Aryl Pyrazoles as Potent Inhibitors of Arginine Methyltransferases: Identification of the First PRMT6 Tool Compound

Abstract

A novel aryl pyrazole series of arginine methyltransferase inhibitors has been identified. Synthesis of analogues within this series yielded the first potent, selective, small molecule PRMT6 inhibitor tool compound, EPZ020411. PRMT6 overexpression has been reported in several cancer types suggesting that inhibition of PRMT6 activity may have therapeutic utility. Identification of EPZ020411 provides the field with the first small molecule tool compound for target validation studies. EPZ020411 shows good bioavailability following subcutaneous dosing in rats making it a suitable tool for in vivo studies. KEYWORDS: PRMT6, protein methyltransferase, oncology, tool compound PRMT6 is a member of the protein arginine methyltransferase (RMT) family, which comprises 45 enzymes, nine of which are known to catalyze protein arginine N-methylation reactions. These post-translational modifications are important regulators of RNA processing, transcriptional regulation, signal transduction, and other cellular processes. 1,2 PRMT6 is a nuclear-localized RMT capable of creating omega-N(G)-monomethylarginine and asymmetric omega-N(G),N(G)-dimethylarginine derivatives on histone and other protein substrates containing a GAR motif; 3 it is the only RMT known to methylate the H3R2 mark. 4,5 This mark can act in opposition to the activating H3K4me3 mark, effectively acting as a transcriptional repressor. 6 PRMT6 has been reported to play a role in a variety of cellular processes including maintenance of stem cell pluripotency, 7 regulation of cell cycle, 8 DNA repair, 9 regulation of nuclear receptor-mediated transcription, 10 and viral transactivation. 11 PRMT6 overexpression has been reported in several cancer types including melanoma 12 and bladder, lung, 13 and prostate 14 carcinoma, suggesting that inhibition of PRMT6 may have therapeutic utility and supporting development of small molecule inhibitors for use as tool compounds for in vitro and in vivo target validation studies.An aryl pyrazole bearing a diamine side-chain, 1, was found to have potent PRMT1, PRMT6, and PRMT8 inhibitory activity through screening of the Epizyme internal library.A 2.4 Å resolution crystal structure of a ternary complex of 1, SAH, and PRMT6 was obtained and is shown in Figure 1a,b (4Y2H). The diamine side-chain occupies the putative site of the substrate arginine side-chain. The terminal nitrogen atom is 3.4 Å away from the sulfur atom of SAH. The terminal NH 2 group makes multiple direct hydrogen bonds to the Glu155 side-chain and backbone carbonyl and water-mediated hydrogen bond interactions with the backbone carbonyl of Trp156

A Potent, Selective, and Cell-Active Inhibitor of Human Type I Protein Arginine Methyltransferases

“…29, 36 We rationalized that the ethylenediamino group is an arginine mimetic and a major contributor to PRMT6 and PRMT4 (both of which are type I PRMTs) inhibitory activities of these compounds. Based on this analysis, we hypothesized that an ethylenediamino group would be an excellent moiety for targeting type I PRMTs.…”

“…We also explored two additional regions of the EPZ020411 scaffold. 36 Because the substituted cyclobutoxy group on the phenyl ring likely contributed to the selectivity for PRMT6, we replaced this group with a smaller functional group such as trifluoromethyl or isopropoxy group (compounds 1 – 3 in Table 1) to gain inhibitory activities for other type I PRMTs. In addition, we probed the electronic nature of the main heteroaromatic core by replacing the pyrazole ring with a 1,2,3-triazole or pyrrole ring (compounds 1 – 3 in Table 1).…”

“…Inspired by the recent discoveries of EPZ020411 (Figure 1), 36 a potent and selective PRMT6 inhibitor, and CMPD-1 (Figure 1), 29 a PRMT4 (CARM1) inhibitor, we designed and synthesized MS023 (Figure 1) and its close analogs, and discovered MS023 as a selective inhibitor of type I PRMTs. We also discovered a close analog of MS023 as a negative control for cell-based studies.…”

Small Molecule Inhibitors of Protein Arginine Methyltransferases

“…As mentioned in Section 2, compounds with the core structure of 17 , represented by compound 51 (Table 6), were found to show promising potency to inhibit PRMT1, -6 and -8 via the SPA measurement [99, 126, 127, 153]. Extension of the para -position of the phenyl group of 51 with alkoxy group retains the activity.…”

“…Further extension of the length of the group at para -position of the phenyl group results retained activity to PRMT6 and decreased activity to PRMT1 and -8, while large substitution on the meta -position yields opposite effect: the activity is diminished toward PRMT6 but not PRMT1 and -8. During the structural optimization, some analogues even show low nanomolar level of potency (IC 50 ranging 5 – 20 nM) and remarkable selectivity for PRMT6 [99]. Out of them is compound 52 (EPZ020411, IC 50 = 10 nM) that exhibits desired pharmacokinetic property as well as excellent selectivity (> 100 fold) for PRMT6 over other tested PRMTs (PRMT3, -4, -5 and -7) and PKMTs.…”