A high-throughput, competitive fluorescence polarization immunoassay has been developed for the detection of methyltransferase activity. The assay was designed to detect Sadenosylhomocysteine (AdoHcy), a product of all S-adenosylmethionine (AdoMet)-utilizing methyltransferase reactions. We employed commercially available anti-AdoHcy antibody and fluorescein-AdoHcy conjugate tracer to measure AdoHcy generated as a result of methyltransferase activity. AdoHcy competes with tracer in the antibody/tracer complex. The release of tracer results in a decrease in fluorescence polarization. Under optimized conditions, AdoHcy and AdoMet titrations demonstrated that the antibody had more than a 150-fold preference for binding AdoHcy relative to AdoMet. Mock methyltransferase reactions using both AdoHcy and AdoMet indicated that the assay tolerated 1 to 3 μM AdoMet. The limit of detection was approximately 5 nM (0.15 pmol) AdoHcy in the presence of 3 μM AdoMet. To validate the assay's ability to quantitate methyltransferase activity, the methyltransferase catechol-Omethyltransferase (COMT) and a known selective inhibitor of COMT activity were used in proofof-principle experiments. A time-and enzyme concentration-dependent decrease in fluorescence polarization was observed in the COMT assay that was developed. The IC 50 value obtained using a selective COMT inhibitor was consistent with previously published data. Thus, this sensitive and homogeneous assay is amenable for screening compounds for inhibitors of methyltransferase activity. KeywordsFluorescence polarization; Methyltransferase; S-Adenosylhomocysteine; S-Adenosylmethionine; Catechol-O-methyltransferase Methyltransferases are a diverse class of enzymes that catalyze the transfer of a single methyl group from a methyl donor molecule to a methyl acceptor. The vast majority use Sadenosylmethionine (AdoMet) 1 as the methyl donor [1,2]. AdoMet is the second most HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptwidely used substrate molecule in the cell after ATP. Methylation is an essential and highly choreographed regulatory metabolic process. Small organic molecules and peptides and most types of cellular macromolecules, including DNA, RNA, protein, and lipids, can serve as substrates for methylation (for detailed reviews, see Refs. [2][3][4][5][6][7]).A rapidly growing area of methyltransferase research focuses on methylation of histone tails by histone methyltransferases (HMTs) (for reviews, see Refs. [8,9]). Histone methylation is an epigenetic modification that has a role in formation of heterochromatin, X chromosome inactivation, and modification of gene expression patterns. Histone methylation occurs on arginine and lysine residues and is catalyzed by methyltransferases belonging to three distinct families of proteins: the protein arginine N-methyltransferase 1 (PRMT1) family, the SET-domain-containing protein family, and the non-SET-domain proteins DOT1/DOT1L. This histone methylation alters chromatin structure and, thus, regulates...

PON1 has been demonstrated to be the serum enzyme responsible for detoxifying organophosphate chemical weapons and plays a protective role against atherosclerosis. In order to identify small molecules that modulate PON1 activity in serum, we developed a high throughput kinetic absorbance assay using mouse serum and the organophosphate paraoxon. The IC50 value obtained for the known PON1 inhibitor, 2-hydroxyquinoline, matched the value reported for purified PON1. A compound library was screened resulting in no confirmed activators, but 12 confirmed inhibitors. Seven of these hits also inhibited purified human PON1. One compound was only two-fold less potent than 2-hydroxyquinoline in the serum assay, but 10-fold more potent against purified PON1. This compound (IC50 = 420 nM) may be useful towards a chemical probe for PON1. Therefore, this assay has utility as a high throughput assay for discovery of small molecule modulators of PON1 activity that maintain activity in serum.

No abstract