Small Molecule Inhibitors
Victoria Korboukh, Ph.D., post-doctoral scientist at the University of North Carolina’s Center for Integrative Chemical Biology & Drug Discovery, described her team’s research on finding small molecule inhibitors for histone methyltransferase G9a, an enzyme of the protein lysine methyltransferases (PKMTs) group.
These enzymes mediate methylation of histone proteins in nucleosomes, the structures that comprise the basic units of chromosomes. Nucleosomes consist of double-stranded DNA wrapped around a protein octamer containing two copies each of the histone proteins H2A, H2B, H3, and H4.
Histone proteins undergo a variety of post-translational modifications including methylation, occurring within the histone core region as well as on the N-terminal tails that protrude from the core region. Methylation, among other modifications, affects DNA regulatory processes including replication, repair, and transcription.
In particular, the PKMTs and PRMTs that modify lysine and arginine residues within histone proteins have been correlated with a variety of human disease states including rheumatoid arthritis, cancer, heart disease, diabetes, as well as neurodegenerative disorders such as Parkinson and Alzheimer diseases.
As part of understanding the catalytic mechanisms of these enzymes, Dr. Korboukh and her colleagues are looking for small molecule chemical probes that could be used to validate targets either in cell-based or in vivo experiments. In light of the importance of these enzymes in a large variety of human disease states, it is critical to elucidate their catalytic mechanisms, in particular G9a and GLP, both of which modulate transcriptional repression of a variety of genes via dimethylation of Lys9 on histone H3 (H3K9me2) as well as dimethylation of nonhistone targets.
To facilitate chemical exploration of these proteins, Dr. Korboukh and her colleagues, working with Caliper technology, developed a highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes, as well as the kinetics of their inhibition. This technology separates small biomolecules, in this case peptides, based on their charge-to-mass ratio.
But since methylation doesn’t change the charge of peptide substrates, the investigators used a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules.
Because methylation produces only a minor change in the substrate’s mass and itself bears no charge, separation of the substrate and the product has been impossible, Dr. Korboukh noted.
Typically, she said, researchers use radiolabeled co-factor, s-adenosylmethionine, to study this class of enzymes, as well as coupled fluorescent assay. In the fluorescent assay, SAHH (s-adenosylhomocysteine hydrolase) and adenosine deaminase convert the methyltransferase reaction by-product (s-adenosyl homocysteine) to homocysteine and inosine. Homocysteine is then quantified using ThioGlo.
Dr. Korboukh explained that her group has developed a high-throughput assay employing Endo LysC proteinase, which cleaves proteins on the C-terminal side of the lysine residues. Activity of the Endo LysC is inhibited if the lysine is methylated.
“In our assays we use short fluorescently labeled peptides as substrates that are subjected to Endo LysC proteolysis after methylation reaction. If the PKMT is active—the methyl mark on the lysine will protect the peptide from cleavage, if the activity is inhibited—unmethylated peptide will be cleaved with Endo LysC.
“Now we have a significant change in mass-to-charge ratio of the product vs. substrate, and the two can be separated and quantified using the Caliper EZ Reader platform. Although coupling methylation reaction with Endo LysC makes it an endpoint assay, this platform still allows detailed kinetic studies of the enzyme of interest.”
Use of this assay to study enzyme activity and kinetics allowed the development of a probe, UNC638, which Dr. Korboukh and her colleagues said provides high potency, excellent selectivity, low cell toxicity, and robust on-target activities in cells, making it the preferred chemical probe of G9a/GLP34.