REACTION BIOLOGY CORPORATION — Department of Health and Human Services SBIR Phase I: 102

Description

DESCRIPTION provided by applicant Post translational modification PTM of the lysines and other residues in the N terminal tails of histones are known to have profound effects on the structure and function of chromatin perhaps most notably on the regulation of gene expression One of these andapos epigeneticandapos histone modifications the PTM lysine acetylation is generally associated with gene activation Epigenetic regulation of gene expression plays significant roles in normal development as well as in diseases including cancer neurodegeneration and metabolic disease The andapos perceptionandapos of the chromatin acetylation state is mediated by proteins that contain a type of modular andquot epigenetic readerandquot domain a bromodomain The human bromodomains different domains present in distinct proteins are the principal known class of acetyllysine readers The bromodomains particular a subclass known as the BET family are implicated in various disease states including cancer inflammation type II diabetes atherosclerosis and viral infections Investigation of the biological roles and disease connections of the BET family has been aided tremendously by the development of high affinity inhibitors sometimes termed chemical andquot probesandquot which also show promise as possible therapeutic agents for cancer and other diseases The development of probes for many of the non BET bromodomains however has lagged and to some extent this may be due to deficiencies in the available assay systems which can on the one hand be simple to use and relative artefact free albeit not very quantitative thermal melt shift aka Differential Scanning Fluorimetry and on the other hand highly sensitive and quantitative assays AlphaScreen that are expensive relative difficult to implement and prone to numerous andapos false hitandapos artefact when screening compound libraries We propose to address this problem by developing a suite of simple to perform quantitative but inexpensive fluorescence polarization FP assays based on fluorophor conjugated bromodomain inhibitor compounds Our goal is for these assays to be broadly applicable to the human bromodomains In order to accomplish that efficiently we propose to screen for andapos promiscuousandapos bromodomain inhibitors so that relatively few of these in fluorophor conjugated form will be needed to allow convenient assay of the entire set of human bromodomains We argue and have some preliminary evidence to suggest that phenolic plant natural products particularly the chalcones and flavones may be relatively rich in suitable inhibitor compounds Hits from screening these and other compounds classes along with already developed probe compounds will be coupled to fluorophors to produce FP probes and these will be used to develop the assays PUBLIC HEALTH RELEVANCE Histones proteins which are packaged together with DNA into chromatin can be chemically modified in a number of ways including methylation on specific lysines in their amino acid sequences Such modifications can regulate gene expression and are termed andquot epigeneticandquot because while they sometimes can be inherited they do not alter the cellandapos s genetics the underlying DNA sequence Epigenetic regulation of gene expression is essential to normal development and when dysfunctional can be involved in the development and maintenance of disease states including cancer neurodegeneration and metabolic disorders This has led to intense research into epigenetic modifications e g NIHandapos s Epigenomics Program as well as efforts to discover drugs that affect the enzymes that add or remove these modifications Two FDA approved drugs for cutaneous T cell lymphoma CTCL target enzymes histone deacetylases HDACs that reverse acetylation of histone lysines The andapos perceptionandapos of the chromatin acetylation state is mediated by proteins that contain a type of modular andquot epigenetic readerandquot domain a bromodomain The human bromodomains different domains present in distinct proteins are the principal known class of acetyllysine readers The bromodomains particularly a subclass known as the BET family are implicated in various disease states including cancer inflammation type II diabetes atherosclerosis and viral infections While many high affinity inhibitors andquot probesandquot for the BET family have been developed the development of probes for many of the non BET bromodomains however has lagged and to some extent this may be due to deficiencies in the available assay systems We propose to address this problem by developing a suite of simple to perform quantitative but inexpensive fluorescence polarization FP assays based on fluorophor conjugated bromodomain inhibitor compounds