Rise of the machines for fragment optimization

Our latest poll (please vote on the right-hand side of the page!) is about fragment libraries. Once you have your library, you can screen it using a variety of approaches. But what do you do once you get hits? Computational methods are increasingly being adopted; just this year we’ve discussed two approaches: growing via merging and AutoCouple. A new paper in J. Med. Chem. by Philippe Roche, Xavier Morelli, and collaborators at Aix-Marseille University and several other institutions describes a method that combines virtual screening with automated real-world synthesis in a platform called diversity-oriented target-focused synthesis (DOTS).

The process is best described with an example, and the test case presented is the first bromodomain of BRD4, BRD4(BD1). The researchers, who had previously identified a xanthine-containing series of inhibitors, pared this back to fragment-sized compound F1. Crystallography revealed a nearby pocket, which the researchers attempted to target with DOTS.

The researchers built a virtual library of 576 sulfonamides extending off the para position of the phenyl ring of compound F1. These were then virtually screened against BRD4(BD1) using the S4MPLE molecular modeling tool in which the F1 portion was constrained in the crystallographically observed conformation while the variable bits were allowed to move. The 100 top-scoring molecules were examined more closely, and 17 representatives were chosen to be synthesized on an automated robotic platform. This was actually a fairly modest set, as the Chemspeed system they used can run up to 96 parallel reactions. The crude products were then tested in a fluorescence assay, and all of them showed improved activities compared to the initial fragment. The majority, such as compound 17, showed high nanomolar inhibition.

The 13 submicromolar compounds were then resynthesized, purified, and validated in thermal shift and isothermal titration calorimetry (ITC) assays; these orthogonal methods confirmed their activities. The crystal structure of compound 17 bound to BRD4(BD1) was also solved, and this revealed that – as designed – the initial fragment retained its binding mode while the added portion makes new interactions with the protein.

The fact that 14 of the 17 molecules synthesized were at least an order of magnitude more potent than the initial fragment is satisfying, though it is worth noting that bromodomains are not the most difficult targets. Also, all of the new molecules have lower ligand efficiencies than the initial fragment. Still, advances and combinations of computational and robotic approaches will certainly increase the throughput of synthesis and testing, and I expect to see more of these examples.