Abstract
Epigenetic therapies are being investigated for the treatment of cancer, cognitive disorders, metabolic alterations and autoinmune diseases. Among the different epigenetic target families, protein lysine methyltransferases (PKMTs), are especially interesting because it is believed that their inhibition may be highly specific at the functional level. Despite its relevance, there are currently known inhibitors against only 10 out of the 50 SET-domain containing members of the PKMT family. Accordingly, the identification of chemical probes for the validation of the therapeutic impact of epigenetic modulation is key. Moreover, little is known about the mechanisms that dictate their substrate specificity and ligand selectivity. Consequently, it is desirable to explore novel methods to characterize the pharmacological similarity of PKMTs, going beyond classical phylogenetic relationships. Such characterization would enable the prediction of ligand off-target effects caused by lack of ligand selectivity and the repurposing of known compounds against alternative targets. This is particularly relevant in the case of orphan targets with unreported inhibitors. Here, we first perform a systematic study of binding modes of cofactor and substrate bound ligands with all available SET domain-containing PKMTs. Protein ligand interaction fingerprints were applied to identify conserved hot spots and contact-specific residues across subfamilies at each binding site; a relevant analysis for guiding the design of novel, selective compounds. Then, a recently described methodology (GPCR-CoINPocket) that incorporates ligand contact information into classical alignment-based comparisons was applied to the entire family of 50 SET-containing proteins to devise pharmacological similarities between them. The main advantage of this approach is that it is not restricted to proteins for which crystallographic data with bound ligands is available. The resulting family organization from the separate analysis of both sites (cofactor and substrate) was retrospectively and prospectively validated. Of note, three hits (inhibition > 50% at 10 µM) were identified for the orphan NSD1.
Highlights
Protein lysine methyltransferases (PKMTs) are proteins that methylate histone and nonhistone proteins by catalyzing the transfer of the methyl group of the cofactor S-adenosyl-l-methionine (SAM or AdoMet) to a lysine residue of its corresponding substrate and yielding S-adenosyl-l-homocysteine (SAH or AdoHcy)
Cofactor binding site Analysis of bound ligand interactions A comprehensive study of the cofactor binding cavity of 10 SET protein lysine methyltransferase (PKMT), 3 classical protein arginine methyltransferase (PRMT) and the non-SET PKMT DOT1L was done by Campagna-Slater et al in 2011 using GRID maps [19]
Our analysis of explicit interactions for the 23 SETdomain containing PKMTs reveals that most of the 1320 residue-based contacts detected with Protein ligand interaction fingerprints (PLIF) correspond to hydrogen–bond interactions (86%), with a preference for the ligand being hydrogen bond donor (HBD) (50.6%) versus hydrogen bond acceptor (HBA) (35.4%)
Summary
Protein lysine methyltransferases (PKMTs) are proteins that methylate histone and nonhistone proteins by catalyzing the transfer of the methyl group of the cofactor S-adenosyl-l-methionine (SAM or AdoMet) to a lysine residue of its corresponding substrate and yielding S-adenosyl-l-homocysteine (SAH or AdoHcy). PKMTs. Rabal et al J Cheminform (2018) 10:32 the C-terminal segment of SET [4, 5]. Rabal et al J Cheminform (2018) 10:32 the C-terminal segment of SET [4, 5] This pseudoknotmotif contains the highly conserved NHS/CxxPN motif, where x is any amino acid, and is in close proximity with the loop having the second highly conserved ELxF/YDY motif (the last Y being the catalytic residue). A third highly conserved motif is the GxG triplet at the N-terminal region. The core SET domain forms part of the catalytic domain and is flanked by non-conserved set of regions like the i-SET and post-SET (cSET) domain that form the binding groove for the substrate peptide. The cofactor binds at a different pocket, partially contributed by the post-SET domain and connected by a narrow hydrophobic binding channel to the substrate binding site. Depending on the PKMT subfamily, SET domains can be flanked by Pre-SET, N-SET, MYND and CTD domains [5]
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