title: Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex.
abstract: Dcp1/2 is the major eukaryotic RNA decapping complex, comprised of the enzyme Dcp2 and activator Dcp1, which removes the 5' m(7)G cap from mRNA, committing the transcript to degradation. Dcp1/2 activity is crucial for RNA quality control and turnover, and deregulation of these processes may lead to disease development. The molecular details of Dcp1/2 catalysis remain elusive, in part because both cap substrate (m(7)GpppN) and m(7)GDP product are bound by Dcp1/2 with weak (mM) affinity. In order to find inhibitors to use in elucidating the catalytic mechanism of Dcp2, we screened a small library of synthetic m(7)G nucleotides (cap analogs) bearing modifications in the oligophosphate chain. One of the most potent cap analogs, m(7)GpSpppSm(7)G, inhibited Dcp1/2 20 times more efficiently than m(7)GpppN or m(7)GDP. NMR experiments revealed that the compound interacts with specific surfaces of both regulatory and catalytic domains of Dcp2 with submillimolar affinities. Kinetics analysis revealed that m(7)GpSpppSm(7)G is a mixed inhibitor that competes for the Dcp2 active site with micromolar affinity. m(7)GpSpppSm(7)G-capped RNA undergoes rapid decapping, suggesting that the compound may act as a tightly bound cap mimic. Our identification of the first small molecule inhibitor of Dcp2 should be instrumental in future studies aimed at understanding the structural basis of RNA decapping and may provide insight toward the development of novel therapeutically relevant decapping inhibitors.
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Ziemniak
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Mugridge
authors.affiliation: Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kowalska
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Rhoads
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Gross
authors.affiliation: Centre of New Technologies, University of Warsaw, 02-097 Warsaw, Poland.
authors.forename: Jacek
authors.identifier: http://orcid.org/0000-0001-7633-788X
authors.initials: J
authors.lastname: Jemielity
mesh_terms: D004353:Drug Evaluation, Preclinical; D012314:RNA Cap Analogs / Q000737:chemistry*; D059367:RNA Cleavage; D012333:RNA, Messenger / Q000737:chemistry; D012568:Schizosaccharomyces / Q000201:enzymology; D029702:Schizosaccharomyces pombe Proteins / Q000037:antagonists & inhibitors*
chemical_list: C527678:Dcp1 protein, S pombe; C508496:Dcp2 protein, S pombe; D012314:RNA Cap Analogs; D012333:RNA, Messenger; D029702:Schizosaccharomyces pombe Proteins
keywords: Dcp1/Dcp2; RNA metabolism; decapping enzymes; inhibitors; mRNA cap
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