Fluorescent HTS Assay for Methyltransferases in Neurodegenerative Diseases

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DESCRIPTION (provided by applicant): Project Summary/Abstract: Methylation is a ubiquitous covalent modification used to control the function of diverse biomolecules including hormones, neurotransmitters, xenobiotics, proteins, nucleic acids and lipids. More than 50 distinct methyltransferase (MTs) enzymes are present in humans, and they are being targeted for a broad range of diseases, but their involvement in neurodegenerative disease pathways is of special relevance. Modulation of neurotransmitter methylation is an emerging therapeutic strategy for the treatment of several neurodegenerative diseases, most notably Parkinson's and Alzheimer's diseases, and DNA and protein MTs are also being targeted for neurodegenerative diseases and cancers of the CNS. Moreover, the involvement of some MT family members in disease pathways is intertwined with their role in metabolizing commonly prescribed drugs. The development of highly selective MT modulators is clearly a compelling medical priority. However, efforts to achieve this are being hampered by a lack of flexible enzyme assay methods adaptable to high throughput screening (HTS). The current use of isozyme-specific assay methods that rely on radioactivity and cumbersome post reaction processing steps is preventing facile incorporation of diverse MT enzymes into pharmaceutical drug discovery programs. To accelerate efforts to identify selective MT inhibitors we propose to develop a universal catalytic assay that will enable screening of all members of the MT family using a single set of detection reagents. The method is based on detection of the invariable MT reaction product S-adenosyl homocysteine-using a competitive fluorescence polarization immunoassay. The Phase I work includes development of antibodies and fluorescent nucleotide conjugates that enable highly selective detection of S-adenosyl homocysteine in the presence of S-adenosyl methionine, which differs only in the addition of a methyl group. Project Narrative: Aberrant behavior by several proteins in the methyltransferase family is a possible cause of debilitating neurodegenerative disorders like Alzheimer's disease and Parkinson's disease. To accelerate the discovery of improved therapies for these diseases, we are developing screening kits to find drug molecules that correct the malfunctioning methyltransferase proteins.