Methylation is a ubiquitous and reversible covalent modification used to control the function of diverse biomolecules including their activity, stability and localization. Like phosphorylation, methylation is used to modulate protein function, but in addition many small molecules are regulated by methylation including hormones, neurotransmitters, xenobiotics, and lipids. More than 50 distinct methyltransferase (MT's) enzymes are present in humans, and not surprisingly they are emerging as therapeutic targets for a broad range of diseases. Their involvement in neurodegenerative disease pathways is of special relevance because of the central role that methylation plays in regulating neurotransmitter function. 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 robust, flexible enzyme assay methods adaptable to high throughput screening (HTS). To address this need, in Phase I BellBrook Labs developed a novel fluorescence polarization immunoassay for S-adenosylhomocysteine (SAH), the invariant product of all MT reactions. This required development of a highly selective antibody that is able to differentiate between SAH and the substrate S-adenosylmethionine on the basis of a single methyl group. The Transcreener MT assay enables detection of any MT with any acceptor substrate in a homogenous format - and without the use of coupling enzymes - thus it eliminates all of the shortcomings associated with alternative methods. In Phase II we propose to 1) rapidly commercialize a first generation, fluorescence polarization-based Transcreener MT assay based of the polyclonal antibodies developed in Phase I, 2) increase flexibility and ease-of-use for the MT assay by using targeted mutagenesis of recombinant IgG clones to increase antibody selectivity for SAH, 3) increase HTS market penetration by formatting the assay for a second commonly used fluorescent detection mode, TR-FRET, and 4) use the novel MT assay combined with in silico screening to develop isoform selective inhibitors for five small molecule methyltransferases. The availability of robust, generic fluorescent HTS assay methods and selective chemical probes for MT's should greatly accelerate their validation as drug targets and movement into the pharma drug discovery pipeline.