Polycomb repressive complex 1 (PRC1) is an epigenetic regulatory complex that is responsible for the deposition of monoubiquitin onto lysine 119 on histone H2A (H2AK119). This repressive mark results in chromatin compaction and transcriptional repression. The canonical core of PRC1 is a heterodimeric complex comprised of RING1B and BMI1. This heterodimeric E3 ligase interacts with the nucleosome and E2-conjugating enzymes to mitigate the transfer of ubiquitin onto H2AK119. Emerging data implicates RING1B in various types of cancers, as well as the importance of PRC1 function in the maintenance and proliferation of leukemic stem cells. Recent studies demonstrate that loss of PRC1 activity through RING1B knockdown combats leukemic stem cell proliferation, causes leukemic stem cell differentiation, and enhances survival in MLL-AF9 mouse models. Therefore, the development of well-characterized small molecules targeting the E3 ligase activity of PRC1 might be beneficial to explore the role of PRC1 in cancer, as well as to investigate the therapeutic potential of targeting PRC1. We performed an NMR-based fragment screen using RING1B-BMI1, which identified a fragment hit with low millimolar binding affinity. Through extensive medicinal chemistry efforts, we developed a series of compounds with substantially improved binding affinity to RING1B-BMI1, including RB-3 and more recent analogs with more pronounced inhibitory activity. Structural studies revealed that these compounds elicit a conformational change in the RING domain of RING1B, generating a hydrophobic binding pocket. Furthermore, these compounds interact with residues in the RING domain of RING1B that associate with the nucleosome, and our PRC1 inhibitors block the protein-protein interaction between RING1B and the nucleosome. In vitro ubiquitination studies demonstrated that these compounds inhibit H2A ubiquitination activity of PRC1 E3 ligase cores comprised of either RING1B or the paralog RING1A. The activity of PRC1 inhibitors was also evaluated in several leukemia cell lines and primary AML samples, resulting in inhibition of H2A ubiquitination and induction of cellular differentiation. In summary, we developed first-in-class inhibitors directly targeting the core E3 ligase unit of PRC1, which inhibit the ubiquitination activity in leukemic cell lines. Additionally, these inhibitors serve as chemical probes to further investigate the role of PRC1 in leukemia.

Development of small-molecule inhibitors targeting RING1B of polycomb repressive complex 1

ACS Spring 2022

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