title: Asparaginyl-tRNA synthetase pre-transfer editing assay.
abstract: Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-β-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 mM. The assay sensitivity was enhanced by the thiol agents, DTT and L-Cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.
authors.affiliation: Basilea Pharmaceutica International Ltd., Gren Zacherstrass 487, CH-4058 Basel, Switzerland. Franck.Danel@basileapharma.com
authors.forename: Franck
authors.initials: F
authors.lastname: Danel
authors.forename: Patrick
authors.initials: P
authors.lastname: Caspers
authors.forename: Claude
authors.initials: C
authors.lastname: Nuoffer
authors.forename: Michael
authors.initials: M
authors.lastname: Härtlein
authors.forename: Michael A
authors.initials: MA
authors.lastname: Kron
authors.forename: Malcolm G P
authors.initials: MG
authors.lastname: Page
mesh_terms: D000596:Amino Acids / Q000502:physiology; D000604:Amino Acyl-tRNA Synthetases / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D000818:Animals; D001216:Asparagine / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D001226:Aspartate-tRNA Ligase / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017178:Brugia malayi / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D004396:Coloring Agents; D003545:Cysteine / Q000378:metabolism; D055808:Drug Discovery; D057166:High-Throughput Screening Assays; D010936:Plant Extracts / Q000032:analysis*; D017393:RNA Editing / Q000502:physiology*; D012343:RNA, Transfer / Q000235:genetics / Q000502:physiology; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012394:Rosaniline Dyes; D013212:Staphylococcus epidermidis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004396:Coloring Agents; D010936:Plant Extracts; D012346:RNA, Transfer, Amino Acyl; D012394:Rosaniline Dyes; C005095:malachite green; C024848:beta-aspartylhydroxamic acid; D001216:Asparagine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase; D003545:Cysteine