Analogues with Fluorescent Leaving Groups for Screening and Selection of Enzymes That Efficiently Hydrolyze Organophosphorus Nerve Agents Luis Brisen ˜o-Roa, ² Jim Hill, § Stuart Notman, § David Sellers, § Andy P. Smith, § Christopher M. Timperley,* ,§ Janet Wetherell, § Nichola H. Williams, § Gareth R. Williams, § Alan R. Fersht, ² and Andrew D. Griffiths ‡ Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, UK, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK, and Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire, SP4 0JQ, UK ReceiVed June 2, 2005 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 organophos- phatases. Introduction Organophosphorus (OP) compounds are used as pesticides and chemical warfare (CW) agents. 1 The discovery of the pesticide dimefox occurred around the time of the development of the first nerve agent (NA) tabun, which was weaponised but not used during the Second World War (Figure 1). Diisopropyl fluorophosphate (DFP) was investigated as a potential war gas in the same period, 2 while the development of the more potent NAs sarin, soman, and cyclosarin followed later. 3 By the late- 1950s, more toxic NAs with a different structure were synthe- sized, including VX and its isomer Russian-VX. 4 These and other related NAs pose a threat to military personnel and civilian populations. OP compounds can inhibit acetylcholinesterase (AChE), 5 an enzyme that controls nerve impulse transmission by hydrolyzing acetylcholine to acetic acid and choline. Hydrolysis of acetyl- choline by AChE involves an active-site serine residue initiating a nucleophilic attack on the carbonyl carbon of acetylcholine to form a covalent acetyl-enzyme intermediate, concurrent with the release of free choline from the active site. The free enzyme is regenerated in a second step via a hydrolytic attack by water and the release of acetate. The NAs mimic the natural substrate of AChE, they phosphorylate the active site serine residue while losing either a cyanide, fluoride, or N,N-dialkylaminoethanethi- olate group. This first step is fast, but the regeneration of the free enzyme through the nucleophilic attack by water is extremely slow, creating a phosphorylated AChE unable to hydrolyze acetylcholine. 6 Inhibitors having a secondary ester group, once bound to AChE, are prone to ‘aging’, a term describing cleavage of the PO-C bond with loss of a carbenium ion, sometimes within minutes (half-life < 2 min in the case of soman). 2,3 The negative charge on the bound inhibitor renders the phosphorus atom resistant to attack by nucleophiles such as oximes, thus preventing reactivation of AChE. A number of enzymes have been identified that can catalyze the hydrolysis of OP compounds, including CW agents. Two of the best characterized are the phosphotriesterase (PTE) from Pseudomonas diminuta 7 and PON1, a member of the serum paraoxonase family. 8 With its best substrate, the pesticide paraoxon, the turnover rate (k cat ) of PTE is high (>2280 s -1 ) and its catalytic efficiency (k cat /K M ) of 6.2 × 10 7 M -1 s -1 is close to the limit set by the diffusion-controlled encounter of the enzyme and the substrate. 1,9 PTE can also catalyze the hydrolysis of the NAs sarin and soman, and VX-type NAs. However, the catalytic efficiency of PTE toward NAs is 10 3 to 10 5 -fold lower than for paraoxon (Table 1). If it was possible to engineer a variant PTE which could efficiently hydrolyze NAs, it would have several potential * To whom correspondence should be sent. E-mail: cmtimperley@ dstl.gov.uk. Fax: 44 (0) 1980 613834. Tel: 44 (0) 1980 613566. ² Medical Research Council Centre for Protein Engineering. ‡ Medical Research Council Laboratory of Molecular Biology. § Defence Science and Technology Laboratory. Figure 1. Structures of the main military nerve gases, their prototypes dimefox and diisopropyl fluorophosphate (DFP), and the pesticides paraoxon and coumaphos. 246 J. Med. Chem. 2006, 49, 246-255 10.1021/jm050518j CCC: $33.50 © 2006 American Chemical Society Published on Web 12/15/2005