title: A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase.
abstract: Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.
authors.affiliation: Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Str. 24, 50931, Cologne, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Hauser
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstr. 400, 01328, Dresden, Germany.|Department of Chemistry and Food Chemistry, Technical University Dresden, Mommsenstraße 4, 01062, Dresden, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Wodtke
authors.affiliation: Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Bautzner Landstr. 400, 01328, Dresden, Germany. r.loeser@hzdr.de.|Department of Chemistry and Food Chemistry, Technical University Dresden, Mommsenstraße 4, 01062, Dresden, Germany. r.loeser@hzdr.de.
authors.forename: Reik
authors.initials: R
authors.lastname: Löser
authors.affiliation: Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Str. 24, 50931, Cologne, Germany. markus.pietsch@uk-koeln.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Pietsch
mesh_terms: D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D002364:Caseins / Q000737:chemistry; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry*; D019793:Fluorescein / Q000138:chemical synthesis; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry; D006168:Guinea Pigs; D057166:High-Throughput Screening Assays; D006801:Humans; D007460:Iodoacetamide / Q000737:chemistry; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011994:Recombinant Proteins / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry; D060327:Solid-Phase Synthesis Techniques; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D002364:Caseins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012235:Rhodamines; D006160:Guanosine Triphosphate; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; C029773:rhodamine B; D002103:Cadaverine; D019793:Fluorescein; D007460:Iodoacetamide
keywords: Active-site titration; Cadaverine; Enzyme inhibition; Fluorescent labeling; Transglutaminases; Xanthene dyes