The kinetics of inhibition of human acetylcholinesterase and butyrylcholinesterase by two series of novel carbamates

ABSTRACT Controlled inhibition of brain acetyl-and butyrylcholinesterases (AChE and BChE, respectively) and of monoamine oxidase-B (MAO-B) may slow neurodegeneration in Alzheimer's and Parkinson's diseases. It was postulated that certain carbamate esters would inhibit AChE and BChE with the concomitant release in the brain of the OH-derivatives of rasagiline or selegiline that can serve as inhibitors of MAO-B and as antioxidants. We conducted a detailed in vitro kinetic study on two series of novel N-methyl, N-alkyl carbamates and compared them with rivastigmine, a known anti-Alzheimer drug. The rates of carbamylation (k i ) and decarbamylation (k r ) of recombinant human AChE were mainly determined by the size of the N-alkyl substituent and to a lesser extent by the nature of the leaving group. k i was highest when the alkyl was methyl, hexyl, cyclohexyl, or an aromatic substituent and lowest when it was ethyl. This suggested that k i depends on a delicate balance between the length of the residue and its degree of freedom of rotation. By contrast, presumably because of its wider gorge, inhibition of human BChE was less influenced by the size of the alkyl group and more dependent on the structure of the leaving group. The data show how the degree of enzyme inhibition can be manipulated by structural changes in the N-methyl, N-alkyl carbamates and the corresponding leaving group to achieve therapeutic levels of brain AChE, BChE, and MAO-B inhibition

Evolved Stereoselective Hydrolases for Broad-Spectrum G-Type Nerve Agent Detoxification

SummaryA preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic SP isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 107 M−1 min−1. Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 × 10−4 in favor of the RP isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the SP isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics