TP-064, a potent and selective small molecule inhibitor of PRMT4 for multiple myeloma

Abstract

Protein arginine methyltransferase (PRMT) 4 (also known as coactivator-associated arginine methyltransferase 1; CARM1) is involved in a variety of biological processes and is considered as a candidate oncogene owing to its overexpression in several types of cancer. Selective PRMT4 inhibitors are useful tools for clarifying the molecular events regulated by PRMT4 and for validating PRMT4 as a therapeutic target. Here, we report the discovery of TP-064, a potent, selective, and cell-active chemical probe of human PRMT4 and its co-crystal structure with PRMT4. TP-064 inhibited the methyltransferase activity of PRMT4 with high potency (half-maximal inhibitory concentration, IC50 < 10 nM) and selectivity over other PRMT family proteins, and reduced arginine dimethylation of the PRMT4 substrates BRG1-associated factor 155 (BAF155; IC50= 340 ± 30 nM) and Mediator complex subunit 12 (MED12; IC50 = 43 ± 10 nM). TP-064 treatment inhibited the proliferation of a subset of multiple myeloma cell lines, with affected cells arrested in G1 phase of the cell cycle. TP-064 and its negative control (TP-064N) will be valuable tools to further investigate the biology of PRMT4 and the therapeutic potential of PRMT4 inhibition.

A Chemical Probe of CARM1 Alters Epigenetic Plasticity against Breast Cancer Cell Invasion

“…Competitive assays with SAM cofactor and peptide substrate showed that 2a and 5a act To the best of our knowledge, EZM2302, TP-064, SKI-73 (www.thesgc.org/chemicalprobes/SKI-73) and their derivatives are the only selective and cell-active CARM1 inhibitors. (Drew et al, 2017;Nakayama et al, 2018) While the potency, selectivity, on-target engagement and potential off-target effects associated with these compounds have been examined in vitro and in cellular contexts as chemical probes, EZM2302, TP-064, SKI-73 are distinct by their molecular scaffolds and modes of interaction with CARM1 (www.thesgc.org/chemical-probes/SKI-73). (Drew et al, 2017;Nakayama et al, 2018) SKI-73 is a cofactor analog inhibitor embedding a N6'homosinefungin moiety to engage the SAM binding site of CARM1 in a cofactor-competitive, substrate-noncompetitive manner; EZM2302 and TP-064 occupy the substrate-binding pocket of CARM1 in a SAH-uncompetitive or SAM-noncompetitive manner.…”

“…(Drew et al, 2017;Nakayama et al, 2018) While the potency, selectivity, on-target engagement and potential off-target effects associated with these compounds have been examined in vitro and in cellular contexts as chemical probes, EZM2302, TP-064, SKI-73 are distinct by their molecular scaffolds and modes of interaction with CARM1 (www.thesgc.org/chemical-probes/SKI-73). (Drew et al, 2017;Nakayama et al, 2018) SKI-73 is a cofactor analog inhibitor embedding a N6'homosinefungin moiety to engage the SAM binding site of CARM1 in a cofactor-competitive, substrate-noncompetitive manner; EZM2302 and TP-064 occupy the substrate-binding pocket of CARM1 in a SAH-uncompetitive or SAM-noncompetitive manner. (Drew et al, 2017;Nakayama et al, 2018) In particular, the prodrug property of SKI-73 allows its ready cellular uptake, followed by rapid conversion into its active forms inside cells.…”

“…(Blanc and Richard, 2017;Yang and Bedford, 2013) The requirement of CARM1 is implicated in multiple cancers with its methyltransferase activity particularly addicted by hematopoietic malignancies and metastatic breast cancer. (Drew et al, 2017;Greenblatt et al, 2018;Nakayama et al, 2018;Wang et al, 2014) Our prior efforts using in vivo mouse and in vitro cell models uncovered the role of CARM1 in promoting breast cancer metastasis. (Wang et al, 2014) Mechanistically, CARM1 methylates Arg1064 of BAF155 and thus facilitates the recruitment of the BAF155-containing SWI/SNF complex to a specific subset of gene loci essential for breast cancer metastasis.…”

“…(Wang et al, 2014) While this cancer relevance inspired the development of CARM1 inhibitors, (Kaniskan et al, 2018;Scheer et al, 2019) many small-molecule CARM1 inhibitors lack target selectivity or cellular activity (Kaniskan et al, 2018)---two essential criteria of chemical probes. (Frye, 2010) To the best of our knowledge, EZM2302, (Drew et al, 2017;Greenblatt et al, 2018) TP-064 (Nakayama et al, 2018) and SKI-73 (www.thesgc.org/chemical-probes/SKI-73) are the only selective and cell-active CARM1 chemical probes, which were developed by Epizyme, Takeda/SGC(Structural Genomic Consortium), and our team, respectively. EZM2302 and TP-064 were developed through conventional small-molecule scaffolds occupying the substrate-binding pocket of CARM1.…”

“…(Drew et al, 2017;Greenblatt et al, 2018;Nakayama et al, 2018) The potential utility of EZM2302 and TP-064 is implicated by their selective anti-proliferative effects on hematopoietic cancer cells, in particular multiple myeloma cells. (Drew et al, 2017;Greenblatt et al, 2018;Nakayama et al, 2018) However, definitive molecular mechanisms of the CARM1 addiction in these contexts remain elusive. (Greenblatt et al, 2018) Here we report the characterization and novel utility of SKI-73---a chemical probe of CARM1 with pro-drug properties.…”