title: High-throughput screen identifies cyclic nucleotide analogs that inhibit prostatic acid phosphatase.
abstract: The secretory and transmembrane isoforms of prostatic acid phosphatase (PAP) can dephosphorylate extracellular adenosine 5'-monophosphate (AMP) to adenosine, classifying PAP as an ectonucleotidase. Currently, there are no compounds that inhibit PAP in living cells. To identify small-molecule modulators of PAP, we used a 1536-well-based quantitative high-throughput fluorogenic assay to screen the Library of Pharmacologically Active Compounds (LOPAC(1280)) arrayed as eight-concentration dilution series. This fluorogenic assay used difluoro-4-methylumbelliferyl phosphate as substrate and collected data in kinetic mode. Candidate hits were subsequently tested in an orthogonal absorbance-based biochemical assay that used AMP as substrate. From these initial screens, three inhibitors of secretory human (h) and mouse (m)PAP were identified: 8-(4-chlorophenylthio) cAMP (pCPT-cAMP), calmidazolium chloride, and nalidixic acid. These compounds did not inhibit recombinant alkaline phosphatase. Of these compounds, only pCPT-cAMP and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) inhibited the ectonucleotidase activity of transmembrane PAP in a cell-based assay. These cyclic nucleotides are structurally similar to AMP but cannot be hydrolyzed by PAP. In summary, we identified two cyclic nucleotide analogs that inhibit secretory and transmembrane PAP in vitro and in live cells.
authors.affiliation: University of North Carolina, Chapel Hill, NC 27599-7545, USA.
authors.forename: Eric S
authors.initials: ES
authors.lastname: McCoy
authors.forename: Wendy A
authors.initials: WA
authors.lastname: Lea
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Mott
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Zylka
mesh_terms: D000135:Acid Phosphatase; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003199:Computer Systems; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D020128:Inhibitory Concentration 50; D051379:Mice; D009708:Nucleotidases / Q000378:metabolism; D009712:Nucleotides, Cyclic / Q000737:chemistry* / Q000494:pharmacology*; D063065:Organophosphonates / Q000737:chemistry / Q000494:pharmacology; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D009712:Nucleotides, Cyclic; D063065:Organophosphonates; D054852:Small Molecule Libraries; C472310:alpha-benzylaminobenzylphosphonic acid; D000249:Adenosine Monophosphate; D009708:Nucleotidases; D000135:Acid Phosphatase; C428439:prostatic acid phosphatase; D017027:Protein Tyrosine Phosphatases