18 PI3K fragments

As we’ve noted before, kinases are a fertile field for fragment finding, but most of the targets have been protein kinases. Lipid kinases such as the phosphatidylinostide 3-kinases (PI3Ks), which mediate signal transduction by transferring a phosphate group to lipids, are also popular targets for a variety of diseases, but less has been disclosed about their suitability for fragment-based lead discovery. A paper in a recent issue of Bioorg. Med. Chem. Lett. remedies that.

Fabrizio Giordanetto and colleagues at AstraZeneca started with a homology model of p110beta (no crystal structure of this enzyme has been reported). They then used commercial software to dock 183,330 fragments selected from their corporate collection. All fragments that made at least two hydrogen bonds with the protein were organized into clusters of similar molecules and representatives of each cluster were visually inspected. This led to the selection of 210 fragments to be screened against the protein, of which 18 showed measurable activity. Structures of these fragments are provided in the paper; they range from kinase workhorses such as compound 1 to known PI3K motifs such as compound 10 to more unusual molecules such as compound 18. These hits were also tested on other members of the PI3K family, and while most showed activity across the board, others (such as compound 18) showed some selectivity.

There are some interesting structures in here; if I were starting a PI3K program I would definitely take a close look at them. Although the researchers have likely developed some of these into attractive leads, one of the virtues of fragments is that they are often so protean that different teams can start with the same fragment and end up in very different places.

Fragment linking in crystallo

Of the many ways to link fragments, one of the most intriguing is when the protein itself catalyzes or templates the assembly of two fragments (see for example here and here). The latest example of such target-directed fragment linking was published in last month’s issue of J. Appl. Cryst.

The researchers, led by Isao Tanaka at Hokkaido University, were interested in ligating fragments together in protein crystals. They first took crystals of the model protein trypsin and soaked these with an “anchor molecule,” in this case one of two benzamidine-containing aldehydes (benzamidines are classic trypsin binders). The crystals were then transferred to a second solution containing a “tuning molecule,” each of which contained either an aminooxy or hydrazine moiety that could react covalently with the aldehyde of the anchor molecule. Finally, the crystals were analyzed by X-ray and structures of any bound ligands solved.

A total of 33 different tuning molecules were examined, and two of these produced clear electron density in the active site showing that ligation with the anchor molecule had occurred (for example ALD2 and OXA9). Three others produced structures that suggested some disorder in the binding mode of the tuning molecule, and a fourth showed an assembled product that extended from the active site to a second trypsin molecule in the crystal lattice.

A study similar to this was published a number of years ago, but in that case it was not clear whether the ligation occurred in the crystal or in solution. In the present case, soaking pre-assembled molecules into the crystals produced inferior electron density to the two-step process. More excitingly, time-resolved experiments actually showed structural snapshots of the complex forming, both in the active site (which occurred in under a minute) as well as at the dimer interface (which took over an hour).

Unfortunately, the assembled products are not notably better binders than the initial fragment. The authors attribute this to the fact that their library of tuning molecules was very small. However, it is also possible that the approach selects not for the best binders but for those that can best form complexes within a fairly rigid crystal lattice. As we’ve seen before, protein crystals are far from physiological. It will be interesting to see whether in-crystal chemical ligation can generate superior binders.

Ligand efficiency in action

At an introductory talk I was giving recently on FBLD, someone asked how useful ligand efficiency (LE) really is. A paper published online in J. Med. Chem. by Daisuke Tanaka and colleagues at Dainippon Sumitomo Pharma illustrates how the metric can guide medicinal chemistry to superior molecules.

The enzyme soluble epoxide hydrolase (sEH) is a potential anti-inflammatory target. Inhibitors have been reported, but these tend to be quite lipophilic, so the researchers sought to find smaller, less hydrophobic inhibitors that bound with high-affinity to the target. A virtual screen against multiple ligand-bound crystal structures led to the selection of 735 diverse compounds which were tested in a biochemical assay, resulting in 68 compounds with IC50 values better than 1 micromolar. Most of these were relatively hydrophobic amides or ureas.

After removing known chemotypes and obviously unattractive molecules, the researchers were left with 42 compounds. They decided to eliminate compounds with MW > 380 or logP > 3.5, leaving 17 compounds; as might be expected, these smaller molecules turned out to be the most ligand-efficient of the bunch. Despite being one of the weakest hits identified, fragment-like compound 1 was the most ligand efficient and was chosen for lead optimization. A crystal structure of this compound bound to sHE guided parallel synthesis of 155 analogs, all with low molecular weights. Many of these compounds were very potent, and compound 11 not only showed a nice improvement in affinity but also demonstrated good ADME properties.

The authors conducted a retrospective analysis using some of the other ligand-efficiency-like indices such as %LE and fit-quality, which allow looser standards for affinity as molecule size increases. Interestingly, compound 1 was not an obvious starting point using these metrics. It is impossible to say what would have happened had these measures been prioritized over ligand efficiency, but the success of the simpler LE suggests that taking size into account would not have been useful, and could even have been misleading.

This paper is not a traditional fragment paper in which a low affinity lead is optimized; the initial hit was already quite potent. Rather, as the authors note, this is a nice example of “fragment-inspired medicinal chemistry, in which the essence and advantages of FBDD are faithfully respected.” It also provides another example of how focusing on ligand efficiency, rather than just potency, can lead to attractive chemotypes.