title: Identification of PDE6D as a molecular target of anecortave acetate via a methotrexate-anchored yeast three-hybrid screen.
abstract: Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.
authors.affiliation: Alcon, a Novartis Company, Fort Worth, TX 76134, USA. allan.shepard@novartis.com
authors.forename: Allan R
authors.initials: AR
authors.lastname: Shepard
authors.forename: Raymond E
authors.initials: RE
authors.lastname: Conrow
authors.forename: Iok-Hou
authors.initials: IH
authors.lastname: Pang
authors.forename: Nasreen
authors.initials: N
authors.lastname: Jacobson
authors.forename: Mandana
authors.initials: M
authors.lastname: Rezwan
authors.forename: Katrin
authors.initials: K
authors.lastname: Rutschmann
authors.forename: Daniel
authors.initials: D
authors.lastname: Auerbach
authors.forename: Rohitha
authors.initials: R
authors.lastname: Sriramaratnam
authors.forename: Virginia W
authors.initials: VW
authors.lastname: Cornish
mesh_terms: D000818:Animals; D002460:Cell Line; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D008727:Methotrexate / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D015394:Molecular Structure; D011244:Pregnadienediols / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D020798:Two-Hybrid System Techniques
chemical_list: D011244:Pregnadienediols; C517432:PDE6D protein, human; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C418047:anecortave acetate; D008727:Methotrexate