Amino acid residues involved in stereoselective inhibition of cholinesterases with bambuterol

Bambuterol is a chiral carbamate known as selective inhibitor of butyrylcholinesterase (BChE). In order to relate bambuterol selectivity and stereoselectivity of cholinesterases to the active site residues, we studied the inhibition of recombinant mouse BChE, acetylcholinesterase (AChE) and six AChE mutants, employed to mimic BChE active site residues, by bambuterol enantiomers. Both enantiomers selectively inhibited BChE about 8000 times faster than AChE. The largest inhibition rate increase in comparison to AChE w.t. was observed with the F295L/Y337A mutant, showing that leucine 295 and alanine 337 are crucial residues in BChE for high bambuterol selectivity. All studied enzymes preferred inhibition by the R- over the S-bambuterol. The enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations (Y337A, F295L/Y337A and F297I/Y337A) increased, in comparison to w.t. enzymes, inhibition rate constants of R- bambuterol more than that of S- bambuterol resulting in four times higher stereoselectivity. Peripheral site mutations (Y124Q and Y72N/Y124Q/Y337A) increased inhibition rate by S- more than R-bambuterol and consequently diminished the stereoselectivity.     

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.     

Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP

We used mouse recombinant wild-type acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8), and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.     

Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents

Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.     

New diterpenoid alkaloids from Aconitum heterophyllum Wall: Selective butyrylcholinestrase inhibitors

Two new diterpenoid alkaloids, heterophyllinine-A (1) and heterophyllinine-B (2), along with two known alkaloids dihydroatisine (3) and lycoctonine (4) were isolated from the roots of Aconitum heterophyllum Wall. The structure of (1) and (2), were deduced on the basis of spectral data. Compounds 1-2 inhibited acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes in a concentration-dependent manner with percent inhibition ranging between 4.24% and 6.94 % and 79.1 % and 82.75 % for AChE and BChE, respectively indicating that compounds 1 and 2 are about thirteen times more specific to BChE than AChE.     

Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.     

Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.     

Interaction of human butyrylcholinesterase variants with bambuterol and terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylamino-ethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085min (-1)microM(-1). Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3 mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Interaction of Human Butyrylcholinesterase Variants with Bambuterol and Terbutaline

Bambuterol, a dimethylcarbamate, carbamoylates butyrylcholinesterase (BChE; EC 3.1.1.8). The carbamoylated enzyme is not very stable and the final product of the two-step hydrolysis is a bronchodilator drug, terbutaline (1-(3,5-dihydroxyphenyl)-2-t-butylaminoethanol sulphate). Both bambuterol and terbutaline inhibit BChE, but their affinities differ in human serum BChE variants (U, A, F, K and S) due to their positive charge. Bambuterol inhibition rate constants for the homozygous usual (UU), Kalow (KK), fluoride-resistant (FF) or atypical (AA) variant ranged from 4.4 to 0.085?min-1?μM-1. Terbutaline showed competitive reversible inhibition for all BChE variants. The dissociation constants for UU, FF and AA homozygotes were 0.18, 0.31 and 3.3?mM, respectively. The inhibition rate or dissociation constants for heterozygotes were distributed between the respective constants for the corresponding homozygotes. A 50-fold difference in inhibition between the UU and AA enzyme might affect terbutaline release in humans. The affinity of all studied BChE variants for terbutaline was low, which suggests that terbutaline originating from bambuterol hydrolysis should not affect the hydrolysis of bambuterol by BChE.     

Organophosphorus Compound O,O-Dimethyl-O-(2,2-dichlorovynyl)-Phosphate as Selective Inhibitor for Separate Determination of Acetylcholinesterase and Butyrylcholinesterase Activities in the Roach Rutilus rutilus

A comparative study is carried out on dependence of degree of activity inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of a freshwater bony fish, the roach Rutilus rutilus L., on concentration of organophosphates: O,O-dimethyl-O-(2,2-dichlorovynyl)phosphate (DDVP) and tetraisopropylamidopyrophosphate (iso-OMPA). It has been shown that both in roach and in horse the both inhibitors are selective for BChE in comparison with AChE. Their selectivity degree was 2000-fold and 80-fold, respectively. The ranges of effective DDVP concentrations are overlapped for horse AChE and BChE, while they do not for the roach enzymes. A similar regularity is revealed at action of iso-OMPA. It is established that DDVP has a higher inhibitory potency and selectivity in relation to roach BChE, than iso-OMPA. It is suggested to use DDVP as a new selective inhibitor for separate evaluation of AChE and BChE activities in fish tissues.     

Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K(i) was 0.01mM for AChE and 0.06mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000min(-1)M(-1), which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K(i)), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20h, the reactivation maximum was 60% for 1mM K074 and K075, and only 20% for 1mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K_i was 0.01 mM for AChE and 0.06 mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000 min⁻¹ M⁻¹, which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20 h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K_i), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20 h, the reactivation maximum was 60% for 1 mM K074 and K075, and only 20% for 1 mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases.

E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.     

The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase,butyrylcholinesterase, glutathione S-transferase,lactoperoxidase, and carbonic anhydrase isoenzymes I,II, IX,and XII

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates including humans as many diverse isoforms. Acetylcholinesterase (AChE) is responsible for acetyl choline (ACh) hydrolysis and plays a fundamental role in nerve impulse transmission by terminating the action of the ACh neurotransmitter at cholinergic synapses and neuromuscular junctions. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effect of CAPE on human carbonic anhydrase (hCA) isoforms I, II, IX, and XII, AChE, BChE, LPO, and GST was evaluated. CAPE inhibited these enzymes with K_is in the range between micromolar to picomolar. The best inhibitory effect was observed against AChE and BChE.     

Inhibitory effects of isatin Mannich bases on carbonic anhydrases,acetylcholinesterase, and butyrylcholinesterase

The effects of isatin Mannich bases incorporating (1-[piperidin-1-yl (P1)/morpholin-4-yl (P2)/N-methylpiperazin-1-yl (P3)]methyl)-1H-indole-2,3-dione) moieties against human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoenzymes hCA I and hCA II, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) enzymes were evaluated. P1–P3 demonstrated impressive inhibition profiles against AChE and BChE and also inhibited both CAs at nanomolar level. These inhibitory effects were more powerful in all cases than the reference compounds used for all these enzymes. This study suggests that isatin Mannich bases P1–P3 are good candidate compounds especially for the development of new cholinesterase inhibitors since they were 2.2–5.9 times better inhibitors than clinically used drug Tacrine.     

Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms.

The pattern of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) separated by density gradient centrifugation was investigated in the brain and cerebrospinal fluid in Alzheimer's disease (AD), in human embryonic brain and in rat brain after experimental cholinergic deafferentation of the cerebral cortex. While a selective loss of the AChE G4 form was a rather constant finding in AD, a small but significant increase of G1 for both AChE and BChE was found in the most severely affected cases. Both in normal human brain and in AD a significant relationship could be established between the AChE G4/G1 ratio in different brain regions and the activity of choline acetyltransferase (ChAT). A similar decrease of the AChE G4 form as observed in AD can be induced in rat by experimental cholinergic deafferentation of the cerebral cortex. The increase in G1 of both AChE and BChE in different brain regions in AD is quantitatively related to the local density of neuritic plaques which are histochemically reactive for both enzymes. In human embryonic brain, a high abundance of G1 and a low G4/G1 ratio for both AChE and BChE was found resembling the pattern observed in AD. Furthermore, both in embryonic brain and in AD AChE shows no substrate inhibition which is a constant feature of the enzyme in the adult human brain. It is, therefore, concluded that the degeneration of the cholinergic cortical afferentation in AD as reflected by a decrease of AChE G4 is accompanied by the process of a neuritic sprouting response involved in plaque formation which is probably associated with the expression of a developmental form of the enzyme.     

Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities and in vitro inhibition by DDVP, an organophosphorus pesticide

A comparative study of specific activities and in vitro inhibition of brain and serum acetylcholinesterase (AChE; EC 3.1.1.7) and serum butyrylcholinesterase (BChE; EC 3.1.1.8) by DDVP, an organophosphorus pesticide, was conducted in 11 freshwater teleost species belonging to four families (Cyprinidae; common carp Cyprinus carpio, bream Abramis brama, blue bream A. ballerus, white bream Blicca bjoerkna, roach Rutilus rutilus, bleak Alburnus alburnus, ide Leuciscus idus; Percidae: perch Perca fluviatilis, pikeperch Stizostedion lucioperca; Esocidae: pike Esox lucius and Coregonidae: whitefish Coregonus albula). Specific AChE and BChE activities in brain and serum of fish were determined. Brain AChE activity varied among fish species approximately 10-fold, ranging from 192.6 to 1353.2 micromol g(-1) h(-1), respectively in perch and whitefish. All cyprinids had higher brain AChE activity than those of other fish families. Serum AChE activity was 100-fold lower than in brain. Serum BChE activity was found only in cyprinids with the exception of the common carp. It varied from 163.8 to 970.3 micromol g(-1) h(-1), respectively in roach and bleak. The bimolecular enzyme inhibition rate constants (kIIs) and pI50) values for DDVP were calculated. Sensitivity of fish AChEs both in brain and serum is similar to those of typical AChEs in mammals. The range of kIIs was 3.4-51.7 x 10(3) mol(-1) 1 min(-1) (pI50s were 5.3-6.5), respectively in white bream and ide. In contrast, fish serum BChE was more sensitive to inhibition than typical BChE and AChE in mammals. Values of kII for BChE were 1.0-2.5 x 10(7) mol(-1) 1 min(-1) (pI50 was 8.8-9.2), respectively in ide and bleak.     

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer's disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE-/- mice). Extracellular ACh levels in the hippocampus were 60-fold elevated in AChE-/- mice compared with wild-type (AChE+/+) animals. In AChE-/- mice, calcium-free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose-dependent, up to 16-fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE-/- mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE-/- mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis-norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.     

Design,synthesis and biological evaluation of novel carbamates as potential inhibitors of acetylcholinesterase and butyrylcholinesterase

Rivastigmine, a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), has been approved by U.S. Food and Drug Administration to treat Alzheimer’s disease (AD) and Parkinson’s disease (PD) dementia. In the current work, a bambuterol derivative lacking one of the carbamoyloxy groups on the benzene ring (BMC-1) and its analogues were synthesized using 1-(3-hydroxyphenyl) ethan-1-one and 1-(4-hydroxyphenyl) ethan-1-one as starting materials. In-vitro cholinesterase assay established that nine compounds were more potent to inhibit both electric eel AChE and equine serum BChE than rivastigmine under the same experimental conditions. Further study confirmed that among the nine carbamates, BMC-3 (IC_50(AChE) = 792 nM, IC_50(BChE) = 2.2 nM) and BMC-16 (IC_50(AChE) = 266 nM, IC_50(BChE) = 10.6 nM) were excellent cholinesterase inhibitors with potential of permeating through the blood-brain barrier. These carbamates could be used as potential dual inhibitors of AChE and BChE and to discover novel drugs for the treatment of AD and PD dementia.     

Synthesis,in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators

Abstract

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.