19 June 2017

Fragments vs BRD4, two ways

Bromodomains, epigenetic targets that recognize acetylated lysine residues, have received considerable attention from the fragment community. (I devoted all of last July to the topic, and covered it more recently here.) Of the dozens of bromodomain-containing proteins, the four BET-family members have been highly studied, and the second bromodomain of BRD4 (BRD4-BRDII) in particular has been implicated in cancer and inflammation. In two new papers, researchers from AbbVie describe inhibitors of this target.

In the first Bioorg. Med. Chem. Lett. paper, George Sheppard and colleagues briefly describe a protein-detected (13C-HSQC) NMR screen of BRD4 in which the methyl groups of isoleucine, leucine, valine, and methionine were 13C-labeled. About 18,000 fragments were screened in pools of 30, and hits were then tested individually in NMR and time-resolved fluorescence resonance energy transfer (TR-FRET) assays. Despite extensive work on this target by multiple groups, these screens were able to identify several new fragments, such as the related compounds 1 and 2.


Crystallography of each fragment bound to BRD4 revealed that they bind in the acetyl lysine recognition site and make contacts with the conserved asparagine residue as well as a nearby water molecule. Merging the fragments led to compound 5, with a slight increase in affinity.

Comparison with other BRD4 inhibitors suggested a growth strategy, leading to compound 15, with nanomolar activity in the TR-FRET assay and two cell-based assays. The compound was orally bioavailable but had relatively high clearance, so further medicinal chemistry focused on changing the original core. This ultimately led to compound 38, with improved oral bioavailability, lower clearance, good selectivity against non-BET bromodomains, and activity in a mouse xenograft assay.

The second paper, by Le Wang, John Pratt, and colleagues in J. Med. Chem., starts with a different fragment from the original screen, compound A1. This molecule was even weaker than the fragments described above, but crystallography confirmed that it binds in the same acetyl lysine binding pocket.


Again, comparison with known inhibitors provided ideas for fragment growing, rapidly leading to compound A11. Further medicinal chemistry – which is extensively described in the paper – led to compound A30a, which bears considerable resemblance to the series reported in the previous paper.

Crystal structures of compounds bound to the protein suggested that it might be possible to make a macrocycle, which would in theory increase the affinity by locking the molecule in a low energy conformation. This proved to be synthetically challenging but ultimately worthwhile in the form of compound A74b. (Incidentally, this is the first case I can recall where a fragment led to a macrocycle. It won’t be the last.) Not only was this molecule more potent than the open form, it also showed excellent oral bioavailability and pharmacokinetics, good selectivity against non-BET bromodomains, and even better activity in a mouse xenograft model. 

One lesson from these papers is that fragments can generate new ideas even for heavily pursued targets. A second is that, as we saw in the recent poll, crystallographic information can be critical for advancing fragments to leads. The discovery of new moieties along with clear data on their binding modes can be a powerful combination for creative medicinal chemists.

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