Protein Lysine methyltransferases (PKMTs) are group of histone modifiers that are responsible for the transfer of one to three methyl groups from S-adenosyl-L-methionine (AdoMet) to the ε-amino group of the target lysine residues in histones and some non-histone targets. To date more than 50 PKMTs have been identified and EHMT1 (Euchromatic Histone Methyltransferase 1, GLP, G9a like proteins) and EHMT2 (Euchromatic Histone Methyltranferase 2, G9a) are amongst the most studied ones. Genetic variations of EHMT1/2 have been associated with human diseases such as cancer, inflammatory diseases and neuro-generative disorders. As a consequence, there has been a growing interest to identify potent inhibitors of these enzymes.
Materials and methods
We employed Sinefungin as a lead structure for the design and synthesis of a series of methyltransferase inhibitors and tested them for inhibition of EHMT1/2. The α-amino acid moiety of Sinefungin was exchanged to obtain two different series of compounds- one with the additional amino group and one without amino group. Screening of compounds were done by using a FRET-based LANCE ultra G9a histone H3-Lysine N-methyltransferase assay that measures the dimethylation of a biotinylated histone H3 (1-21) peptide at lysine 9.
A series of analogues of the natural product Sinefungin was designed and synthesized, and probed for their ability to inhibit EHMT1 and EHMT2. This led to a highly potent inhibitor 4d with a Ki of 5 nM at EHMT1 and 24 nM at EHMT2. There was variation in the activity of the compounds and most of the compounds displayed little inhibition.
Here we exchanged the α-amino acid moiety and demonstrated that it is not essential for inhibitory activity at EHMT1/2. Our results indicate that these scaffolds upon further modifications can lead to selective, potent inhibitors of EHMTs and possibly other PKMTs.
We are thankful to Novo Nordisk Foundation Center for Protein Research and Department of Health Sciences, University of Copenhagen for the Ph.D. fellowship, Dr. Thomas Frimurer for valuable inputs in designing the analogues, Ms. Huili Lu and Ms. Jie Zhang for technical assistance, the Danish Cancer Society, the University of Copenhagen Programme of Excellence and the Ministry of Science and Technology of China (2009ZX09302-001, 2012ZX09304011 and 2013ZX09507002) for grant support.
Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T: Regulation of chromatin structure by site-specific histone H3 methyltransferases.
Rouleau N, Pedro L, Gauthier N, Labonté A, Paquet V, Rodenbrock A, Roy M, Marcil A, Plante H, Beaudet L, Rodriguez-Suarez R: Development of High-Throughput Assays to Study Histone H3K4 Methyltransferases & H3K9 Methyl- and Acetyltransferases.
Zheng WH, Ibáñez G, Wu H, Blum G, Zeng H, Dong AP, Li FL, Hajian T, Allali-Hassani A, Amaya MF, Siarheyeva L, Yu WY, Brown PJ, Schapira M, Vedadi M, Min JR, Luo MK: Sinefungin Derivatives as Inhibitors and Structure Probes of Protein Lysine Methyltransferase SETD2.