1. F A C U L T Y O F H E A L T H A N D M E D I C A L S C I E N C E S U N I V E R S I T Y O F C O P E N H A G E N Discovery of potent inhibitors of the epigenetic cancer target PRMT4 Kamilla Meyer & Brian Lohse Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen Future work The peptides have to be synthesized and further tested in competition assay to obtain an IC50 value and in affinity assays as ITC to obtain the KD value. PRMT4 as a target in cancer PRMT4 regulates a number of cellular processes, including transcription [1], mRNA splicing [2], cell cycle progression [3], and DNA damage response [4] through epigenetic mechanisms. The overexpression of the PRMT4 activates a multitude of oncogenic pathways and promotes a favorable microenvironment for tumor growth and metastasis [5]. It is reported overexpressed in prostate [6], colorectal [7] and in aggressive breast cancers [8]. To this date, few PRMT4 inhibitors have been discovered among which EPZ011652 is an example [9]. Unfortunately, EPZ011652, similarly to other discovered PRMT inhibitors, unselectively inhibits both PRMT1, -4, -6, and -8. A new approach could thus be to target the surface for allosteric sites of the PRMTs to obtain selective inhibitors. This can be achieved using phage display biotechnology. Ligand screening Phage display is based on a M13 phage vector, modified for pentavalent display of DNA-encoded ligands on its surface toward a coated protein. These are stored in different libraries, and after several washing steps unbound and weak binders will be outcompeted by strong binders, and the pool of strong binders will increase. The strong binders can afterwards be eluted and isolated. Promising peptide binders to PRMT4 Clones showing the highest absorbance, were sequenced and translated into the amino acid sequence The obtained panning rounds and isolated clones have been tested on Enzyme-linked immunosorbent assay (ELISA) to confirm binding. 3 1 4 6 5 7 Washing Elution References [1] Chen D, Ma H, Hong H, Koh SS, Huang SM, Schurter BT, et al. Regulation of transcription by a protein methyltransferase. Science (New York, NY). 1999;284(5423):2174-7. [2] Cheng D, Cote J, Shaaban S, Bedford MT. The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing. Molecular cell. 2007;25(1):71-83. [3] El Messaoudi S, Fabbrizio E, Rodriguez C, Chuchana P, Fauquier L, Cheng D, et al.Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(36):13351-6. [4] Lee Y-H, Stallcup MR. Roles of protein arginine methylation in DNA damage signalingpathways: Is CARM1 a life-or-death decision point? Cell Cycle. 2011;10(9):1343-4. [5] Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumormicroenvironment. Cell. 2010;141(1):52-67. [6] Majumder S, Liu Y, Ford OH, 3rd, Mohler JL, Whang YE. Involvement of argininemethyltransferase CARM1 in androgen receptor function and prostate cancer cell viability. The Prostate. 2006;66(12):1292-301 [7] Kim Y-R, Lee BK, Park R-Y, Nguyen NTX, Bae JA, Kwon DD, et al. DifferentialCARM1 expression in prostate and colorectal cancers. BMC Cancer. 2010;10:197. [8] El Messaoudi S, Fabbrizio E, Rodriguez C, Chuchana P, Fauquier L, Cheng D, et al.Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(36):13351-6. [9] Rioux N, Mitchell LH, Tiller P, Plant K, Shaw J, Frost K, et al. Structural and KineticCharacterization of a Novel N-acetylated Aliphatic Amine Metabolite of the PRMT Inhibitor, EPZ011652. Drug Metabolism and Disposition. 2015;43(7):936-43. Epigenetic mechanism of PRMT4 Clone 16 Clone 19 Contact information Kamilla Meyer Kamilla_meyer@hotmail.com/lcs879@alumni.ku.dk (+45) 5174 0053 Acknowledgements A huge thanks to Andreas H. Laustsen, Line P. Lauridsen and Saioa Oscoz for scientific discussion. Repeat 2 DNA-encoded libraries in M13 phages PRMT4 Panning round Abs, 490 nm Clone number PRMT4 Reference PRMT4 Reference Phage Display Peptide binding