Fragments in the Clinic: AT13387

We recently discussed BACE, a target that has been tackled by FBDD due to its intractability to other methods. The subject of this post is quite the opposite: the anticancer target Hsp90 has proven very amenable to a variety of approaches, including fragment methods (see here and here); close to a dozen compounds targeting Hsp90 are in the clinic. Now Astex has detailed their work in this area with two back-to-back papers in a recent issue of J. Med. Chem. describing the discovery of AT13387.

The first paper, by Christopher Murray and colleagues, actually presents the discovery of two separate series of inhibitors. The researchers started with a library of about 1600 fragments and used NMR techniques (water LOGSY) to identify hits against Hsp90. Competition with ADP allowed them to identify molecules that bind to the nucleotide binding site. In all, 125 fragments were taken into crystallography, using both co-crystallography and soaking, resulting in 26 co-crystal structures. Four of these structures are described in some detail, with two leading to potent inhibitors. Throughout the process, isothermal titration calorimetry was used to measure dissociation constants.

In the first series, compound 1 was identified as a weak hit (see Figure 1). Virtual screening led to the purchase of a few variants, including compound 5, with roughly 100-fold improved affinity. Interestingly, the crystal structure of compound 1 bound to Hsp90 showed that the molecule was twisted around the bond connecting the two aromatic rings, despite this not being energetically optimal for the unbound molecule. By substituting the phenyl ring of compound 5 to stabilize this twisted conformation the researchers were able to improve the potency another 20-fold (compound 9), along with a boost in ligand efficiency. Further structural work suggested adding another chlorine to fill a lipophilic site as well as adding a solubilizing group, ultimately leading to compound 14, with low nanomolar binding affinity and low micromolar cell activity.

Figure 1

In the second series, compound 3 (which is actually itself a drug, ethamivan) had only modest ligand efficiency, but crystallography suggested that replacing the methoxy group with something slightly larger and more lipophilic would improve the interactions, a hypothesis borne out by the increased activity of compound 17 (see Figure 2). Increasing the lipophilicity of the amide side chain to take advantage of protein flexibility led to a further two orders of magnitude increase in potency (compound 28). Finally, the researchers were able to use the known binding mode of a natural product to add an additional hydroxyl group, leading to compound 31, with sub-nanomolar affinity (more than a million-fold more potent than the initial fragment!) and mid-nanomolar cell activity.

Figure 2

An impressive feature of both these examples is that, through the use of elegant medicinal chemistry, the researchers were able to improve ligand efficiency throughout the course of affinity improvement. Of course, it helps that they were working on a crystallographically friendly target for which several other groups had published extensive SAR, but these are nonetheless beautiful case studies. As the researchers point out, “in terms of the efficiency of the added groups, the two fragment to lead campaigns… are among the most efficient ever reported.”

But the story doesn’t end there. The second paper, by Andrew Woodhead and colleagues, describes the further optimization of compound 31 to the clinical candidate AT13387. Despite its impressive biochemical and cell potency, compound 31 had only modest activity in a mouse xenograft model, as well as a short plasma half-life. Not surprisingly the hydroxyl groups were found to be points of metabolism, but initial efforts at capping these or changing their electronics either proved detrimental to activity or did not improve the pharmacokinetics. This led to a medicinal chemistry focus on the isoindoline portion of the molecule: a number of positively charged moieties were added at various positions to try to change the overall properties of the molecule. Several substituents were tolerated, and seven related molecules were taken into preclinical candidate selection to look for optimal in vivo properties, solubility, and selectivity against P450 and hERG. AT13387 (see Figure 2) was chosen as the molecule having the best overall profile and entered human clinical trials for solid tumors.

This second paper is a valuable companion to the first: it is particularly notable that, on the simple measures of biochemical and cell potency, AT13387 is no better than compound 31. This emphasizes yet again that affinity is only the first step in drug discovery – it’s a long road from a good lead to the clinic, and an even longer road from there to a marketed drug.