Human carboxylesterase 1 stereoselectively binds the nerve agent cyclosarin and spontaneously hydrolyzes the nerve agent sarin

Abstract

Organophosphorus (OP) nerve agents are potent toxins that inhibit cholinesterases and produce a rapid and lethal cholinergic crisis. Development of protein-based therapeutics is being pursued with the goal of preventing nerve agent toxicity and protecting against the long-term side effects of these agents. The drug-metabolizing enzyme human carboxylesterase 1 (hCE1) is a candidate protein-based therapeutic because of its similarity in structure and function to the cholinesterase targets of nerve agent poisoning. However, the ability of wild-type hCE1 to process the G-type nerve agents sarin and cyclosarin has not been determined. We report the crystal structure of hCE1 in complex with the nerve agent cyclosarin. We further use stereoselective nerve agent analogs to establish that hCE1 exhibits a 1700- and 2900-fold preference for the P(R) enantiomers of analogs of soman and cyclosarin, respectively, and a 5-fold preference for the P(S) isomer of a sarin analog. Finally, we show that for enzyme inhibited by racemic mixtures of bona fide nerve agents, hCE1 spontaneously reactivates in the presence of sarin but not soman or cyclosarin. The addition of the neutral oxime 2,3-butanedione monoxime increases the rate of reactivation of hCE1 from sarin inhibition by more than 60-fold but has no effect on reactivation with the other agents examined. Taken together, these data demonstrate that hCE1 is only reactivated after inhibition with the more toxic P(S) isomer of sarin. These results provide important insights toward the long-term goal of designing novel forms of hCE1 to act as protein-based therapeutics for nerve agent detoxification.

Fig. 1.

Organophosphate nerve agents and nerve agent analogs. Nerve agents contain a methyl, O-alkoxy, and good leaving group built on a central chiral phosphonate. The stereogenic OP nerve agent analogs contain a thiomethyl leaving group in place of the fluoride.

Fig. 2.

hCE1 monomer. Each protein monomer has three domains; the α/β domain (blue) forms the trimer interface and caps the active site; the regulatory region (orange) contains the secondary surface binding site (Z-site) and Glu354 of the catalytic triad; and the catalytic domain (green) contains the central β-sheet conserved in serine hydrolases and the two catalytic residues His468 and Ser221. There are two disulfide bonds (cyan), one in the α/β domain and the other in the catalytic domain, and one glycosylation site, at Asn79 (yellow).

Fig. 3.

hCE1-cyclosarin complex. A, chemical scheme of hCE1 reacting with cyclosarin. B, cut-away view of the hCE1-cyclosarin active site. The catalytic triad and cyclosarin molecule are shown in purple, oxyanion hole in white, and the surrounding residues in green. Hydrogen bonds between the phosphoryl oxygen and the backbone nitrogen atoms in the oxyanion hole are shown in red.

Fig. 4.

hCE1-cyclosarin adduct stereochemistry. Ser221 and His468 of the catalytic triad and the cyclosarin adduct are shown in purple. The oxyanion hole is colored white. A, stereoview of a 3.1-Å resolution F_o − F_c simulated annealing omit map (blue, contoured to 5 σ) calculated for the P_R-cyclosarin adduct. B, stereoview of initial difference density maps for the incorrect P_S-cyclosarin adduct (green for positive, shown at 3 σ; red for negative, shown at −3 σ).

Fig. 5.

Bimolecular rates of inhibition (k_i) for hCE1 and nerve agent analogs. Plotted as log k_i, hCE1 exhibits strong enantiomeric P_R preference for soman and cyclosarin analogs, with only slight P_S sarin analog selectivity. Dissociation constants (K_d) are similar to authentic nerve agents, whereas phosphorylation rates (k₂) are 2 to 3 orders of magnitude slower. (n = 3, S.E.)

Fig. 6.

hCE1 reactivation after inhibition by racemic bona fide OP nerve agents or stereospecific nerve agent analogs. A, spontaneous reactivation of racemic bona fide OP agents. Reactivation only occurs with sarin (■), with a half-time of reactivation of 45 h (n = 5, S.D.). Soman (●) and cyclosarin (♦) remained permanently inhibited. B, DAM-assisted reactivation with racemic bona fide nerve agents. Reactivation is only measured against sarin (■) with a half-time of reactivation of 41 min (n = 3, S.E.). Soman (●) and cyclosarin (♦) did not reactivate. C, DAM-assisted reactivation of sarin analogs. The P_S isomer (■) reactivates with a half-time of 30 min (n = 3, S.E.). P_R sarin (□) analog did not reactivate. D, DAM-assisted reactivation of soman analogs. The greatest reactivation was measured in the P_S (○) enantiomer, whereas the P_R (●) analog remained inhibited. E, DAM, assisted reactivation of cyclosarin analogs. Negligible reactivation was measured for either P_S (♢) or P_R (♦) cyclosarin analog. F, determination of aging with P_S nerve agent analogs; no aging was measured for the P_S species.

Fig. 7.

hCE1-sarin model. A, P_R and P_S enantiomers of sarin modeled in the hCE1 active site. The O-isopropyl group on P_S sarin makes hydrophobic contacts with Phe101 and Leu97, whereas P_R does not have any additional interactions. B, model of proposed mechanism of P_S sarin reactivation in hCE1. In AChE, His468 has been observed to rotate away from Glu354 and interact with Glu221 after acylation. Modeling this shift in the hCE1 active site, there is an electronic network formed between Tyr152 (green) and His468 (yellow) that may either deprotonate Nε2 or allow Nδ1 of His468 to act as a general base for water activation. P_S sarin (blue) was modeled into the hCE1-soman structure (RCSB PDB access code 2hrq; Fleming et al., 2007).