Fragments vs HSP90: Nerviano’s turn

HSP90, an oncology target, is one of those proteins that seems tailor-made for fragments: it has an active site with a predilection for small molecules, it’s easy to work with, and it crystallizes readily. Indeed, at least two fragment-derived molecules targeting this protein have advanced to Phase 2 clinical trials. In a recent Bioorg. Med. Chem. paper, Elena Casale, Francesco Casuscelli, and colleagues at Nerviano describe their efforts against this target.

The researchers started by identifying a fluorinated probe molecule that they could use in a Fluorine chemical shift Anisotropy and eXchange for Screening (FAXS) assay. This is an NMR-based competition method, in which fragments are screened to find those that displace a known ligand, in this case one that binds in the active site. A total of 1200 fragments were screened in pools of 10, each at the relatively low concentration of 50 micromolar. Nonetheless, 23 hits were found, four of which were characterized crystallographically bound to the protein.

Fragment 3 was among the more interesting, both because of its high ligand efficiency as well as its structural novelty. SAR-by-catalog failed to find anything better from 20 compounds tested, and initial fragment growing also proved disappointing. However, a closer inspection of the crystal structure (cyan) revealed the possibility of linking the fragment to the well-known HSP90 fragment resorcinol. This led to compound 8b, which binds about 5-fold more tightly. Crystallography revealed that the molecule (magenta) also binds as expected.

However, the team wisely chose to test synthetic intermediate 7h (in which the hydroxyl groups were still methylated) and this turned out to be even more active than the designed compound. Since the hydroxyls of the resorcinol are essential for binding in other lead series, the team solved the crystal structure of compound 7h (green) and was surprised to find that it binds in a completely different manner than compound 8b; the ligand essentially flips over.

This discovery led to a change in direction for medicinal chemistry, leading ultimately to the low nanomolar compound 12a. Unfortunately this molecule had only modest cell-based activity and was metabolically unstable.

This is a solid, nuts-and-bolts sort of story. Although it does not conclude with a clinical candidate, it does provide a useful window into how fragment-based methods are applied in industry. It is also a reminder to screen all your intermediates and to remember that even subtle changes to a molecule may have dramatic effects on its binding mode. Those surprising shifts can point the way to promising chemical space.