Interactions of human acetylcholinesterase with phenyl valerate and acetylthiocholine: Thiocholine as an enhancer of phenyl valerate esterase activity

Phenyl valerate (PV) is a neutral substrate for measuring the PVase activity of neuropathy target esterase (NTE), a key molecular event of organophosphorus-induced delayed neuropathy. This substrate has been used to discriminate and identify other proteins with esterase activity and potential targets of organophosphorus (OP) binding. A protein with PVase activity in chicken (model for delayed neurotoxicity) was identified as butyr ylcholinesterase (BChE). Further studies in human BChE suggest that other sites might be involved in PVase activity. From the theoretical docking analysis, other more favorable sites for binding PV related to the Asn289 residue located far from the catalytic site (“PVsite”) were deduced.In this paper, we demonstrate that acetyl cholinesterase is also able to hydrolyze PV. Robust kinetic studies of interactions between substrates PV and acetylthiocholine (AtCh) were performed. The kinetics did not fit the classic competition models among sub strates. While PV interacts as a competitive inhibitor in AChE activity, AtCh at low concentrations enhances PVase activity and inhibits this activity at high concentrations. Kinetic behavior suggests that the potentiation effect is caused by thiocholine released at the active site, where AtCh could act as a Trojan Horse. We conclude that the products released at the active site could play an important role in the hydrolysis reactions of different substrates in biological systems

Inhibition studies of serine hydrolases by cyclic phosphates and phosphonates

The serine hydrolase superfamily is one of the largest known enzyme families comprising approximately 1% of the predicted protein product in human genome. This family of enzymes contains a catalytic triad that is mainly consists of serine, aspartic acid/glutamic acid and histidine residues in their active sites. It has been proposed that the potential drug targets for Alzheimer’s disease and diabetes type 2 are enzymes that belong to this enzyme family. Acetylcholinesterase is an enzyme that catalyses the breakdown of acetylcholine, a neurotransmitter that helps transport information from one nerve cell to another. Breakdown of acetylcholine in Alzheimer’s disease patients enhances memory loss, which could be reduced if AChE is inhibited. Cyclophostin, a bicyclic phosphate, is a natural product inhibitor of AChE having an IC50, of 8 e-4 μM. The laboratory synthesized mono- and bicyclic analogs of phosphonate analog of cyclophostin exhibited low μM potency against human AChE. It is established that these analogs covalently modify the active site of AChE and do not dissociate from the active site upon treatment with oximes. From a comparative analysis of kinetic data it is revealed that these compounds are less toxic and milder than the existing AChE inhibitors and can be used as potential chemotherapeutic agent against Alzheimer’s disease. Hormone-sensitive lipase (HSL) is another serine hydrolase enzyme that hydrolyzes lipids in the form of triglycerides. It is a homodimer of 84 kDa subunits and is mostly found in adipose tissues. HSL is a potential drug target for diabetes type 2. The activity of HSL must be inhibited in insulin deficient patients to lower the risk of associated cardiovascular disease. Cyclipostin is a natural product inhibitor of HSL. Laboratory synthesized monocyclic phosphonate analogs of cyclipostin having varying C-chain length exhibited μM potency against rat HSL. The potency of these analogs improved upon introducing longer C-chain like C16. This class of compounds showed an aggregation property that affected their potency against the enzyme. The attachment of the C-chain at the P-center of the monocyclic phosphonate analog considerably improved the potency (almost 10 fold)

Cholinesterase Research

This collection of 10 papers includes original as well as review articles focused on the cholinesterase structural aspects, drug design and development of novel cholinesterase ligands, but also contains papers focused on the natural compounds and their effect on the cholinergic system and unexplored effects of donepezil

Biochemical mechanisms and target drugs in neurodegenerative diseases

Neurodegenerative diseases, namely Alzheimer’s and Parkinson’s diseases are a major challenge for medicine and public health due to their prevalence in developed countries. Thus, the research for therapies for neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, should be based on understanding their molecular and biochemical pathogenesis. The research conducted in this thesis involves screening of different families of compounds (isoquinolinones, azepanones, indolinones, diether-esters, chromanones, chromanols and rivastigmine derivatives) based on their ability to inhibit the activity of the therapeutic targets acetylcholinesterase, butyrylcholinesterase and monoamine oxidase B. These targets were chosen for their importance in the neuropathology of Alzheimer’s and Parkinson’s diseases. The most promising compounds were then selected, and the determination of their action at the molecular level was studied via STD-NMR. These studies allow us to understand the importance of different functionalities within the inhibitor molecule on the inhibition of the selected targets, and thus direct the investigation in the sense of developing compounds that can be better inhibitors. Toxicological and pharmacological evaluation of the most promising synthesized compounds was performed using two different biological models, A. salina and Swiss mouse model. Compound 4-[(3-hydroxy-2-oxo-3-phenylindolin-1-yl)methyl]piperidin-1-ium chloride was tested ex vivo against hepatic AChE and BuChE, showing IC50 values of 594.64 μM and 434.51 μM, respectively. This compound was also assayed in vivo after intraperitoneal administration of 3 mg kg-1 and 6 mg kg-1 in Swiss mice, using donepezil (3 mg kg-1) as a benchmark. This synthetic compound gave better brain AChE inhibition than donepezil, indicating that this compound might have a similar brain uptake mechanism to that of donepezil; Resumo: Mecanismos bioquímicos e alvos terapêuticos em patologias neurodegenerativas As doenças neurodegenerativas, nomeadamente as doenças de Alzheimer e Parkinson são um enorme desafio quer para a medicina quer em termos de saúde pública devido à sua prevalência nos países desenvolvidos. Assim, a pesquisa de terapias para doenças neurodegenerativas, como o Parkinson e o Alzheimer, deve ser baseada na compreensão da sua patogénese molecular e bioquímica. Este trabalho envolve screening de diferentes famílias de compostos; derivados de isoquinolinonas, azepanonas, indolinonas, dieter-ester, cromanonas, cromanois e de rivastigmina, baseado na sua capacidade de inibir a atividade de alvos terapêuticos de algumas doenças neurodegenerativas como Alzheimer e Parkinson, nomeadamente: acetilcolinesterase, butirilcolinesterase e monoamino oxidase B. Os compostos promissores foram selecionados para determinação da sua ação a nível molecular por STD-NMR. Este estudo permite compreender a importância dos diferentes grupos funcionais na inibição dos alvos selecionados, e desta forma, direcionar a investigação no sentido de desenvolver compostos com maior atividade inibitória. A avaliação toxicológica e farmacológica dos compostos sintetizados mais promissores foi efetuada utilizando dois modelos biológicos diferentes, A. salina e ratinho Swiss. O composto cloreto de 4-[(3-hidroxi-2-oxo-3-fenilindolin-1-il) metil] piperidin-1-ium foi testado ex vivo em homogenatos de hepatócito de murganho Swiss, tendo apresentado valores de IC50 de 594.64 μM e de 434.51 μM para as atividades de AChE e BuChE, respetivamente. A capacidade inibitória deste composto foi também avaliada in vivo após administração interperitoneal de 3 mg kg-1 e 6 mg kg-1 em ratinhos Swiss, utilizando donepezilo (3 mg kg-1) como padrão. Os resultados mostraram que o composto sintetizado apresentou valores de inibição de atividade de AChE no cérebro superiores aos observados para o donepezilo, podendo indicar que a sua captação pelos tecidos cerebrais poderá ser efetuada de modo semelhante à do donepezilo

Development of rapid and inexpensive derivatisation methods for methylphosphonic acid

This study probed alternative methods towards rapid and inexpensive in-field derivatisation of methylphosphonic acid (MPA), the final hydrolysis product of several chemical warfare nerve agents, for detection by gas chromatography-mass spectrometry (GC-MS). Initially, research focused on acetylation as a derivatisation method, with attempted preparation of a reference sample of diacetyl methylphosphonate. This was unsuccessful with reduction of the acetyl carbonyl group during synthesis and detection of diethyl methylphosphonate instead. The research subsequently focused on esterification of methylphosphonic acid with methanol or ethanol. Preparations employed the use of dicyclohexyl carbodiimide as a coupling reagent, and silica chloride or sulfuric acid as catalysts. The coupling reagents and catalysts utilised had limited success, with no dialkyl methylphosphonate ester derivates detected via GC-MS. Dialkyl derivatives were successfully prepared by conversion of MPA to methylphosphonyl dichloride using thionyl chloride, and subsequent reaction with alcohols. Conversion of methylphosphonyl dichloride to corresponding dialkyl methylphosphonates occurs rapidly with derivatives being detected within 10 minutes for primary alcohols (methanol, ethanol, and butan-1-ol) and within 20 minutes for secondary alcohols (propan-2-ol), while benzyl alcohol did not react. Dialkyl methylphosphonates were identified by analysis of reaction products using GC-MS and nuclear magnetic resonance (NMR) spectroscopy. This method applied here acts as a proof of concept, providing a promising platform for future exploration. Alkylation of methylphosphonic acid with alcohols provides an inexpensive and relatively non-hazardous alternative to current widely accepted derivatisation methods in supporting the rapid in-field detection of chemical warfare nerve agents

Snake Venom

Venomous snakes belonging to the family Viperidae, Elapidae, Colubridae and Hydrophidae, produces snake venom in order to facilitate immobilization and digestion of prey, act as defense mechanism against threats. Venom contains zootoxins which is a highly modified saliva that is either injected via fangs during a bite or spitted. The modified parotid gland, encapsulated in a muscular sheath, present on each side of the head, below and behind the eye, have large alveoli which temporarily stores the secreted venom and later conveyed by a duct to tubular fangs through which venom is injected. Venoms are complex mixtures of more than 20 different compounds, mostly proteins and polypeptides, including proteins, enzymes and substances with lethal toxicity which are either neurotoxic or haemotoxic in action and exert effects on nervous/muscular impulses and blood components. Lots of research are directed to use venoms as important pharmacological molecules for treating various diseases like Alzheimer’s disease, Parkinson’s disease etc