Application of Recombinant DNA Methods for Production of Cholinesterases as Organophosphate Antidotes and Detectors
| Autor: | Palmer Taylor, Elsa Reiner, Zrinka Kovarik, Zoran Radić |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2007 |
| Předmět: |
acetylcholinesterase
fluorescent acetylcholinesterase organophosphate inhibitors oxime antidotes oxime-assisted catalysis reactivation kinetics remote detection in situ - click chemistry template-guided drug design acetilkolinesteraza antidoti detekcija fluorescentna acetilkolinesteraza in situ - klik kemija kalup-dizajn lijekova kataliza potpomognuta oksimom kinetika reaktivacije organofosfatni inhibitori |
| Zdroj: | Arhiv za higijenu rada i toksikologiju Volume 58 Issue 3 |
| ISSN: | 1848-6312 0004-1254 |
| Popis: | To develop new avenues for synthesizing novel antidotes for organophosphate poisoning and for detection of the organophosphates, we have turned to recombinant DNA methods to synthesize cholinesterases with unusual properties. For antidotal therapy we describe mutations of the native mouse and human enzymes that allow for enhanced rates of oxime reactivation. Such enzymes, when localized in the circulation, would enable the circulating cholinesterase to become a catalytic rather than simply a stoichiometric scavenger. Hence, “oxime-assisted catalysis” provides a means for scavenging the organophosphates in the circulation thereby minimizing their tissue penetration and toxicity. Accordingly, the oxime antidote or prophylactic agent has a dual action within the circulation and at the tissue level. Second, through a novel chemistry, termed freeze-frame, click chemistry, we have used organophosphate conjugates of acetylcholinesterase as templates for the synthesis of novel nucleophilic reactivating agents. Finally, acetylcholinesterase can be modified through cysteine substitution mutagenesis and attachment of fluorophores at the substitution positions. When linked at certain locations in the molecule, the attached fluorophore is sensitive to organophosphate conjugation with acetylcholinesterase, and thus the very target of insecticide or nerve agent action becomes a detection molecule for organophosphate exposure. Razvijajući novi pristup sintezi antidota pri otrovanju organofosfatima kao i njihovu detekciju, primijenili smo metode rekombinantne DNA za pripremu kolinesteraza s neuobičajenim svojstvima. Za antidotsku terapiju istražili smo mutacije prirodnih enzima miša i čovjeka koje povećavaju brzine reaktivacije oksimom. Takvi enzimi bi po unosu u cirkulaciju postali katalitički, a ne samo stehiometrijski odstranjivači organofosfata. Na taj način “oksimom potpomognuta kataliza” omogućava čišćenje organofosfata iz cirkulacije umanjujući prodiranje organofosfata u tkiva i njihovu toksičnost. Prema tome, oksim kao antidot ima dvojaku ulogu: u cirkulaciji i na razini tkiva. S druge strane, uporabom novog sintetskog pristupa u oblikovanju biološki aktivnih spojeva poznatog kao “klik kemija” diskretnih proteinskih konformacija, organofosforilirani konjugati acetilkolinesteraze služe kao kalup u sintezi novih nukleofilnih reaktivatora. Naposljetku, acetilkolinesteraza se može mutagenezom modificirati uvo|enjem cisteina na koje se mogu vezati fluorofori. Fluorofori uvedeni na određena mjesta u molekuli acetilkolinesteraze mijenjaju svoja fluorescentna svojstva pri konjugaciji organofosfata s enzimom koji na taj način od objekta djelovanja insekticida i živčanih bojnih otrova postaje molekula za detekciju izloženosti organofosfatima. |
| Databáze: | OpenAIRE |
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