Download Discovery of potent inhibitors of the epigenetic cancer target PRMT4

FACULTY OF HEALTH AND M EDI CAL SCI ENCES
U N IVE R SITY O F C O PEN HAG 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
1
PRMT4 as a target in cancer
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Epigenetic mechanism of PRMT4
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.
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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.
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Phage Display
Peptide binding
The obtained panning rounds and isolated clones have been tested on Enzyme-linked
DNA-encoded libraries in
M13 phages
Abs, 490 nm
immunosorbent assay (ELISA) to confirm binding.
Washing
PRMT4
PRMT4
Reference
PRMT4
Repeat
Elution
Panning round
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Abs, 490 nm
PRMT4
Reference
Promising peptide binders to PRMT4
Clones showing the highest absorbance, were sequenced and translated into the amino acid
sequence
Clone number
Clone 16
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Future work
Clone 19
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.
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.
Contact information
Kamilla Meyer
[email protected]/[email protected]
(+45) 5174 0053
Acknowledgements
A huge thanks to Andreas H. Laustsen, Line P. Lauridsen and Saioa Oscoz for scientific discussion.