SAR by Crystallography – minus the “A”

Practical Fragments has highlighted at least a couple papers (here and here) where fragments were identified by crystallography but where functional activity was not reported. Indeed, most experienced fragment hunters will be able to point to cases where they observed well-defined electron density but were unable to measure inhibition. Although this is often a source of frustration, researchers from Johnson and Johnson decided to avoid measuring activity altogether until after a couple cycles of crystallography-guided library design, as they describe in a paper published this month in J. Med. Chem.

The researchers were interested in the protein ketohexokinase, an ATP-dependent enzyme that is a potential target for metabolic diseases. They assembled a primary fragment screening library of 900 compounds with the following characteristics:

• 6-15 non-hydrogen atoms
• < 3 hydrogen bond acceptors
• < 3 hydrogen bond donors
• < 2 rings
• No unspecified chiral centers
• No unpleasant functionalities (peroxides, acyl halides, etc.)
• Prioritization given to molecules found in known pharmaceutical compounds (similar to the Fragments of Life)

This is a fairly standard set of criteria for assembling a fragment library. The researchers broke with convention by pooling fragments into groups of 5, with the members of a given fragment pool as structurally similar as possible. Normally when researchers pool libraries for crystallography-based fragment screening the idea is that each member of a given pool will be structurally unique to facilitate identification. In this case, though, the researchers were interested in observing general features of how fragments bind to the target protein.

Crystals of hexokinase were soaked with the pools, and 60 of these pools yielded electron density in the active site – a very high hit rate of 30%. A number of generalizations could be made about what molecular features were preferred in different parts of the binding site, and this information was used to build a secondary library of about 350 compounds based on 6 scaffolds; the idea was to merge or grow fragments found in the primary screen. These compounds were similarly pooled and screened crystallographically but not functionally, yielding hits from 4 of the scaffolds. The structures of these hits were then used to generate a third library of several hundred compounds around at least 4 separate scaffolds. These compounds were then tested individually to see if they could inhibit ketohexokinase, and were also characterized crystallographically. Gratifyingly, a number of these were active at submicromolar potency, including compound 6.

In addition to showing decent potency against ketohexokinase, compound 6 exhibited respectable drug-like properties as well, including lack of activity against the CYP450 enzymes, high oral bioavailability, and good selectivity against receptors, ion channels, and protein kinases, though other ribokinases were not tested.

This is an interesting and potentially useful approach, but it would be fun to see how it compares with a more “traditional” strategy, where individual fragments are advanced. It should be relatively straightforward to assay fragments from the first and second library for affinity; was fragment 3 (precursor to compound 6) one of the most potent or ligand efficient? Or was the information from fragments with no detectable activity instrumental in delivering the best compounds?