Inhibitors Tethered Near the Acetylcholinesterase Active Site Serve as Molecular Rulers of the Peripheral and Acylation Sites

Abstract

The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants K I2 for propidium and tacrine, inhibitors specific for the P-and A-sites, respectively. Values of K I2 for propidium increased 30-to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of K I2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.The primary physiological role of acetylcholinesterase (AChE) 1 is to hydrolyze the neurotransmitter acetylcholine at cholinergic synapses. The AChE structure has evolved to carry out this hydrolysis at rates that are among the highest known for enzyme-catalyzed reactions (1). One feature of the AChE catalytic pathway is the formation of an intermediate acyl enzyme that is hydrolyzed by water. It has long been known that AChE forms an acyl enzyme not only with carboxyl esters like acetylcholine but also with carbamic acid esters and phosphoric acid esters (also called organophosphates or OPs) and that these intermediates differ dramatically in their deacylation rate constants (2-5). In particular, organophosphorylated AChEs are hydrolyzed some 10 10 times slower than acetylated AChE and are effectively inactivated. OP inactivation of AChE results in failure of cholinergic synaptic transmission, deterioration of neuromuscular junctions, flaccid muscle paralysis, an...

Control of protein functional dynamics by peptide linkers

“…Such polypeptides have been termed ''molecular rulers'' because of their ability to measure the effect of different depths of binding pockets on various properties of proteins of interest. 43 Consequently, Stryer and Haugland used a poly-L-proline ''spectroscopic ruler'' to measure distances of energy transfer between attached moieties. Poly-L-proline peptides were of defined length to separate an energy donor and acceptor by distances ranging from 12 to 46 Å .…”

“…Because of their rigidity, predictable changes in length are observed (up to a limiting point) as additional monomers are added between functional groups. 43 For instance, in electron transfer experiments, proline peptides provide predictable separation distance based on the number of proline residues. 45 The major concern in the design of a molecular ruler is the possibility of softening and structural failure that arises when the ruler is unable to provide a predictable separation distance between its bound moieties.…”

A Docking Score Function for Estimating Ligand−Protein Interactions: Application to Acetylcholinesterase Inhibition

“…38 Sitedirected mutagenesis in huAChE indicated that Asp74, Tyr337, Phe338, Phe295, Phe297, Tyr133, and Glu450 can affect the affinity although it has been difficult to determine experimentally whether these residues contribute mainly electrostatic or VDW interactions. 39,40 The docked ligand structures generally support the above analysis regarding the identity of principle residues. In our current docking calculations, molecules 2-7 all share similar conformations in the PAS.…”

Isolation and characterization of N98-1272 A, B and C, selective acetylcholinesterase inhibitors from metabolites of an actinomycete strain

“…A number of studies have focused on the structural basis of the catalytic efficiency of AChE [23,24,25,26]. X-ray crystallography of Torpedo california AChE (TcAChE) reveals that the active site of AChE lies at the bottom of a deep (20 Å ) and narrow gorge, named the "active site gorge" or "aromatic gorge".…”