25 July 2016

Multiple bromodomains, multiple methods, and even more fragment hits

All this month Practical Fragments has been focused on bromodomains, highlighting chemical probes against BRD9, CBP and EP300, and family VIII bromodomains. Today’s post covers three earlier-stage programs on three different bromodomains.

In Acta Pharm. Sinica, Bing Xiong, Nai-xia Zhang, and colleagues at the Chinese Academy of Sciences discuss their work on BRD4, an anti-cancer target about which we’ve written previously. The researchers describe the construction of a fragment library designed for NMR screening; this is a good resource for people undertaking similar efforts. Interestingly, of 800 compounds purchased, only 539 were soluble to at least 100 µM in aqueous buffer. These were pooled into 56 groups of 8-10 compounds and screened at 200 µM (total fragments) using STD and T1ρ. This yielded 10 hits, of which three had measurable IC50 values from 110 to 440 µM. Five of the hits were characterized in more detail using two dimensional NMR (1H-15N HSQC), and three by X-ray crystallography. Some of these fragments are less-precedented as bromodomain ligands, and could be useful starting points for further work.

In contrast to BRD4, for which multiple ligands have been reported, the bromodomain on BRPF1 is less explored. In a recent paper in J. Med. Chem., Jian Zhu and Amedeo Caflisch (University of Zürich) provide 20 new co-crystal structures, all of which have been deposited in the protein data bank. The researchers performed a computational screen of 24,133 molecules using a program called SEED, which was able to crank through the entire set in just a day. Crystal soaking was attempted with thirteen of the top 30 hits, resulting in five structures, of which three bound in the manner predicted. Crystal structures of another 15 analogs and other bromodomain inhibitors were also determined. Some of the molecules are reasonably potent, with double-digit micromolar affinities and good ligand efficiencies.

Finally, while most bromodomains have a conserved asparagine residue that makes hydrogen bonds to the substrate (or inhibitor), 13 of the 61 known human bromodomains do not, and these tend to be more difficult targets. The second bromodomain of the pleckstrin homology domain-interacting protein (PHIP(2)), which has been implicated in melanoma, is one of these “atypical” bromodomains. Researchers at the Structural Genomics Consortium (SGC) led by Frank von Delft (Diamond Light Source) and Paul Brennan (University of Oxford) took a crystallography-first approach toward this target, as they report in an open-access paper in Chemical Science.

The researchers started by assembling what they call a “poised fragment library”. This is essentially a library designed for rapid follow-up chemistry, in which each library member can be deconstructed into individual components, which can be systematically varied. For example, a fragment might consist of two moieties connected by an amide bond, so that analogs could be easily made using parallel synthesis. The initial 2347 fragments were a subset of the 11,677 fragments available in-house or through collaborators, but the researchers also identify a set of 10,448 commercially available poised fragments. Commendably, they also provide full identities of both sets of fragments, which could be useful for folks building or adding to their own collections.

The Diamond Light Source is able to crystallographically screen 1000 fragments per week, but in this case only 406 diverse fragments were tested. Rather than using the nearly universal DMSO as a solvent, the researchers dissolved their fragments in ethylene glycol, since DMSO actually binds to bromodomains. Previous solution-phase screens of PHIP(2) at the SGC had come up empty, so the crystallographic screen was done at the very high concentration of 200 mM. Not surprisingly, this yielded just four hits.

Each of the hits bound in the acetyl-lysine recognition pocket, and three of them even showed high-micromolar activity in an AlphaScreen assay, with impressive ligand efficiency values. A few dozen analogs were made, which led to slight increases in activity in all cases, and measurable activity for analogs of the fragment which had shown no activity by itself. Although there is still a long way to go to find chemical probes for PHIP(2), at least there are now good starting points.

And that concludes bromodomain month. The number of papers and chemical probes that have come out just this year are a testament to the power of fragments to tackle this class of targets, perhaps equaled only by kinases. And while I'm not aware of any clinical candidates targeting bromodomains that started as fragments, I'm sure these will be coming soon.

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