Yael Loewenstein

Engineering of human cholinesterases explains and predicts diverse consequences of administration of various drugs and poisons

The acetylcholine hydrolyzing enzyme, acetylcholinesterase, primarily functions in nerve conduction, yet it appears in several guises, due to tissue-specific expression, alternative mRNA splicing and variable aggregation modes. The closely related enzyme, butyrylcholinesterase, most likely serves as a scavenger of toxins to protect acetylcholine binding proteins. One or both of the cholinesterases probably also plays a non-catalytic role(s) as a surface element on cells to direct intercellular interactions. The two enzymes are subject to inhibition by a wide variety of synthetic (e.g., organophosphorus and carbamate insecticides) and natural (e.g., glycoalkaloids) anticholinesterases that can compromise these functions. Butyrylcholinesterase may function, as well, to degrade several drugs of interest, notably aspirin, cocaine and cocaine-like local anesthetics. The widespread occurrence of butyrylcholinesterase mutants with modified activity further complicates this picture, in ways that are only now being dissected through the use of site-directed mutagenesis and heterologous expression of recombinant cholinesterases.

Excavations into the active-site gorge of cholinesterases

Acetyl- and butyrylcholinesterase (ACHE, BCHE) from evolutionarily distant species display a high degree of primary sequence homology and have biochemically similar catalytic properties, yet they differ in substrate specificity and affinity for various inhibitors. The biochemical information derived from analyses of ACHE and BCHE from human, Torpedo, mouse, and Drosophila, as well as that from the recombinant forms of their natural variants and site-directed mutants, can currently be re-examined in view of the recent X-ray crystallography data revealing the three-dimensional structure of Torpedo ACHE. The picture that emerges deepens the insight into the biochemical basis for choline ester catalysis and the complex mechanism of interaction between cholinesterases and their numerous ligands.

Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.

Abstract: Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle‐specific proteins, among them the acetylcholine‐hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DMA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over‐expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell‐associated rHAChE accumulated in myotomes of 2‐and 3‐day‐old embryos within the same sub‐cellular compartments as native Xenopus AChE. NMJs from 3‐day‐old DNA‐injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.

Site-Directed Mutagenesis of Active Site Residues Reveals Plasticity of Human Butyrylcholinesterase in Substrate and Inhibitor Interactions

Abstract: In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site‐directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso‐OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10‐fold decreased IC50 values for DFP, iso‐OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.

Molecular dissection of cholinesterase domains responsible for carbamate toxicity

Carbamate compounds marked for their cholinesterase (ChE) inhibition are widely used as therapeutics and as insecticides. Groups of closely related carbamate molecules provide an important tool in the understanding of the domains responsible for binding these ligands to ChEs. Comparative inhibition profiles were derived for five N-methyl carbamates, mostly carbofuran derivatives, differing in length and branching of their hydrocarbonic chain towards human erythrocyte acetylcholinesterase (H.AChE), human serum butyrylcholinesterase (H.BChE) in its normal form or in a mutant form containing the point mutation Asp70-->Gly, and Drosophila nervous system ChE. Carbofuran was more toxic to all three ChEs than any of the other derivatives, with IC50 values which differed by more than 1000-fold. Drosophila ChE appeared to be most sensitive to all of the examined carbamates, and H.AChE was consistently more sensitive than H.BChE. Moreover, inhibition efficiency for H.BChE decreased more effectively than it did for H.AChE with increased length and complexity of the side chain, indicating less flexible carbamate binding site in BChE as compared with AChE. The Asp70-->Gly mutation had no apparent effect on H.BChE inhibition by N-methyl carbamates, suggesting that the Asp70 domain localized near the rim of the active site groove is not important in carbamate binding. Comparison of the carbamate IC50 values with published LD50 values demonstrated correlation between the in vivo toxicity and inhibition of BChE by carbamates, suggesting a biological in addition to scavenging importance for BChE in mammals. Pinpointing different domains characteristic of carbamate binding in each member of the ChE family can thus shed light on the variable toxicity of these inhibitors to insects and mammals, predict the toxicity of yet untested inhibitor molecules and help in designing novel and improved ChE inhibitors.

Mutations and impaired expression in the ACHE and BCHE genes: neurological implications

The acetylcholine hydrolysing cholinesterases control the termination of cholinergic signalling in multiple tissues and are targets for a variety of drugs, natural and man-made poisons and common insecticides. Molecular cloning and gene mapping studies revealed the primary structure of human acetyl- and butyrylcholinesterase and localized the corresponding ACHE and BCHE genes to the chromosomal positions 3q26-ter and 7q22, respectively. Several different point mutations in the coding region of BCHE were found to be particularly abundant in the Israeli population. Analytical expression studies in microinjected Xenopus oocytes have demonstrated that the biochemical properties of cholinesterases may be modified by rationalized site-directed mutagenesis and in chimeric ACHE/BCHE constructs. These properties are differently altered in the various allelic BCHE variants, conferring resistance to several anti-cholinesterases, which may explain the evolutionary emergence of these multiple alleles. At the clinical level, abnormal expression of both ACHE and BCHE and the in vivo amplification of the ACHE and BCHE genes has been variously associated with abnormal megakaryocytopoiesis, leukemias and brain and ovarian tumors. Moreover, antisense oligonucleotides blocking the expression of these genes were shown to interfere with hemocytopoiesis in culture, implicating these genes in cholinergic influence on cell growth and proliferation.

Population Diversity of Point Mutations in the Human AChE and BCHE Genes Predicts Variable Responses to Anticholinesterase Drugs

On an experimental basis, patients with neurodegenerative diseases receive anti-cholinesterases (Iversen, 1993; Enz et al., 1993; Giacobini, 1993) to improve cognitive function (Alzheimer’s disease) or to reduce muscle spasms (Parkinson’s disease). These drugs are directed toward the nervous system acetyl- and butyrylcholinesterase (AChE, BuChE). However, the response of specific individuals to such drugs was found to be highly variable. Molecular genetics findings strongly suggest that this variability may be due to genomic diversity in the corresponding genes, primarily in the BuChE gene, BCHE.

Molecular Dissection of Functional Domains in Human Cholinesterases Expressed in Microinjected Xenopus Oocytes

The two classes of human cholinesterases (CHEs), acetylcholinesterase(acetylcholine acetyl hydrolase, ACHE, EC 3.1.1.7) and butyrylcholinesterase (acylcholine acyl hydrolase, BCHE, EC 3.1.1.8) are highly homologous proteins capable of rapidly hydrolyzing choline estersl. Despite their similar mechanisms of action, they differ in substrate specificity and sensitivity to various inhibitors1,2. Recent advances including cloning,3–6 expression,5,7–10 and 3 dimensional structural analysis of members of the CHE superfamily,11,12 now enable the dissection of functional domains thereof. Within these domains, key amino acids are found which may be implicated in catalysis or in binding of various ligands. The disclosing of such key residues could lead to designing of novel therapeutic agents as well as to the unravelling of the molecular mechanisms underlying the functioning of ChEs.

Genetic Predisposition for Variable Response to Anticholinesterase Therapy Anticipated in Carriers of the Butyrylcholinesterase “Atypical” Mutation

Anticholinesterases were recently approved for treating patients suffering from Alzheimer’s disease (AD) in an attempt to balance their cholinergic system. These drugs are targeted at acetylcholinesterase (AChE) but also inhibit butyrylcholinesterase (BuChE), known for its numerous genetic variants. The most common of these is the “atypical” phenotype created through a replacement of Asp70 by Gly (D70G) due to a point mutation. The “atypical” enzyme causes prolonged postanesthesia apnea following succinylcholine administration for muscle relaxation and displays a considerably reduced sensitivity to various other inhibitors. The allelic frequency of “atypical” BuChE was studied in different populations and revealed distinct patterns particular to various ethnic groups. Recently, a relatively high allelic frequency of 0.06 was found in a population of Georgian Jews, differing by up to 4-fold from the incidence in other populations (Ehrlich et al., Genomics, in press). This implies that in groups of AD patients from diverse ethnic origins, a significant fraction of carriers of at least one allele of this mutation should be expected. To predict their responsiveness to anticholinesterase treatment, we examined the susceptibility of AChE, as compared to that of BuChE and the “atypical” BuChE variant, towards several anticholinesterases in use for AD treatment. IC50 values and rate constants reflecting inhibitor susceptibilities were calculated for various recombinant human cholinesterases produced in Xenopus oocytes and immobilized on microtiter plates through selective monoclonal antibodies. The reversible amino acridinium compound Tacrine, currently in use for AD therapy, displayed a 300-fold higher IC50 value for the “atypical” enzyme than for BuChE (1mM BtCh as substrate). Pseudo first order rate constants for inhibition of BuChEs by the carbamates heptyl-physostigmine (0.139 min−1, lOnM inhibitor), physostigmine (0.3 min−1, 1 μM inhib.) and SNZ-ENA713 (0.139 min−1, 10 μM inhib.) were found to be higher than or equal to those of AChE, suggesting that BuChE serves as a second primary target for these drugs. Moreover, the “atypical” variant of BuChE displayed considerably slower inactivation rates to these drugs (0.01 min−1, 0.025 min−1, and 0.01 min−1, respectively) as compared with the wild type BuChE. These findings predict that carriers of the D70G BuChE mutation would vary from other patients in their susceptibility to the above drugs, which potentially contributes to the wide variability of responses observed in clinical trials. Screening patients for D70G carriers should therefore precede anticholinesterase treatment.

STRUCTURE-FUNCTION RELATIONSHIP STUDIES IN HUMAN CHOLINESTERASES AS AN APPROACH FOR EVALUATING POTENTIAL PHARMACOTHERAPEUTIC AND/OR TOXICITY EFFECTS OF CHOLINERGIC DRUGS

Attempts to restore cholinergic deficits in Alzheimer’s disease patients involve the use of various drugs inhibiting cholinesterases (CHEs; Bartus et al., 1982). In addition, CHE inhibitors are clinically employed in the treatment of other common syndromes, including Parkinson’s disease, myasthenia gravis and multiple sclerosis (Taylor, 1990). The efficacy and specificity of such drugs on the one hand, and their toxicity factor on the other, largely depend on their inhibitory effects on CHEs in the treated individuals. Therefore, updated evaluation methods for these parameters should be valuable to pharmacological research focused on the development and use of cholinergic drugs.