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A novel protein engineering tool for rapid manufacturing of designer nucleosomes

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R41CA214076-01
Agency Tracking Number: R41CA214076
Amount: $224,999.00
Phase: Phase I
Program: STTR
Solicitation Topic Code: 103
Solicitation Number: PA15-270
Solicitation Year: 2016
Award Year: 2016
Award Start Date (Proposal Award Date): 2016-09-20
Award End Date (Contract End Date): 2017-09-19
Small Business Information
Chapel Hill, NC 27514-4617
United States
DUNS: 078882699
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (919) 348-6601
Business Contact
Phone: (919) 923-3716
Research Institution

Nucleosomes are the basic building blocks of chromatin and composed of bp DNA wrapped
around a core histone octamer Chromatin function is controlled by the dynamic addition removal of histone
post translational modifications PTMs Significantly alterations in specific PTMs are associated with changes
in gene expression driving the emergence progression of many cancer pathologies Mounting evidence
indicates that screening chromatin modifiers in the context of nucleosomal substrates is essential to
recapitulate specificity toward appropriate PTM sites and thus biologically relevant activity However PTM
regulators continue to be studied via modified histone fragments which poorly mimic chromatin regulation in
vivo EpiCypher is pioneering the commercial development of nucleosomes carrying disease relevant PTMs
`designer nucleosomesandapos or `dNucsandapos for innovative drug discovery development The current leading
technology to generate modified histones for dNuc assembly is native chemical ligation NCL which permits
the scarless incorporation of diverse PTMs e g methylation acetylation and phosphorylation on a single
histone subunit However NCL is a multistep and labor intensive process that requires weeks of dedicated
effort to synthesize a single dNuc in the milligram scale Therefore a new methodology is greatly needed to
support the rapid generation of dNucs with diverse PTM layouts To meet this need EpiCypher is joining with
Dr Matthew Levy from The Albert Einstein College of Medicine to develop a novel protein engineering tool for
accelerated dNuc manufacturing The Staphylococcus aureus Sortase A SrtA transpeptidase can be modified
by directed evolution to alter its recognition sequence LPXTG where X any amino acid to seamlessly ligate
`unnaturalandapos protein substrates Dr Levyandapos s group recently developed a powerful directed evolution approach to
rapidly screen large mutant libraries generating a robust SrtA SrtA variant with improved activity
over wild type enzyme In Aim Dr Levyandapos s team will further evolve this highly active SrtA variant toward
histone H to rapidly incorporate PTMs into this substrate In Aim EpiCypher will demonstrate how this tool
can be used for accelerated dNuc manufacturing First we will optimize a single step ligation reaction to
synthesize large quantities of ultrapure dNucs for high throughput assay development We will also optimize
ligation of modified peptides directly to pre assembled nucleosomes providing a powerful diversity
manufacturing platform capable of multiplex synthesis In Phase we will utilize the SrtA H variant
developed here to scale up manufacturing of a diverse catalog of dNucs with disease associated PTMs In
addition we will optimize multiplexed dNuc manufacturing to develop dNuc based arrays for drug discovery
Finally we will continue to generate new SrtA variants that selectively target histone H The breakout
technology described herein will provide transformational opportunities for the development of next generation
cancer therapeutics and epigenetic tool development
Semi synthetic nucleosomes carrying specific post translational modifications known as designer
nucleosomes represent powerful substrates for novel drug discovery and development However their
potential is limited by the large investment of time and resources currently required for their synthesis Here
EpiCypher outline plans to vastly increase dNUC manufacturing throughput The innovative technology we
propose will enable the rapid generation of a diverse catalog of nucleosomes carrying disease relevant
modifications for high throughput drug screening applications or novel tool development

* Information listed above is at the time of submission. *

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