A fragment library designed for merging: application to PKCζ

Advancing fragments in the absence of structural information has a reputation for being so challenging that some people do not even attempt it. Modeling can help, but what if you could improve your odds by designing your library strategically? This approach has been demonstrated in a recent J. Med. Chem. paper by Masakazu Atobe and colleagues at Asahi Kasei Pharma.

To facilitate fragment merging, the researchers synthesized a library of 5000 substituted isoquinoline fragments. As illustrated by the drug fasudil, isoquinoline is a privileged pharmacophore for binding to the hinge region of kinases. Importantly, isoquinoline has 7 different positions from which to grow: screening monosubstituted versions would potentially allow rapid merging of hits. This approach is conceptually similar to that used to discover vemurafenib.

The target of interest was protein kinase C ζ (PKCζ – that’s a zeta, by the way), one of the 11 members of the PKC family that has been implicated in diseases ranging from diabetes to cancer. Previously reported inhibitors are insufficiently potent or selective, in part because no crystal structure of the kinase has been reported. The researchers were interested in developing a chemical probe to better understand the biology.

A biochemical screen of the 5000-member isoquinoline library at 100 µM yielded just a dozen hits, with IC₅₀values ranging from low to mid-micromolar. Importantly, substituents were found at four different positions, thus facilitating fragment merging. The researchers first merged fragment 6 with fragment 8, resulting in mid nanomolar inhibitor 10. Further optimization yielded compound 21, which is highly selective for PKCζ in a panel of 216 kinases and also has good pharmacokinetic properties in mice. However, cell potency is relatively modest.

Next, the researchers merged fragment 7 with fragment 9 to generate sub-nanomolar compound 26. This molecule also inhibited protein kinase A, but further optimization led to compound 37, which showed excellent selectivity in a panel of 381 kinases as well as good mouse pharmacokinetic properties and mid-nanomolar activity in a cellular assay. Encouragingly, the compound also showed good activity in a collagen-induced arthritis mouse model. The aniline – which adds about ten-fold to the affinity – may ultimately need to be removed, but clearly this molecule is well-suited for further optimization. 

This paper provides two lovely examples of fragment merging by design, but how general is the approach? One of the key advantages of fragment-based screening is the ability to survey huge swaths of chemical space. Building an entire library around a single fragment obviously constricts this. The fact that the hit rate (0.24%) was so low perhaps illustrates this point; it would be interesting to know how ligandable PKCζ is, or whether a library built around a different privileged pharmacophore would yield a higher hit rate. Lower expected hit rates necessitate larger libraries; 5000 fragments is already more than average according to our poll. And of course, if you are going to build a library of thousands of similar fragments, you had better be certain you choose one that has good pharmaceutical properties, further limiting your choices. Despite all these cavaeats, clearly the investment paid off for PKCζ. It will be fun to see what else comes out of this effort.