Primjena tehnologije rekombinantne DNA za pripravke kolinesteraza kao antidota i detektora organofosfata

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

Design, expression and characterization of mutants of fasciculin optimized for interaction with its target, acetylcholinesterase

Predicting mutations that enhance protein–protein affinity remains a challenging task, especially for high-affinity complexes. To test our capability to improve the affinity of such complexes, we studied interaction of acetylcholinesterase with the snake toxin, fasciculin. Using the program ORBIT, we redesigned fasciculin's sequence to enhance its interactions with Torpedo californica acetylcholinesterase. Mutations were predicted in 5 out of 13 interfacial residues on fasciculin, preserving most of the polar inter-molecular contacts seen in the wild-type toxin/enzyme complex. To experimentally characterize fasciculin mutants, we developed an efficient strategy to over-express the toxin in Escherichia coli, followed by refolding to the native conformation. Despite our predictions, a designed quintuple fasciculin mutant displayed reduced affinity for the enzyme. However, removal of a single mutation in the designed sequence produced a quadruple mutant with improved affinity. Moreover, one designed mutation produced 7-fold enhancement in affinity for acetylcholinesterase. This led us to reassess our criteria for enhancing affinity of the toxin for the enzyme. We observed that the change in the predicted inter-molecular energy, rather than in the total energy, correlates well with the change in the experimental free energy of binding, and hence may serve as a criterion for enhancement of affinity in protein–protein complexes

Odnos strukture i aktivnosti u reaktivaciji tabunom fosforilirane ljudske acetilkolinesteraze bispiridinijevim para-aldoksimima

We investigated interactions of bispyridinium para-aldoximes N,N’-(propano)bis(4-hydroxyiminomethyl) pyridinium bromide (TMB-4), N,N’-(ethano)bis(4-hydroxyiminomethyl)pyridinium methanosulphonate (DMB-4), and N,N’-(methano)bis(4-hydroxyiminomethyl)pyridinium chloride (MMB-4) with human erythrocyte acetylcholinesterase phosphorylated by tabun. We analysed aldoxime conformations to determine the flexibility of aldoxime as an important feature for binding to the acetylcholinesterase active site. Tabun-inhibited human erythrocyte acetylcholinesterase was completely reactivated only by the most flexible bispyridinium aldoxime - TMB-4 with a propylene chain between two rings. Shorter linkers than propylene (methylene or ethylene) as in MMB-4 and DMB-4 did not allow appropriate orientation in the active site, and MMB-4 and DMB-4 were not efficient reactivators of tabun-phosphorylated acetylcholinesterase. Since aldoximes are also reversible inhibitors of native acetylcholinesterase, we determined dissociation constants and their protective index against acetylcholinesterase inactivation by tabun.Proučavali smo interakcije bispiridinijevih para-oksima N,N’-(propano)bis(4-hidroksiiminometil)piridinijeva bromida (TMB-4), N,N’-(etanano)bis(4-hidroksiiminometil)piridinijeva metanosulfonata (DMB-4) i N,N’- (metano)bis(4-hidroksiiminometil)piridinijeva klorida (MMB-4) s ljudskom eritrocitnom acetilkolinesterazom fosforiliranom tabunom. Da bismo odredili fleksibilnosti aldoksima, što je važna osobina kod njihova vezanja u aktivno mjesto acetilkolinesteraze, analizirali smo i konformacijske odlike aldoksima. Ljudska acetilkolinesteraza inhibirana tabunom bila je potpuno reaktivirana samo najfleksibilnijim bispiridinijevim aldoksimom – TMB-4. Aldoksimi MMB-4 i DMB-4 nisu bili efikasni reaktivatori acetilkolinesteraze fosforilirane tabunom jer je kod tih spojeva lanac koji povezuje dva prstena kraći od propilena (metilen u MMB-4 i etilen u DMB-4), što ne dopušta povoljnu orijentaciju tih aldoksima unutar aktivnog mjesta enzima. S obzirom na to da su aldoksimi i reverzibilni inhibitori nativne acetilkolinesteraze, odredili smo njihove disocijacijske konstante, kao i zaštitu acetilkolinesteraze od inhibiranja tabunom reverzibilnim vezanjem aldoksima

Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms in its ability to produce complex biologics with simple chemically defined media and reduced biosafety concerns. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous technoeconomic models or analyses have been published. Here we present technoeconomic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4–11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes