Aryl pyrazoles as potent inhibitors of protein arginine methyltransferases: identification of the first PRMT6 tool compound Allison Drew 1 , Lorna Mitchell 1 , Nathalie Rioux 1 , Kerren Swinger 1 , Scott Ribich 1 , Suzanne L. Jacques 1 , Trupti Lingaraj 1 , Tim Wigle 1 , Lei Jin 3 , Margaret Porter Scott 2 , Ann Boriak-Sjodin 1 , Nigel Waters 1 , Tom Riera 1 , Richard Chesworth 1 , Jesse Smith 1 , Robert Copeland 1 PRMT6 has been reported to play a role in a variety of cellular processes including maintenance of stem cell pluripotency 6 , regulation of cell cycle 7 , DNA repair 8 , regulation of nuclear receptor-mediated transcription 9 , and viral transactivation 10 . Overexpression of PRMT6 has been reported in several cancer types including melanoma 11 and bladder, lung 12 , and prostate 13 carcinoma, suggesting that PRMT6 inhibition may have therapeutic utility. Until now, no small molecule PRMT6 inhibitor has been available for use as tool compound for in vitro or in vivo target validation studies. Background Results Results • The aryl pyrazole EPZ020411 is a potent and selective small molecule inhibitor of PRMT6 with a biochemical IC 50 of 10nM • The binding mode of EPZ020411 was characterized by protein crystallography • EPZ020411 inhibits cellular methylation of the PRMT6 substrate H3R2 • EPZ020411 has suitable PK properties for in vivo use • EPZ020411 is a suitable tool compound for future in vitro and in vivo target validation studies Pharmacokinetics parameters for EPZ020411 following single IV and SC bolus administration to male Sprague-Dawley rats. Expressed as mean ± SD, n=3. Studies were performed in accordance with the AAALAC International and NIH guidelines standards. Mean total blood concentration-time profiles of EPZ020411 after an IV dose of 1 mg/kg and an SC dose of 5 mg/kg in male Sprague-Dawley rats, n=3, mean ± SD. PRMT6-induced H3R2 methylation is inhibited by 48 hour exposure to EPZ020411 (IC 50 = 0.637 ± 0.241 µM) but not by compound 7 (IC 50 > 20 µM) www.epizyme.com The aryl pyrazole EPZ020411 is a potent inhibitor of PRMT6 EPZ020411 in vitro ADME and pharmacokinetics EPZ020411 inhibits methylation of PRMT6 substrates in cells • Overexpression of PRMT6 induces methylation of the canonical PRMT6 substrate H3R2 • EPZ020411 dose- dependently inhibits H3R2 methylation in PRMT6-overexpressing cells • PRMT6-inactive compound 7 does not affect H3R2 methylation References 1. Richon, V. M.; Johnston, D.; Sneeringer, C. J.; Jin, L.; Majer, C. R.; Elliston, K.; Jerva, L. F.; Scott, M. P.; Copeland, R. A., Chem Biol Drug Des 2011, 78 (2), 199-210. 2. Bedford, M. T.; Richard, S. Mol Cell 2005, 18 (3), 263-7 3. Frankel, A.; Yadav, N.; Lee, J.; Branscombe, T. L.; Clarke, S.; Bedford, M. T. J Biol Chem 2002, 277 (5), 3537-43. 4. Hyllus, D.; Stein, C.; Schnabel, K.; Schiltz, E.; Imhof, A.; Dou, Y.; Hsieh, J.; Bauer, U. M. Genes Dev 2007, 21 (24), 3369-80 5. Guccione, E.; Bassi, C.; Casadio, F.; Martinato, F.; Cesaroni, M.; Schuchlautz, H.; Lüscher, B.; Amati, B. Nature 2007, 449 (7164), 933-7. 6. Lee, Y. H.; Ma, H.; Tan, T. Z.; Ng, S. S.; Soong, R.; Mori, S.; Fu, X. Y.; Zernicka-Goetz, M.; Wu, Q. Stem Cells Dev 2012, 21 (14), 2613-22. 7. Kleinschmidt, M. A.; de Graaf, P.; van Teeffelen, H. A.; Timmers, H. T. PLoS One 2012, 7 (8), e41446. 8. El-Andaloussi, N.; Valovka, T.; Toueille, M.; Steinacher, R.; Focke, F.; Gehrig, P.; Covic, M.; Hassa, P. O.; Schär, P.; Hübscher, U.; Hottiger, M. O. Mol Cell 2006, 22 (1), 51-62. 9. Harrison, M. J.; Tang, Y. H.; Dowhan, D. H. Nucleic Acids Res 2010, 38 (7), 2201-16. 10. Boulanger, M. C.; Liang, C.; Russell, R. S.; Lin, R.; Bedford, M. T.; Wainberg, M. A.; Richard, S. J Virol 2005, 79 (1), 124-31. 11. Limm, K.; Ott, C.; Wallner, S.; Mueller, D. W.; Oefner, P.; Hellerbrand, C.; Bosserhoff, A. K. Eur J Cancer 2013, 49 (6), 1305-13. 12. Yoshimatsu, M.; Toyokawa, G.; Hayami, S.; Unoki, M.; Tsunoda, T.; Field, H. I.; Kelly, J. D.; Neal, D. E.; Maehara, Y.; Ponder, B. A.; Nakamura, Y.; Hamamoto, R. Int J Cancer 2011, 128 (3), 562-73 13. Vieira, F. Q.; Costa-Pinheiro, P.; Ramalho-Carvalho, J.; Pereira, A.; Menezes, F. D.; Antunes, L.; Carneiro, I.; Oliveira, J.; Henrique, R.; Jerónimo, C. Endocr Relat Cancer 2014, 21 (1), 51-61. These data can also be found in: Mitchell, L. H. et al. ACS Med Chem Lett 2015. DOI: 10.1021/acsmedchemlett.5b00071 HN N N H N O O O EPZ020411 IC 50 (PRMT6) = 10 nM PRMT6 is a member of the protein arginine methyltransferase (RMT) family which comprises 45 enzymes, 9 of which are known to catalyze protein arginine N-methylation reactions 1 . These post-translational modifications regulate RNA processing, transcription, signal transduction and other cellular processes 2 . A nuclear-localized RMT, PRMT6 creates 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 . This mark can act in opposition to the activating H3K4me3 mark, effectively behaving as a transcriptional repressor 5 . • The aryl pyrazole 1 was identified to have potent PRMT6, PRMT8, and PRMT1 activity through screening of the Epizyme proprietary HMT-biased library • Selectivity towards PRMT6 was achieved by introduction of bulky substituents in the para-position of the aryl ring (EPZ020411 and compounds 2-6) • Except for PRMT1 and 8, EPZ020411 shows greater than 100X selectivity for PRMT6 over all tested HMT including 4 RMT (PRMT3, 4, 5 and 7) • Analoging efforts showed that PRMT6 activity could be decreased with di- meta substitution of the aryl ring as seen with compound 7 • EPZ020411 has a free fraction of 0.13 ± 0.01 in rat blood • Sprague-Dawley rats administered EPZ020411 by IV bolus showed a moderate clearance (CL) of 19.7 mL/min/kg, translating to a long mean terminal half-life (t 1/2 ) of 8.5 h • EPZ020411 shows good bioavailability following subcutaneous (SC) dosing in rats making it a suitable tool for in vivo studies a IC 50 ’s were determined from at least two independent experiments. b Compound is an undetermined mixture of isomers at the chiral center. Parameter 1 mg/kg IV 5 mg/kg SC CL (mL/min/kg) 19.7 ± 1.0 V ss (L/kg) 11.1 ± 1.6 t ½ (h) 8.54 ± 1.43 9.19 ± 1.60 t max (h) 0.444 C max (ng/mL) 844 ± 306 AUC 0-τ (h*ng/mL) 745 ± 34 2456 ± 135 AUC 0-inf (h*ng/mL) 846 ± 45 2775 ± 181 F (%) 65.6 ± 4.3 Cmpd #7 • . Structures of 1 (A) and EPZ020411 (C) (green) showing SAH (yellow). And 2Fo-Fc electron density maps for ligands 1 (B) and EPZ020411 (D) at 1σ (blue mesh). Crystal structure of EPZ020411 bound to PRMT6 • Crystal structures of complexes of PRMT6 and SAH with 1 (2.4Å) and EPZ020411 (2.1Å) were obtained (PDB IDs 4Y2H and 4Y30) • The diamine side-chain occupies the putative site of the substrate arginine side-chain • The majority of the interactions between the two ligands and the protein occur via the diamine side-chain and the pyrazole • There is more space in the pocket for expansion of 1 beyond the para position of the aryl group; the R group of EPZ020411 fills this additional space, though the entirety of the oxygen linked alkyl side chain is not well ordered in the structure 1 Epizyme Inc., 400 Technology Square, Cambridge MA 02139, USA 2 Current address: Genentech, Inc., 1 DNA Way, South San Francisco CA 94080, USA 3 Current address: Agile Biostructures, Wellesley, MA, USA #6968 # Pyrazole isomer R1 R2 R3 R4 PRMT6 IC 50 (µM) a PRMT8 IC 50 (µM) a PRMT1 IC 50 (µM) a 1 A F H H H 0.011 0.067 0.018 2 A H H H 0.013 0.45 0.117 3 A H H H 0.020 1.5 0.387 4 A H H Me 0.003 0.179 0.026 5 B H H Me 0.002 0.347 0.060 6 B H H Me 0.005 0.065 0.050 EPZ020411 A H H Me 0.010 0.223 0.119 7 b A H CF 3 Me 1.9 0.044 0.016 N N O Ph O N O O NMe 2 O O O NHMe O O O O NMe 2 O O OH EPZ020411 has > 10-fold selectivity for PRMT6 over PRMT1 and PRMT8 PRMT6 activity is inhibited by EPZ020411 (IC 50 = 0.010 µM) in a biochemical assay measuring transfer of the tritiated methyl group from 3 H-SAM to a biotinylated peptide by full length hPRMT6. EPZ020411 inhibits PRMT1 and PRMT8 activity with IC 50 s of 0.119 and 0.223 µM respectively. H3R2me2a Histone H3 H3R2me2a Histone H3 EPZ020411 H3R2 methylation is induced by 48 hour overexpression of PRMT6, but not an empty vector, in A375 cells. R 4 HN N N H N R 1 R 4 HN N N N H R 1 A B R 2 R 3 R 2 R 3 µM Compound Marker Disclosures: Epizyme authors: Epizyme employment, Epizyme equity ownership Conclusions