Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

Rosenberry, Terrone L.; Sonoda, Leilani K.; Dekat, Sarah E.; Cusack, Bernadette; Johnson, Joseph L.

doi:10.1016/j.cbi.2008.06.002

Cited by 18 publications

(23 citation statements)

References 16 publications

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“…5). This approach can be monitored by either thioflavin T fluorescence or simultaneous acetylthiocholine hydrolysis (24), and reactions monitored by acetylthiocholine hydrolysis are illustrated in Fig. 5A.…”

Section: Resultsmentioning

confidence: 99%

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Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Auletta

Johnson

Rosenberry

2010

Chemico-Biological Interactions

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Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to the catalytic efficiency of substrate hydrolysis by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Ligands that bind to the A-site invariably inhibit the hydrolysis of all AChE substrates, but ligands that bind to the P-site inhibit the hydrolysis of some substrates but not others. To clarify the basis of this difference, we focus here on second order rate constants for substrate hydrolysis (kE), a parameter that reflects the binding of ligands only to the free form of the enzyme and not to enzyme-substrate intermediates. We first describe an inhibitor competition assay that distinguishes whether a ligand is inhibiting AChE by binding to the A-site or the P-site. We then show that the P-site-specific ligand thioflavin T inhibits the hydrolysis of the rapidly hydrolyzed substrate acetylthiocholine but fails to show any inhibition of the slowly hydrolyzed substrates ATMA (3-(acetamido)-N,N,N-trimethylanilinium) and carbachol. We derive an expression for kE that accounts for these observations by recognizing that the rate-limiting steps for these substrates differ. The rate-limiting step for the slow substrates is the general base-catalyzed acylation reaction k2, a step that is unaffected by bound thioflavin T. In contrast, the rate-limiting step for acetylthiocholine is either substrate association or substrate migration to the A-site, and these steps are blocked by bound thioflavin T.

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Section: Resultsmentioning

confidence: 99%

“…To achieve k 12 rate constants that were second order and corresponded to eq. 6, carbachol was maintained at 0.5 mM (where K app ≡ K M was 5.7 mM (24)). Acetylthiocholine concentrations were 50 µM to minimize [S 2 ]/K app2 in eq.…”

Section: Resultsmentioning

confidence: 99%

Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Auletta

Johnson

Rosenberry

2010

Chemico-Biological Interactions

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“…Melanogenesis inhibitors are used to whiten the skin of patients treated with pigmentation disorders, such as overproduction of melanin (Hartong et al, 2006) and Addison's disease (Pandya and Guevara, 2000). Oliveira et al (2011) found the anticholinesterase activity of CNSL constituents, although cardol, cardanol, carbachol and anacardic acids were previously reported for their cholinesterase inhibitory activity (Rosenberry et al, 2008). Some of the biological activities of LCC and its chemical compounds are presented in Table 1.…”

Section: A Occidentale and Its Biological Rolesmentioning

confidence: 99%

Pharmacological properties of cashew (Anacardium occidentale)

Leite¹,

Islam²,

Junior³

et al. 2016

Afr. J. Biotechnol.

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Anacardium occidentale L. is a tree native to Brazil, which is rich in phenolic lipids. Nowadays, the cashew bark (Cashew Nut Shell Liquid) has received great attention in the pharmaceutical industry, due to its economy, abundance and important chemical compounds. Net of cashew nut shell is classified according to the method of production of: (1) net of the shell of natural cashew nut (60-65% anacardic acid; 15-20% cardol and 10% of cardanol) and (2) liquid from the technical cashew nut shell (60-65% of cardanol, 15-20% cardol and 10% of polymeric material). This work aims to report the pharmacological properties of liquids from cashew nut shells. Results suggest that both liquids have antifungal, antibacterial, antiparasitic, anti-tumor, antiulcerogenic, molluscicides, antimutagenic and antioxidant activities. Natural cashew nut liquid is non-genotoxic, whereas technical liquid is genotoxic in prokaryotes and eukaryotes, although there is no evidence of their mutagenic effects on eukaryotic cells. In conclusion, the excellent antioxidant and non-mutagenic activities of cashew nut shell liquid (CNSL) provide opportunities for CNSL in the cosmetic and/or pharmaceutical industries, but continuous study is needed to allow safe and efficacious preparations.

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“…Moreover, the non-ionic character of these oximes should increase the lipophilicity and BBB penetration of the conjugates. Although we know that the structural and functional differences between human, animal and electric eel AChE may result in a different affinity and reactivity of oximes, we have been working with electric eel AChE due to its ready availability, which facilitates screening assays.Some reports have demonstrated that an allosteric enhancement of reactivation of carbamoylated or phosphorylated acetylcholinesterases occurred through PAS occupation by peripheral site ligands [47][48][49] . furthermore, it was also shown that oxime-mediated dephosphorylation was accelerated in the presence of a ligand with affinity for the PAS 50 .…”

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confidence: 99%

“…Some reports have demonstrated that an allosteric enhancement of reactivation of carbamoylated or phosphorylated acetylcholinesterases occurred through PAS occupation by peripheral site ligands [47][48][49] . furthermore, it was also shown that oxime-mediated dephosphorylation was accelerated in the presence of a ligand with affinity for the PAS 50 .…”

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In vitro evaluation of neutral oximes as reactivators of parathion-inhibited electric eel acetylcholinesterase

et al. 2017

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Organophosphorus (OP) compounds are irreversible inhibitors of acetylcholinesterase (AChE) commonly used as pesticides and, unfortunately, as nerve agents in terrorist attacks. These compounds are highly soluble easily crossing the blood-brain barrier (BBB). Clinically, oximes such as pralidoxime and obidoxime are used for the reactivation of AChE. These oximes are not effective to reactivate AChE inhibited by different OPs besides the fact that they are permanently charged and do not readily cross the BBB. This work evaluated the ability of ten neutral oximes to reactivate parathion-inhibited eel AChE. Because oximes can bind to AChE as reversible inhibitors, this property was also evaluated, with pralidoxime (2-PAM) used as a reference compound. Unlike 2-PAM, which inhibited AChE in a concentration-dependent way, neutral oximes did not follow the linear order of AChE inhibition. Neutral ligands can present affinity for the periferic anionic site (PAS) site. Neutral oximes 1 and 2 (200 µM) reactivated parathion-inhibited eel AChE by 9 per cent and 11 per cent, respectively; but neither of them surpassed the reactivation efficacy of 2-PAM (25 per cent). Neutral oximes 1 and 2 reactivated AChE at a safe concentration for humans. Both neutral oximes 1 and 2 are good non-quaternary moieties for the synthesis of conjugates with enhanced reactivation potency and BBB penetration.Keywords: Acetylcholinesterase; Eel AChE; Reactivator; Oxime; Pralidoxime; Parathion RESEARCH PAPERDefence Life Science Journal, Vol. 2, No. 3, July 2017, pp. 363-369, DOI : 10.14429 LIMA, et al.: DEf. LIfE SCI. J., VOL. 2, NO. 3, JULy 2017, DOI : 10.14429/dlsj.2.10730 or allosteric) 14 . Although approved as antidotes, these oximes are not sufficiently effective to reactivate AChE inhibited by the different OPs 15,16 . The mono-quaternary oxime 2-PAM is very efficient in reactivating AChE inhibited with sarin or VX 17 but is not effective against tabun or soman 18 . Obidoxime is the most potent and most efficacious oxime in reactivating AChE inhibited by various classes of OP insecticides and tabun, but was inferior to oxime HI-6 against soman, sarin, cyclosarin and VX 19. A significant drawback to these oximes is they are permanently charged and do not readily cross the blood brain barrier (BBB) 20 . As a result, they show only limited activity in the CNS, which is a major target of OPs. Thus, effective reactivators as antidotes are increasingly needed against a broader spectrum of nerve agents 21 . Introducing non-quaternary organic compounds has been a novel method of efficiently penetrate the BBB for reactivation of brain AChE 22. Non-charged oximes have previously been developed with improved BBB penetration [23][24][25][26][27][28][29] and sufficient reactivation of AChE inhibited by nerve agents and insecticides 24,30 . Even with improved BBB penetration and reactivation of AChE, the specific oxime structures with superior reactivating potency to those in use remain unknown.The work proposed here evaluated the ability of...

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Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

Cited by 18 publications

References 16 publications

Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase

Pharmacological properties of cashew (Anacardium occidentale)

In vitro evaluation of neutral oximes as reactivators of parathion-inhibited electric eel acetylcholinesterase

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