Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.
Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.
Briseño-Roa
Luis
L
Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, UK.
Hill
Jim
J
Notman
Stuart
S
Sellers
David
D
Smith
Andy P
AP
Timperley
Christopher M
CM
Wetherell
Janet
J
Williams
Nichola H
NH
Williams
Gareth R
GR
Fersht
Alan R
AR
Griffiths
Andrew D
AD
D000110:Acetylcholinesterase / Q000187:drug effects*; D000818:Animals; D043303:Aryldialkylphosphatase / Q000737:chemistry; D002619:Chemical Warfare Agents / Q000737:chemistry* / Q000633:toxicity; D002800:Cholinesterase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000633:toxicity; D003374:Coumarins / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D006168:Guinea Pigs; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000138:chemical synthesis / Q000737:chemistry* / Q000633:toxicity; D009946:Organothiophosphorus Compounds / Q000737:chemistry / Q000633:toxicity*; D010575:Pesticides / Q000737:chemistry; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry*; D013329:Structure-Activity Relationship
D002619:Chemical Warfare Agents; D002800:Cholinesterase Inhibitors; D003374:Coumarins; D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D009946:Organothiophosphorus Compounds; D010575:Pesticides; D000110:Acetylcholinesterase; D044345:Phosphoric Triester Hydrolases; D043303:Aryldialkylphosphatase; C451779:S-(N,N-diethylaminoethyl) isobutyl methylphosphothiolate