Mutant Cholinesterases Possessing Enhanced Capacity for Reactivation of Their Phosphonylated Conjugates

Abstract

Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.

Structure-Activity Approach in the Reactivation of Tabun-Phosphorylated Human Acetylcholinesterase with Bispyridinium para-Aldoximes

“…1. This deviation could be due to a fraction of the aged phosphorylated enzyme (due to the post-inhibitory dealkylation known as aging) and/or re-inhibition of the active enzyme by the phosphorylated oxime (13). It is known that the aging of tabun-inhibited human AChE is relatively slow (t 0.5 =13 h; 14).…”

Refinement of Structural Leads for Centrally Acting Oxime Reactivators of Phosphylated Cholinesterases

“…OP-hAChE conjugates were prepared, and oxime reactivation was performed (at 37°C in 0.1 M sodium phosphate buffer, pH 7.4, containing 0.01% BSA) as described earlier (1,16). The first order reactivation rate constant (k obs ) for each oxime ϩ OP conjugate combination was calculated by nonlinear regression (19). The dependence of reactivation rates on oxime concentrations and determination of maximal reactivation rate constant k 2 , Michaelis-Menten type constant K ox , and the overall second order reactivation rate constant k r were conducted as previously described (19 .0, 9.5, and 10 (containing 0.1 M NaCl) were overlaid and aligned using benzene as an external standard placed in a separate capillary tube within the NMR sample probe.…”

“…The first order reactivation rate constant (k obs ) for each oxime ϩ OP conjugate combination was calculated by nonlinear regression (19). The dependence of reactivation rates on oxime concentrations and determination of maximal reactivation rate constant k 2 , Michaelis-Menten type constant K ox , and the overall second order reactivation rate constant k r were conducted as previously described (19 .0, 9.5, and 10 (containing 0.1 M NaCl) were overlaid and aligned using benzene as an external standard placed in a separate capillary tube within the NMR sample probe. Two-dimensional 1 H, 1 H double-quantum filtered COSY spectra were recorded on a Bruker DRX-600 spectrometer.…”

para‐ and ortho‐Pyridinium aldoximes in reaction with acetylthiocholine

“…However, one should keep in mind that the efficacy of aldoxime in AChE reactivation depends not only on its structure, but also on the structure of the OP inhibitor and the source of the enzyme [9,10,22,23]. ) of activation, were calculated using E a and A [17].…”