Inhibitor Report for: DMBMP-thiocholine

Organophosphates inactivate acetylcholinesterase by reacting covalently with the active center serine. We have examined the reactivation of a series of resolved enantiomeric methylphosphonate conjugates of acetylcholinesterase by two oximes, 2-pralidoxime (2-PAM) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium) (HI-6). The S(p) enantiomers of the methylphosphonate esters are far more reactive in forming the conjugate with the enzyme, and we find that rates of oxime reactivation also show an S(p) versus R(p) preference, suggesting that a similar orientation of the phosphonyl oxygen toward the oxyanion hole is required for both efficient inactivation and reactivation. A comparison of reactivation rates of (S(p))- and (R(p))-cycloheptyl, 3,3-dimethylbutyl, and isopropyl methylphosphonyl conjugates shows that steric hindrance by the alkoxy group precludes facile access of the oxime to the tetrahedral phosphorus. To facilitate access, we substituted smaller side chains in the acyl pocket of the active center and find that the Phe295Leu substitution enhances the HI-6-elicited reactivation rates of the S(p) conjugates up to 14-fold, whereas the Phe297Ile substitution preferentially enhances 2-PAM reactivation by as much as 125-fold. The fractional enhancement of reactivation achieved by these mutations of the acyl pocket is greatest for the conjugated phosphonates of the largest steric bulk. By contrast, little enhancement of the reactivation rate is seen with these mutants for the R(p) conjugates, where limitations on oxime access to the phosphonate and suboptimal positioning of the phosphonyl oxygen in the oxyanion hole may both slow reactivation. These findings suggest that impaction of the conjugated organophosphate within the constraints of the active center gorge is a major factor in influencing oxime access and reactivation rates. Moreover, the individual oximes differ in attacking orientation, leading to the presumed pentavalent transition state. Hence, their efficacies as reactivating agents depend on the steric bulk of the intervening groups surrounding the tetrahedral phosphorus.

This paper examines the chiral nature of the covalent conjugates formed upon reaction of acetylcholinesterase (AchE) with enantiomeric cycloheptyl, isopropyl, and 3,3-dimethylbutyl methylphosphonyl thiocholines. With the exception of the conjugate formed from reaction of AchE with RP-cycloheptyl methylphosphonyl thiocholine, all enantiomeric conjugates underwent oxime reactivation at rates that were within 2-3-fold of each other. Oxime reactivation was, therefore, independent of both initial configuration about phosphorus and the alkyl phosphonyl ester (-OR) moiety. Aging of the enantiomeric cyclopheptyl and isopropyl methylphosphonyl conjugates occurred exclusively for the conjugate formed from the SP-enantiomer and therefore displayed an absolute dependence on the initial configuration of the methylphosphonyl group. Equilibrium titrations with decidium, a fluorescent bisquaternary competitive inhibitor of AchE, provided an index of aging and enantiomeric configuration of the conjugates independent of enzyme activity. Decidium association with the enantiomeric conjugates (prior to aging) showed no marked dependence on the initial configuration about phosphorus but was measurably dependent on nature of the -OR moiety. These results are interpreted with respect to symmetry and nonrigidity of the organophosphonyl conjugates and are consistent with formation of final methylphosphonyl conjugates that are enantiomerically pure and of opposite configuration. These studies indicate that the active center of AchE comprises at least two kinetically distinct environments separate from the esteratic region but located within 5 A of the nucleophilic serine and differing in dipolar characteristics that promote charge separation and general acid catalysis.

Enantiomeric cycloheptyl- and isopropyl methylphosphonothioates containing uncharged and cationic leaving groups, and 3,3-dimethylbutyl methylphosphonyl thiocholines were synthesized, and their inhibition of acetylcholinesterase from Torpedo examined. Bimolecular inhibition constants spanned 10(1)-10(9) M-1.min-1, equilibrium dissociation constants 10(-3)-10(-7) M, and phosphonylation constants 1-300 min-1. A general but not absolute preference for the SP-enantiomer, in the range 170-4600 for cycloheptyl-, 0.6-150 for isopropyl-, and 30 for 3,3-dimethylbutyl methylphosphonothioates, varied with nature of the alkyl ester (-OR) and thioic leaving groups (-SR') surrounding phosphorus. While the overall bimolecular reaction constant showed no marked dependence on ionic strength of the medium, the microscopic kp and KD for the RP- but not SP-cycloheptyl methylphosphonyl thiocholine underwent marked reduction with decreases in ionic strength. This result unmasks the interplay between occupation of the active center and productivity of that occupation. These studies reveal that chiral reactions with acetylcholinesterase are dependent more on the nature of the groups surrounding the tetrahedral phosphorus than on the absolute configuration about the phosphorus atom and indicate that the active center comprises partially overlapping subsites that can accommodate the -OR and -SR' groups. The presence of neighboring subsites characterized by different steric, electrostatic, and hydrophobic properties permits a multiplicity of binding orientations, independent of chiral configuration, and which account for the large variation in chiral preference seen among organophosphonates containing different substituents.