FBLD, of course, starts with small libraries of small fragments. DNA-encoded chemical libraries (DEL) usually start from the opposite extreme. Massive numbers of molecules are combinatorially synthesized attached to DNA, screened against a target using affinity selection, and hits identified by sequencing the DNA. A recent paper in J. Med. Chem. by Christopher Wellaway and colleagues at GlaxoSmithKline uses information from both approaches to generate a high-quality candidate.
The researchers were interested in bromodomain and extraterminal (BET) family proteins – the same targets we discussed last week. GlaxoSmithKline had already put molecules into the clinic, but they were looking for structurally different backup candidates, so they performed a DEL screen on the BD1 domain of BRD4. A library of 117 million compounds yielded potent compound 10, and crystallography revealed that the 2,6-dimethylphenol moiety bound in the acetyl-lysine-binding pocket.
Phenols are often metabolic liabilities, and indeed compound 10 was rapidly cleared in mice. However, GlaxoSmithKline has a long and successful history of fragment screening against bromodomains; Teddy first described some of their seminal work back in 2012, when the world didn’t end. Compound 16 had been found in a previous screen as a hit against BRD4, and crystallography revealed that the pyridone binds in a similar fashion to the phenol moiety. (Similar pyridones had been reported by others, for example this one.) Merging the molecules led – after a bit of tweaking – to compound 20a. In addition to BRD4, this molecule binds another bromodomain, BAZ2A, which the researchers wanted to avoid. Structure-based design led them to the more selective compound 20i.
Although compound 20i is potent in cells, it still has moderate clearance in rats. Unsubstituted benzimidazole rings have been reported to be unstable, so the researchers systematically explored a series of substitutions, ultimately arriving at compound 24 (I-BET469). Not only is this compound potent and soluble, it is remarkably stable, with “no detectable turnover in rat, dog, and human microsomal and hepatocyte preparations.” Oral bioavailabilities approach 100%, and the compound proved to be effective in acute and chronic mouse inflammation models. Although selectivity against non-BET family bromodomains members is good, compound 24 does strongly bind to both BD1 and BD2 domains of all four BET family members, and as we saw last week this may lead to toxicity.
Nonetheless, this is a lovely example of using a fragment to replace a problematic moiety in a larger molecule, as we’ve seen previously for chymase, Factor VIIa, and Factor XIa. Throughout the optimization the researchers paid close attention to molecular properties such as lipohilicity and molecular weight, and this resulted in a molecule with excellent pharmacokinetics despite the presence of potentially unstable moieties such as the morpholine. If nothing else, this will be a useful in vivo chemical probe.