Toward antibody-catalyzed hydrolysis of organophosphorus poisons

Abstract

We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-alpha-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-alpha-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M(-1) x min(-1) at pH 7.4 and 25 degrees C, which corresponds to a catalytic proficiency of 14,400 M(-1) toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents.

Figure 1

Hydrolysis reaction, hapten, and organophosphorus poisoning compounds structures.

Figure 2

Hapten synthesis. (i) EtOH, THF, 0°C; (ii) (7), EtN(i-Pr)₂, DME, 50°C; (iii) H₂, Pd/C 5%, EtOH/H₂O 95/5; (iv) (a) acetone, MgSO₄; (b) H₂, Pd/C 5%, MeOH/acetone 50/50; (v) (8) Et₃N, CH₂Cl₂.

Figure 3

Remaining AChE activity of a 0.6 nM AChE solution in EIA buffer treated with a 0.625 nM VX solution pretreated for 18 h with the corresponding mouse serum (1/100 diluted in EIA buffer) (a) before immunization, (b) after immunization, (c) after immunization and in presence of 50 μM hapten Ha, and (d) after immunization and in presence of 150 μM hapten Ha. Continuous and dashed lines represent means ± 2 SD for sera of 18 BALB/c mice preimmune or immunized with Hb, 5a, 5b, or unrelated haptens.

Figure 4

H3 and AChE inhibitors structures.

Figure 5

Hapten analogs.