title: Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.
abstract: Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads.
authors.affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Chormova
authors.affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors.forename: Lenka
authors.initials: L
authors.lastname: Franková
authors.affiliation: Department of Botany and Plant Sciences, Center for Plant Cell Biology, Department of Chemistry (CFM), University of California, 5451 Boyce Hall, Riverside, CA 92521, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Defries
authors.affiliation: Department of Botany and Plant Sciences, Center for Plant Cell Biology, Department of Chemistry (CFM), University of California, 5451 Boyce Hall, Riverside, CA 92521, USA.
authors.forename: Sean R
authors.initials: SR
authors.lastname: Cutler
authors.affiliation: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, School of Biological Sciences, The University of Edinburgh, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK. Electronic address: S.Fry@Ed.Ac.UK.
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Fry
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002473:Cell Wall / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005936:Glucans / Q000378:metabolism; D016695:Glycosyltransferases / Q000037:antagonists & inhibitors*; D057166:High-Throughput Screening Assays / Q000379:methods*; D020128:Inhibitory Concentration 50; D008027:Light; D028528:Petroselinum / Q000201:enzymology; D012256:Riboflavin / Q000494:pharmacology; D027826:Rosa / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013634:Tannins / Q000737:chemistry / Q000494:pharmacology; D015262:Xenobiotics / Q000737:chemistry / Q000494:pharmacology; D014990:Xylans / Q000378:metabolism; D003313:Zea mays / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D005936:Glucans; D054852:Small Molecule Libraries; D013634:Tannins; D015262:Xenobiotics; D014990:Xylans; C029353:xyloglucan; D016695:Glycosyltransferases; C473049:xyloglucan - xyloglucosyltransferase; D012256:Riboflavin
keywords: Anthraquinones; Cell wall; Chemical genetics; Dot-blot assay; Flavonoids; Riboflavin; Singlet oxygen; Sulphydryl reagents; Tannins; Xyloglucan endotransglucosylase (XET)