title: Development of a Scintillation Proximity Assay (SPA) Based, High Throughput Screening Feasible Method for the Identification of PDE12 Activity Modulators.
abstract: The scintillation proximity assay (SPA) technology has been widely used to establish high throughput screens (HTS) for a range of targets in the pharmaceutical industry. PDE12 (aka. 2'- phosphodiesterase) has been published to participate in the degradation of oligoadenylates that are involved in the establishment of an antiviral state via the activation of ribonuclease L (RNAse-L). Degradation of oligoadenylates by PDE12 terminates these antiviral activities, leading to decreased resistance of cells for a variety of viral pathogens. Therefore inhibitors of PDE12 are discussed as antiviral therapy. Here we describe the use of the yttrium silicate SPA bead technology to assess inhibitory activity of compounds against PDE12 in a homogeneous, robust HTS feasible assay using tritiated adenosine-P-adenylate ([3H]ApA) as substrate. We found that the used [3H]ApA educt, was not able to bind to SPA beads, whereas the product [3H]AMP, as known before, was able to bind to SPA beads. This enables the measurement of PDE12 activity on [3H]ApA as a substrate using a wallac microbeta counter. This method describes a robust and high throughput capable format in terms of specificity, commonly used compound solvents, ease of detection and assay matrices. The method could facilitate the search for PDE12 inhibitors as antiviral compounds.
authors.forename: Samuel
authors.initials: S
authors.lastname: Mang
authors.forename: Hannes
authors.initials: H
authors.lastname: Bucher
authors.affiliation: Immunology & Respiratory Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Stra/3e 65, 88400 Biberach an der Riss, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Nickolaus
mesh_terms: D000818:Animals; D016367:Baculoviridae; D001681:Biological Assay; D004121:Dimethyl Sulfoxide; D015226:Dinucleoside Phosphates; D057166:High-Throughput Screening Assays; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012710:Serum Albumin, Bovine; D061987:Sf9 Cells; D017640:Silicates; D018411:Spodoptera; D015019:Yttrium
chemical_list: D015226:Dinucleoside Phosphates; D017640:Silicates; C031130:adenylyl-(2'-5')-adenosine; D012710:Serum Albumin, Bovine; C028301:yttrium silicate; D015019:Yttrium; D010727:Phosphoric Diester Hydrolases; D004121:Dimethyl Sulfoxide