounds will ultimately prove useful as drug leads, they should help to unravel some fiendishly complex biology.

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