Targeted protein degradation (TPD) goes beyond merely inhibiting a protein; it takes a protein out of commission entirely. This is frequently done using a bivalent ligand: one part binds to the protein of interest, while the other part binds to an E3 ligase, which ubiquitinates the protein of interest, targeting it for destruction in the proteasome. Human cells have hundreds of E3 ligase proteins, some of which may work better in certain situations, such as specific cell compartments or tissues. In a recent ACS Med. Chem. Lett. paper, Anna Vulpetti and colleagues at Novartis describe progress against DCAF1.
DCAF1 is one component of the Cullin4-RING E3 ubiquitin ligase complex. The C-terminus of the protein contains a WD40 repeat (WDR) domain, which in this case consists of seven “blades” arranged around a central cavity, or “donut hole”. WDR domains are relatively common, and indeed we wrote about a previous Novartis effort that identified chemical probes against another WDR domain in the protein EED. In the new work, the researchers took 21 EED binders and screened them using both protein-detected and ligand-detected NMR against DCAF1, identifying two hits. Crystallography revealed that compound 1 binds in the central cavity, which previous computational screening had suggested would be ligandable.
Next, the researchers screened 30 related compounds from within Novartis. Two of them, including compound 4, had improved affinity (as assessed both by NMR and SPR) and could be characterized crystallographically. In addition to binding in the central cavity, these compounds also bound to a site in the blade region, which the researchers wanted to avoid. Adding a piperazine to compound 4 both improved affinity and disrupted binding to the blade region; further optimization and growing to better fill the central cavity led to compound 13, the most potent molecule in the paper.
A crystal structure of a closely related molecule reveals that the acetyl group is near the entrance to the donut hole, providing an easy synthetic attachment point to construct bivalent degraders. A separately published preprint revealed this to be successful, with degraders of BRD9, multiple tyrosine kinases, and BTK.
There are several takeaways from this nice fragment to lead story. First, despite the fact that compound 1 is clearly fragment-sized (albeit a bit too lipophilic to be fully rule-of-three compliant), the word fragment never appears in the article. FBLD has become so routine that researchers may not even mention it, which does mean that our list of fragment-derived drugs is destined to be incomplete.
Second, although DCAF1 and EED share less than 25% sequence similarity, screening EED hits turned out to be successful, which could argue for screening specific subsets of fragments (for example kinase-focused or, in this case, WDR-focused). On the other hand, compound 1 binds in a different manner to DCAF1 than it does to EED. Indeed, compound 1 actually binds in two different orientations to DCAF1, consistent with its low affinity. The researchers mention a paper published earlier this year that reports a successful DEL screen against the target. Perhaps DCAF1 is just very ligandable, and a naïve fragment screen would have worked just as well as the pre-selected set.
Finally, the fact that this program yielded bivalent degraders suggests that many E3 ligases might be coopted for drug discovery. The field of targeted protein degradation is just getting started.