FragLites and PepLites meet bromodomains

The last two Practical Fragments posts focused on bromodomains, epigenetic readers that recognize acetylated lysine residues. Today’s post could thus be considered part of a trilogy, though the focus is less on bromodomains themselves than a specific type of fragment library.

 

In 2019 we highlighted FragLites, small fragments containing pairs of hydrogen bond acceptors and/or donors along with a bromine or iodine atom. FragLites were designed to assess ligandability as well as identify what types of interactions would be favorable at various sites. The original test protein was the kinase CDK2. In an open-access paper published late last year in J. Med. Chem. by Martin Noble, Michael Waring, and colleagues at Newcastle University, FragLites are screened against two members of the bromodomain family.

 

The first bromodomain (BD1) of BRD4 is considered highly ligandable, with multiple inhibitors disclosed (see for example here). In contrast, ATAD2, a bromodomain in another subfamily, is more challenging, in part because it lacks a hydrophobic region useful for increasing affinity for small molecules. Thirty-three FragLites were individually soaked at 50 mM into crystals of either bromodomain. The halogen atom on each FragLite facilitates analysis by anomalous dispersion, allowing more sensitive detection of low-occupancy binders. This, along with Pan-Dataset Density Analysis (PanDDA), was used to identify specific protein-ligand “binding events.”

 

In total, 26 binding events at five sites were identified for BRD4; four ligands bound at more than one site. Of these, 17 FragLites bound at the orthosteric site of BRD4 (which recognizes N-acetyl lysine). In contrast, ATAD2 displayed 16 binding events total over seven sites; only three bound at the orthosteric site, consistent with its lower ligandability. ATAD2 had previously been screened crystallographically against the 776-membered DSI-poised fragment library, and this effort also identified seven ligand-binding sites, six of which were common to those discovered here, suggesting that the small FragLite set is able to identify most pockets.

 

As far as specific types of interactions, the average FragLite made 1.1 hydrogen bond, suggesting that the second donor or acceptor is often not engaged. In contrast, the bromine or iodine atom makes protein contacts in 33 of 42 binding events. In half a dozen cases no hydrogen bond to the protein was observed, with the primary interaction being a halogen bond.

 

The FragLites are small, relatively “flat” aromatic molecules, but of course most proteins interact with other proteins. To try to explore such interactions, the researchers developed a library of “PepLites:” N-terminally acetylated amino acid residues with a C-terminal bromopropargyl group. These were also screened crystallographically against the two bromodomains and produced considerably lower hit rates, with six bound to BRD4 (all at the orthosteric site) and nine bound to ATAD2 (of which five bound to the orthosteric site). Reassuringly, the N-acetylated lysine PepLite bound to both proteins in a similar manner as seen in larger peptides.

 

The researchers conclude that FragLites and PepLites “represent highly valuable components of a larger crystallographic screen, and we anticipate that this is where they will fit into most drug discovery programs.” Indeed, this is already happening; last year we wrote about how FragLites were screened against the bromodomain PHIP2 as part of a larger screen, and I was surprised this paper was not mentioned here. Laudably, all the atomic coordinates have been deposited in the Protein Data Bank, so folks are able to do their own analyses.

 

As FragLites and PepLites are screened against ever more targets, it will be fun to see what they can teach us about intermolecular interactions and starting points for new leads.