Evaluation of novel small molecule inhibitors targeting Polycomb repressive complex 1 as chemical probes

Background: Polycomb Repressive Complex 1 (PRC1) is an epigenetic regulatory complex responsible for monoubiquitylation of lysine 119 on histone H2A (H2AK119Ub). This ubiquitylation activity is mediated by a canonical heterodimeric E3 ligase involving Ring1B (or its paralog Ring1A) bound to BMI1. Deposition of H2AK119Ub is associated with gene repression at loci involved in determining cell fate and identity. Notably, genetic depletion of Ring1A and Ring1B results in reduction of H2AK119Ub, derepression of PRC1 target genes, and cell growth arrest in a leukemia model. Therefore, small molecules targeting the ubiquitylation activity of Ring1A/B are useful chemical probes towards studying PRC1 biology. Results: We have previously developed RB-3: a first in class small molecule inhibitor of PRC1. Here we report the next generation of novel small molecule PRC1 inhibitors. Employing structure-based design, we have developed a potent cell permeable next generation inhibitor RB-322. RB-322 is a mixture of two atropisomers which were separated yielding RB-322-1 and RB-322-2. To validate direct binding of our inhibitors to Ring1A/B we used 2D HSQC. Notably, RB-322-1 demonstrates slow exchange while RB-322-2 demonstrates fast exchange indicating strong and weak binding respectively. We then used an AlphaScreen competition assay to evaluate RB-322-1 mediated disruption of the PRC1-nucleosome interaction in vitro in which it shows low nanomolar activity. A novel radiometric ubiquitylation activity assay (RadUb) was then developed to quantitatively assess inhibition of PRC1 E3 ligase activity. Using this assay we found RB-322-1 demonstrates sub-micromolar activity. RB-322-2 is 2 orders of magnitude weaker in these assays. We then investigated the on-target activity of RB-322 in leukemia cell lines. First, we developed a biotinylated RB-322 probe and performed streptavidin-based pull downs followed by proteomics analysis revealing enrichment of multiple PRC1 proteins. We then evaluated the effects of RB-322-1 on cellular H2AK119Ub and gene expression. We found RB-322-1 demonstrates rapid and dose-dependent reduction of the H2AK119Ub mark after 6-hours and derepresses PRC1 target genes in AML cell lines. Crucially, these effects have not been observed for the weak atropisomer RB-322-2. Conclusions: Extensive medicinal chemistry optimization has yielded a potent cell permeable next generation small molecule inhibitor of PRC1: RB-322. RB-322 demonstrates low nanomolar activity in biochemical assays in vitro and demonstrates on-target derepression of PRC1 genes providing novel mechanistic insights. RB-322 represents a useful chemical probe for studying PRC1 biology and serves as the foundation for a potential novel anti-cancer agent.