High-throughput methyltransferase assays using recombinant nucleosome substrates

PROJECT SUMMARYHistone methyltransferasesHMTsare a group of enzymes that represent a critical focal point in the linkage of cellular signals to the regulation of gene expressionThese proteins are not only strongly associated with numerous diseasese gcancersneurodegenerative diseasesand autoimmune disordersbut also have properties that make them excellent targets for drugsThusHMTs provide an exceptional therapeutic opportunityCurrentlythe tests that are used to discover new HMT drugs rely primarily on reagents known as peptidesThese peptides represent only a tiny portion of nucleosomesthe natural HMT substrateThe use of the peptides in tests for HMT activity poorly mimics the physical interactions that occur between HMTs and nucleosomes in a living cellThis can result in findings that are often not recapitulated in living organismsEpiCypher has developed the ability the manufacture and commercialize pure recombinant nucleosomesknown as dNucsthat are highly similar to naturally occurring nucleosomesHerewe propose to develop the reagents and methods needed to manufacture and distribute HMT activity tests that utilize dNucs as key componentsThese kits will allow researchers to efficiently screen up to tens of thousands of potential drug molecules for HMT modulating activityDuring the Phase I portion of this workwe developed the AlphaNucHMT assaya novel nucleosome based HMT activity test using PerkinElmerandapos s AlphaScreen technologyThis study provided strong proof of concept that EpiCypherandapos s dNuc technology will provide the resolution and reproducibility needed for rapidly testing potential drug moleculesDuring this Phase II workEpiCypher will expand the HMT AlphaNucassay for a family of HMTs with known relevance in diseasewhich includes the high sought after drug targetthe NSDenzymeThis work will include an optimization strategy that ensures that the tests are robust and suitable for the high throughput screening of potential drug moleculesAs part of this development processand to check the dNuc reagents and protocols in a real world scenarioEpiCypher will conduct a pilot screen of a chemical library for inhibitors of the NSDHMT using dNucs as substratesThis screen will serve to validate this platform for use in drug screens and to provide insight into how changes in dNuc methylation status alter screening outcomesFinallywe will develop the methods needed to scale up commercial manufacturing of dNucsThese optimizations will enable EpiCypher to generate large quantities of dNucsto orderand at a dramatically reduced price pointPROJECT NARRATIVE Histone methyltransferasesHMTsare enzymes that act as key components of the machinery that allow cells to alter gene expression in response to extracellular stimuliMutations in these proteins are strongly associated with numerous diseasese gcancerneurodegenerative diseasesand autoimmune disordersThe methods under development here will provide researchers with novel and optimized tools for the identification of new drugs that can modulate the activity of these enzymes and eventually be brought to market as lifesaving therapies