Organophosphorus Compounds: Classification and Enzyme Reactions

Organofosforni spojevi su derivati fosfatne, fosfonske ili fosfinske kiseline kod kojih atomi kisika, vezani neposredno na atom fosfora, mogu biti zamijenjeni atomima sumpora ili dušika. Ti spojevi čine veliku skupinu organskih spojeva koji se rabe ponajprije kao pesticidi, neki se rabe kao lijekovi, a najtoksičniji spojevi su živčani bojni otrovi. Glavni uzrok akutne toksičnosti organofosfornih spojeva je inhibicija acetilkolinesteraze, ključnog enzima u prijenosu živčanog impulsa. Organofosforni spojevi koji imaju sumpor vezan koordinatno-kovalentnom vezom na atom fosfora nisu inhibitori acetilkolinesteraze. Da bi ti spojevi postali biološki aktivni, moraju spontanim reakcijama ili u reakcijama biotransformacije prijeći u svoje oksoanaloge. U reakcijama biotransformacije organofosfornih spojeva može doći do transformiranja netoksičnih organofosfornih spojeva u toksične ili pretvorbe jednog toksičnog spoja u drugi, ili do pretvorbe toksičnih organofosfornih spojeva u spojeve koji više nisu inhibitori acetilkolinesteraze. U radu je prikazana klasifikacija organofosfornih spojeva koja se zasniva na prirodi supstituenata koji su vezani neposredno na središnji atom fosfora. Nadalje, u radu su opisani enzimi koji reagiraju s organofosfornim spojevima uz primjere reakcija koje ti enzimi kataliziraju.Organophosphorus compounds are derivatives of phosphoric, phosphonic or phosphinic acids whose oxygen atoms bound directly to the phosphorus atom can be substituted by sulphur or nitrogen atoms. These compounds represent a large group of organic compounds used primarily as pesticides. Some are used as drugs and the most toxic compounds as nerve agents. Acute toxicity of organophosphorus compounds is due to the inhibition of acetylcholinesterase, the critical enzyme in neurotransmission. Organophosphorus compounds whose sulphur atom creates a coordinative covalent bond with the phosphor atom are not acetylcholinesterase inhibitors. To become biologically active these compounds must transform into their oxo analogues, passing through spontaneous or biotransformation reactions. Biotransformation reactions of organophosphorus compounds involve a large number of enzymatic reactions that can make them more or less toxic, or even non-toxic for acetylcholinesterase. The classification of organophosphorus compounds in this paper considers the nature of groups bound directly to the central phosphorus atom. The paper describes the enzymes taking part in biotransformation of organophosphorus compounds and gives examples of their reactions

Aktivnost kolinesteraza u punoj ljudskoj krvi mjerena acetiltiokolinom kao supstratom i etopropazinom kao selektivnim inhibitorom butirilkolinesteraze u plazmi

A procedure is suggested for measuring acetylcholinesterase and butyrylcholinesterase activities in human whole blood using acetylthiocholine as a substrate and ethopropazine as a selective inhibitor of butyrylcholinesterase. The procedure is suitable for screening cholinesterase activities in routine and/or field tests.Predložen je postupak mjerenja aktivnosti acetilkolinesteraze i butirilkolinesteraze u punoj humanoj krvi. Aktivnost se mjeri acetiltiokolinom kao supstratom u prisutnosti i odsutnosti etopropazina. Etopropazin je selektivni inhibitor butirilkolinesteraze te aktivnost mjerena u prisutnosti etopropazina predstavlja aktivnost acetilkolinesteraze. Razlika aktivnosti mjerenih u odsutnosti i prisutnosti etopropazina predstavlja aktivnost butirilkolinesteraze. Predloženi je postupak prikladan za rutinska mjerenja i/ili mjerenja na terenu

Exploring the Active Sites of Cholinesterases by Inhibition with Bambuterol and Haloxon

The paper describes the inhibition of mouse acetylcholinesterase (AChE; EC 3.1.1.7) and mouse, human, and horse butyrylcholinesterase (BChE; EC 3.1.1.8) by 5-[2-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride (bambuterol) and by O,O-bis-(2-chloroethyl)-O-(3-chloro-4-methylcoumarin-7-yl) phosphate (haloxon). The haloxon inhibition rate constant (ki) for mouse BChE was 3.7 × 107 min–1 mol–1 dm3, which was 40-fold higher than the rate constant for mouse AChE. Bambuterol inhibition of horse BChE (ki = 2.1 × 105 min–1 mol–1 dm3) was about 25-fold slower than that of human or mouse BChE, whereas the respective haloxon inhibition of horse BChE (ki = 1.2 × 107 min–1 mol–1 dm3) was about 2-3-fold slower. Sequence alignments and the computational model of the three-dimensional structure of horse BChE suggest that residues inside the active site at positions 69, 277 and 285 are important for the differences in the inhibition of these three BChE species

Exploring the Active Sites of Cholinesterases by Inhibition with Bambuterol and Haloxon

The paper describes the inhibition of mouse acetylcholinesterase (AChE; EC 3.1.1.7) and mouse, human, and horse butyrylcholinesterase (BChE; EC 3.1.1.8) by 5-[2-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride (bambuterol) and by O,O-bis-(2-chloroethyl)-O-(3-chloro-4-methylcoumarin-7-yl) phosphate (haloxon). The haloxon inhibition rate constant (ki) for mouse BChE was 3.7 × 107 min–1 mol–1 dm3, which was 40-fold higher than the rate constant for mouse AChE. Bambuterol inhibition of horse BChE (ki = 2.1 × 105 min–1 mol–1 dm3) was about 25-fold slower than that of human or mouse BChE, whereas the respective haloxon inhibition of horse BChE (ki = 1.2 × 107 min–1 mol–1 dm3) was about 2-3-fold slower. Sequence alignments and the computational model of the three-dimensional structure of horse BChE suggest that residues inside the active site at positions 69, 277 and 285 are important for the differences in the inhibition of these three BChE species

Karbamatna skupina kao strukturni element lijekova – pregledni rad

Due to their very good chemical and proteolytic stability, ability to penetrate cell membranes, and resemblance to a peptide bond, carbamate derivatives have received much attention in recent years and got an important role in modern drug discovery and medicinal chemistry. Today, carbamates make structural and/or functional part of many drugs and prodrugs approved and marketed for the treatment of various diseases such as cancer, epilepsy, hepatitis C, HIV infection, and Alzheimer’s disease. In drugs they can play a role in drug-target interaction or improve the biological activity of parent molecules. In prodrugs they are mainly used to delay first-pass metabolism and enhance the bioavailability and effectiveness of compounds. This brief review takes a look at the properties and use of carbamates in various fields of medicine and provides quick insights into the mechanisms of action for some of them.Zbog svoje vrlo dobre kemijske i proteolitičke stabilnosti, sposobnosti prodiranja kroz stanične membrane i sličnosti s peptidnom vezom, derivati karbamata posljednjih godina sve više privlače pozornost medicinskih kemičara i dobivaju važniju ulogu u modernom načinu otkrivanja lijekova. Tako je u današnje vrijeme karbamatna skupina strukturni i funkcionalni element mnogih odobrenih lijekova, a mnogi se već i koriste kao lijekovi za liječenje raznih vrsta bolesti poput raka, epilepsije, hepatitisa C, infekcije HIV-om, Alzheimerove bolesti i mnogih drugih. U lijekovima, karbamatna skupina može biti važan dio molekule koji ima ulogu u interakciji lijek-meta ili je umetnuta u strukturu spoja kako bi se poboljšala biološka aktivnost temeljne molekule. U protulijekovima, karbamatna se skupina koristi uglavnom zbog mogućnosti smanjenja osjetljivosti spoja na metaboličke enzime, odnosno povećanja hidrolitičke stabilnosti samoga spoja. U ovom je radu dan ne samo kratki pregled karbamata koji se koriste kao lijekovi u raznim područjima primjene, kao i karbamata koji se koriste kao protulijekovi, nego i uvid u mehanizam djelovanja nekih od njih

4-Aminoqionolines as reversible inhibitors of human cholinesterase activity

We synthesised eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) carbon of 4-aminoquinoline, and tested their potency to inhibit human AChE and BChE. All of the compounds reversibly inhibited both enzymes with dissociation inhibition (Ki) constants from 0.50 to 50 µM exhibiting selectivity. In other words, for all compounds, AChE exhibited higher affinity than BChE. The most potent inhibitors of AChE were compounds with an octyl chain or adamantane, regardless of the group in position C(7). The shortening of the chain length caused the AChE inhibition decrease by 5-20 times. Docking studies made it clear that the high AChE affinity resulted from simultaneous interactions of the quinoline group with aromatic residues of both the catalytic active site and the peripheral site. In conclusion, the inhibition potency and selectivity classify several novel compounds as leads for further modification and optimization towards the development of new inhibitors of AChE and potential drugs for treatment of neurodegenerative diseases.13th International Meeting on Cholinesterases and and the 7th International Conference on Paraoxonases, 9th to 14th of September 2018 Hradec Králové, Czech Republi

Resorcinol-, catechol- and saligenin-based bronchodilating beta2-agonists as inhibitors of human cholinesterase activity

We investigated the influence of bronchodilating 2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives, and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with Ki constants ranging from 9.8 µM to 6.4 mM, and had the highest inhibition potency toward usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional - interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases

Enantiomeri kinuklidin-3-ol derivata: Razdvajanje enantiomera i interakcija s ljudskim kolinesterazama

The (R)- and (S)-enantiomers of quinuclidin-3-ol and quinuclidin-3-yl acetate as well as their quaternary N-methyl and N-benzyl derivatives were synthesized in order to study the stereoselectivity of human erythrocyte acetylcholinesterase (EC 3.1.1.7) and plasma butyrylcholinesterase (EC 3.1.1.8). The compounds were tested as substrates and inhibitors of cholinesterases. Both cholinesterases hydrolyze the derivatives of quinuclidin-3-yl acetate with a preference for the (R)- over (S)-enantiomers. In contrast to the hydrolysis of the enantiomers of acetates, the inhibition of acetylcholinesterase and butyrylcholinesterase by the (R)- and (S)-enantiomers of quinuclidin-3-ol derivatives does not reveal enantiomeric preference of the enzymes. The (R)- and (S)-acetates also act as nonstereoselective inhibitors of the enzyme-induced hydrolysis of acetylthiocholine. The best substrate is (R)-N-methyl-3-acetoxyquinuclidinium iodide with kcat = 1.5 x 106 min–1 and kcat = 5.5 x 104 min–1 for acetylcholinesterase and butyrylcholinesterase, respectively. The (R)- and (S)-N-benzylquinuclidinium derivatives are the most potent inhibitors of both enzymes.Priređeni su (R)- i (S)-enantiomeri kinuklidin-3-ola i kinuklidin-3-il-acetata te odgovarajući kvaterni N-metilni i N-benzilni derivati kako bi se proučila njihova interakcija s ljudskom eritrocitnom acetilkolinesterazom (EC 3.1.1.7) i butirilkolinesterazom iz plazme (EC 3.1.1.8). Spojevi su studirani kao supstrati i inhibitori tih enzima. Obje kolinesteraze pokazuju visoku stereoselektivnost pri hidrolizi kinuklidin-3-il acetata preferirajući (R)- u odnosu na (S)-enantiomere. Nasuprot hidrolizi enantiomera acetatnih derivata, inhibicija acetilkolinesteraze i butirilkolinesteraze s (R)- i (S)-enantiomerima kinuklidin-3-ola i kinuklidin-3-il-acetata te njihovih N-metilnih i N-benzilnih derivata, ne pokazuje stereoselektivnost tih enzima. Kao najbolji supstrat za oba enzima pokazao se (R)-N-metilkinuklidinijev acetat, s kcat = 1,5 x 106 min–1 za acetilkolinesterazu, odnosno kcat = 5,5 x 104 min–1 za butirilkolinesterazu. (R)- i (S)-N-benzilkinuklidinijevi derivati bili su najjači inhibitori za te enzime

Flavonoids as Inhibitors of Human Butyrylcholinesterase Variants

Inhibicijom butirilkolinesteraze (BChE, EC 3.1.1.8) moguće je liječiti bolesti kod kojih dolazi do smanjenja koncentracije neuroprijenosnika acetilkolina, poput Alzheimerove bolesti. Međutim, takav pristup terapiji ne smije zanemariti polimorfizam BCHE gena koji može utjecati na njezin konačan ishod. Nekolicina poznatih kolinergičnih lijekova (npr. galantamin, huperzin i rivastigmin) potječe iz biljaka ili su sintetski derivati biljnih spojeva, što opravdava daljnje istraživanje novih terapeutika iz biljaka. Flavonoidi pripadaju velikoj obitelji biološki aktivnih polifenolnih spojeva što se nalaze u mnogim biljkama, a poznata je činjenica da neki inhibiraju BChE. U radu smo se usredotočili na istraživanje flavonoida galangina, kvercetina, fisetina i luteolina kao inhibitora prirodnih inačica ljudske BChE: uobičajene, atipične i fluorid-rezistentne. Pokazano je da ispitani flavonoidi reverzibilno inhibiraju sve inačice BChE s konstantama disocijacije kompleksa enzim-inhibitor (Ki) u rasponu od 10 do 170 μmol/L. Inhibitorna moć ispitanih flavonoida spram svih triju inačica BChE povećavala se sljedećim redoslijedom: luteolin<fisetin<kvercetin<galangin, pri čemu je dokazano da inhibicija ne ovisi o polimorfizmu BChE. Rezultati upućuju na zaključak da flavonoidi mogu pomoći u daljnjem razvoju terapeutika, tj. inhibitora BChE za liječenje simptoma neurodegenerativnih bolesti i demencije.The inhibition of butyrylcholinesterase (BChE, EC 3.1.1.8) appears to be of interest in treating diseases with symptoms of reduced neurotransmitter levels, such as Alzheimer’s disease. However, BCHE gene polymorphism should not be neglected in research since it could have an effect on the expected outcome. Several well-known cholinergic drugs (e.g. galantamine, huperzine and rivastigmine) originating from plants, or synthesised as derivatives of plant compounds, have shown that herbs could serve as a source of novel target-directed compounds. We focused our research on flavonoids, biologically active polyphenolic compounds found in many plants and plant-derived products, as BChE inhibitors. All of the tested flavonoids: galangin, quercetin, fisetin and luteolin reversibly inhibited usual, atypical, and fluoride-resistant variants of human BChE. The inhibition potency increased in the following order, identically for all three BChE variants: luteolin<fisetin<quercetin<galangin. The determined enzyme-inhibitor dissociation constants (Ki) ranged from 10 to 170 mmol/L. We showed that no significant change in the inhibition potency of selected flavonoids exists in view of BChE polymorphism. Our results suggested that flavonoids could assist the further development of new BChE-targeted drugs for treating symptoms of neurodegenerative diseases and dementia

In vitro Evaluation of Aldoxime Interactions with Human Acetylcholinesterase

We related the ability of eleven pyridinium and imidazolium aldoximes to reactivate tabun-inhibited human erythrocyte acetylcholinesterase with their molecular properties. Using molecular mechanics we performed conformational analysis to determine the flexibility of the aldoximes. Semi-empirical calculations show that differences in reactivation rates probably do not origin from different electron density on the oxygen of the oxime group, but can be explained by the steric hindrance within the aldoxime molecule. Tabun-inhibited acetylcholinesterase was efficiently reactivated by flexible bispyridinium para-aldoximes with propylene or butylene linker. Although pyridinium/imidazolium aldoximes with the oxime group in ortho-position did not show significant reactivation ability, they protected acetylcholinesterase against phosphorylation by tabun due to their high affinity for the native acetylcholinesterase. The aldoximes were examined for cytotoxicity on different cell lines and no cytotoxic effect was observed for doses of up to 400 µmol dm–3