Investigating the structural influence of surface mutations on acetylcholinesterase inhibition by organophosphorus compounds and oxime reactivation

Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and to a lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of varying structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. In this study wild type human AChE and human AChE with residue mutations D134H, D134H_E202Q and D134H_F338A were characterized and investigated for inhibition by OPs and consequent oxime reactivation of phosphylated enzymes. The rationale for selecting these substitution positions was based on D134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs. Inhibition of D134H by paraoxon and analogues of cyclosarin was 2-8 times slower than inhibition of wild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Both inhibition and reactivation of D134H_E202Q and D134H_F338A double mutants were up to two orders of magnitude slower than the wt indicating that introduction of the active center substitutions abolished fully the effect of the peripherally located D134H. These results indicate that selected residues outside the active center influence inhibition, reactivation and catalysis rates through longer range interactions. (C) 2010 Elsevier Ireland Ltd. All rights reserved

Investigating the Structural Influence of Surface Mutations on Acetylcholinesterase Inhibition by Organophosphorus Compounds and Oxime Reactivation

Organophosphates (OPs) exert their toxicity by inhibiting primarily acetylcholinesterase (AChE) and to a lesser extent butyrylcholinesterase (BChE). Binary mixtures of mammalian AChE and oximes of varying structure have been recently considered for treatment of OP poisoning as catalytic bioscavengers. In this study wild type human AChE and human AChE with residue mutations D134H, D134H_E202Q and D134H_F338A were characterized and investigated for inhibition by OPs and consequent oxime reactivation of phosphylated enzymes. The rationale for selecting these substitution positions was based on D134H being a naturally occurring single nucleotide polymorphism (SNP) in humans and that E202Q and F338A mutations slow aging of OP inhibited AChEs. Inhibition of D134H by paraoxon and analogues of cyclosarin was 2-8 times slower than inhibition of wild type (wt), while reactivation of the paraoxon inhibited enzyme by 2PAM was 6 times faster. Both inhibition and reactivation of D134H_E202Q and D134H_F338A double mutants were up to two orders of magnitude slower than the wt indicating that introduction of the active center substitutions abolished fully the effect of the peripherally located D134H. These results indicate that selected residues outside the active center influence inhibition, reactivation and catalysis rates through longer range interactions. (C) 2010 Elsevier Ireland Ltd. All rights reserved.Wo

Wnt Inhibition Correlates with Human Embryonic Stem Cell Cardiomyogenesis: A Structure–Activity Relationship Study Based on Inhibitors for the Wnt Response

Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one “lead” (<b>1</b>, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of <b>1</b> is Wnt-dependent, and we developed a structure–activity relationship to optimize the cardiogenic properties of <b>1</b>. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/β-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (<i>r</i>² = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than <b>1</b> at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor β (TGFβ)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules