title: Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies.
abstract: UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.
authors.affiliation: Department of Plant Physiology, Umeå Plant Science Center, Umeå University, Umeå, 90187, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Decker
authors.affiliation: Department of Chemistry, Laboratories for Chemical Biology Umeå, Umeå University, Umeå, 90187, Sweden.
authors.forename: Christopher
authors.initials: C
authors.lastname: Öberg
authors.affiliation: Department of Plant Physiology, Umeå Plant Science Center, Umeå University, Umeå, 90187, Sweden.
authors.forename: Leszek A
authors.initials: LA
authors.lastname: Kleczkowski
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase
keywords: 
  Arabidopsis thaliana
  ; Arabidopsis cell culture; UDP-sugar synthesis; chemical library screening; enzyme kinetics; inhibitors; pollen germination; pyrophosphorylases; reverse chemical genetics