title: A fluorescent HTS assay for phosphohydrolases based on nucleoside 5'-fluorophosphates: its application in screening for inhibitors of mRNA decapping scavenger and PDE-I.
abstract: Several nucleotide-specific phosphohydrolases can cleave P-F bonds in substrate analogues containing a fluorophosphate moiety to release fluoride ions. In this work, by employing a fluoride-sensitive molecular sensor, we harnessed this cleavage reaction to develop a fluorescence assay to screen for phosphohydrolase inhibitors. The assay is rapid, sensitive, and based on simple and synthetically available reagents. The assay was adapted to the high-throughput screening (HTS) format and its utility was demonstrated by screening an 'in-house' library of small nucleotides against two enzymes: DcpS, a metal-independent mRNA decapping pyrophosphatase of the histidine triad (HIT) family; and PDE-I, a divalent cation-dependent nuclease. Our screening results agreed with the known specificities of DcpS and PDE-I, and led to the selection of several inhibitors featuring low-micromolar IC50 values. For DcpS, we also verified the results by using an alternative method with the natural substrate. Notably, the assay presented here is the first fluorescence-based HTS-adaptable assay for DcpS, an established therapeutic target for spinal muscular atrophy. The assay should be useful for phosphohydrolase specificity profiling and inhibitor discovery, particularly in the context of DcpS and other HIT-family enzymes, which play key roles in maintaining cellular functions and have been linked to disease development.
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland. asia@biogeo.uw.edu.pl.
authors.forename: M R
authors.initials: MR
authors.lastname: Baranowski
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland. asia@biogeo.uw.edu.pl and Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Nowicka
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland.
authors.forename: J
authors.initials: J
authors.lastname: Jemielity
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland. asia@biogeo.uw.edu.pl.
authors.forename: J
authors.initials: J
authors.lastname: Kowalska
mesh_terms: D000818:Animals; D017839:Crotalus; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000037:antagonists & inhibitors*; D004722:Endoribonucleases / Q000037:antagonists & inhibitors*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D012315:RNA Caps / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D004791:Enzyme Inhibitors; D012315:RNA Caps; D004722:Endoribonucleases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1