Fragments vs PRC1: toward a chemical probe

E3 ligase proteins conjugate ubiquitin to other proteins, changing their function or targeting them for degradation. Hijacking E3 ligases using PROTACs or molecular glues has become a popular approach for targeted protein degradation, but some E3 ligases can be drug targets themselves. For example, Polycomb Repressive Complex 1 (PRC1) ubiquitylates histone H2A in a process essential for proliferation of acute myeloid leukemia and some other cancer cells. In a new J. Med. Chem. paper, Jolanta Grembecka, Tomasz Cierpicki, and colleagues at University of Michigan Ann Arbor describe their discovery of small molecule inhibitors.

 

As the name implies, PRC1 is a protein complex. The core includes either RING1A or RING1B and one of six other proteins. Thus, the researchers sought to find inhibitors of both RING1A and RING1B. They started with a ¹H-¹⁵N HSQC NMR screen of 1000 fragments in pools of 20, with each compound at 0.25 mM. (This and some other work was described in an earlier Nat. Chem. Biol. paper by the same authors.) Compound RB-1 was found to bind very weakly to RING1B, and chemical shift mapping revealed that it binds in a region important for E3 ligase activity.

 

Scaffold hopping led to the pyrrole-containing compound 1b, which was slightly more potent as well as more soluble than RB-1. Fragment growing on both rings led to compound 1f, and further optimization yielded low micromolar compound RB-2. Crystallography with this compound revealed conformational changes that opened a hydrophobic pocket in the protein. Although no electron density for RB-2 was observed, the researchers used the crystal structure in combination with NMR experiments to develop a binding model. This facilitated further optimization, ultimately yielding RB-4, the most potent compound reported, with low micromolar affinity as assessed by isothermal titration calorimetry.

As noted above, it is important to block both RING1B as well as RING1A, and the researchers tested their compounds against both proteins using an AlphaScreen competition assay. Most compounds were equipotent against both proteins, though some showed around two-fold greater affinity for RING1A.

 

Subsequent experiments demonstrated that some of the compounds could block ubiquitylation of H2A in an in vitro assay. Importantly, compound RB-4 functionally inhibited three different PRC1 complexes having either RING1A or RING1B along with one of two other protein partners. Treatment of leukemia cells with the compound also led to changes in gene expression and lower levels of ubiquitylated H2A consistent with PRC1 inhibition.

 

This is a nice fragment-to-lead story, particularly given the difficult nature of the protein and the absence of co-crystal structures. While compound RB-4 is insufficiently potent to be called a chemical probe, it is nonetheless a well-characterized starting point for further optimization.