<i>In vitro</i> and <i>in vivo</i> efficacy of PEGylated diisopropyl fluorophosphatase (DFPase)

Melzer, Marco; Heidenreich, Anne; Dorandeu, Frédéric; Gäb, Jürgen; Kehe, Kai; Thiermann, Horst; Letzel, Thomas; Blum, Marc‐Michael

doi:10.1002/dta.363

Cited by 21 publications

(13 citation statements)

References 39 publications

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“…One of the ways for the mutant AChE to gain prolonged circulation time is a conjugation with polyethylene glycol. 16,37 Such an enzyme should retain all of the kinetic characteristics of its nonPEGylated form while having 20-fold greater mean residence time. 37 Further analysis of soman scavenging should also be directed to stereoselectivity.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes

Kovarik

Hrvat

Katalinić

et al. 2015

Chem. Res. Toxicol.

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Exposure to the nerve agent soman is difficult to treat due to the rapid dealkylation of soman-acetylcholinesterase (AChE) conjugate known as aging. Oxime antidotes commonly used to reactivate organophosphate inhibited AChE are ineffective against soman, while the efficacy of the recommended nerve agent bioscavenger butyrylcholinesterase is limited by strictly stoichiometric scavenging. To overcome this limitation, we tested ex vivo, in human blood, and in vivo, in soman exposed mice, the capacity of aging-resistant human AChE mutant Y337A/F338A in combination with oxime HI-6 to act as a catalytic bioscavenger of soman. HI-6 was previously shown in vitro to efficiently reactivate this mutant upon soman, as well as VX, cyclosarin, sarin and paraoxon inhibition. We here demonstrate that ex vivo, in whole human blood, 1 μM soman was detoxified within 30 minutes when supplemented with 0.5 μM Y337A/F338A AChE and 100 μM HI-6. This combination was further tested in vivo. Catalytic scavenging of soman in mice improved the therapeutic outcome and resulted in the delayed onset of toxicity symptoms. Furthermore, in a preliminary in vitro screen we identified an even more efficacious oxime than HI-6, in a series of forty-two pyridinium aldoximes, and five imidazole 2-aldoxime N-propyl pyridinium derivatives. One of the later imidazole aldoximes, RS-170B, was a 2–3 –fold more effective reactivator of Y337A/F338A AChE than HI-6 due to the smaller imidazole ring, as indicated by computational molecular models, that affords a more productive angle of nucleophilic attack.

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Section: Discussionmentioning

confidence: 99%

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes

Kovarik

Hrvat

Katalinić

et al. 2015

Chem. Res. Toxicol.

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“…Remarkably, in a preliminary in vivo study, Melzer et al . found that addition of pegylated DFPase minimizes lethality in rats challenged with a subcutaneous 3xLD 50 dose of soman, thereby highlighting its potential for in vivo use (Melzer et al, 2012).…”

Section: Enzymes For Decontaminationmentioning

confidence: 99%

Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges

Thakur

Medintz

Walper

2019

Front. Bioeng. Biotechnol.

101

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Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation.

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“…DFPase also has a much more accessible active site than PON1, 19 lacking the three helices that decorate the top of the β-propeller in PON1 and help it interact with HDL, thus also sequestering the active site from solvent. 19 , 32 , 34 In addition, although DFPase is far less studied than PON1, there has nevertheless also been interest in developing engineered variants of this enzyme that can be used as a biotherapeutic, 45 and the enzyme has been the subject of several biochemical, structural and also more recently computational studies (e.g., refs ( 1 ), ( 36 ), ( 39 ), ( 44 ), and ( 46 − 48 )).…”

Section: Introductionmentioning

confidence: 99%

Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering

2017

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Organophosphate hydrolases are proficient catalysts of the breakdown of neurotoxic organophosphates and have great potential as both biotherapeutics for treating acute organophosphate toxicity and as bioremediation agents. However, proficient organophosphatases such as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzing lactonase SsoPox are unable to hydrolyze bulkyorganophosphates with challenging leaving groups such as diisopropyl fluorophosphate (DFP) or venomous agent X, creating a major challenge for enzyme design. Curiously, despite their mutually exclusive substrate specificities, PON1 and diisopropyl fluorophosphatase (DFPase) have essentially identical active sites and tertiary structures. In the present work, we use empirical valence bond simulations to probe the catalytic mechanism of DFPase as well as temperature, pH, and mutational effects, demonstrating that DFPase and PON1 also likely utilize identical catalytic mechanisms to hydrolyze their respective substrates. However, detailed examination of both static structures and dynamical simulations demonstrates subtle but significant differences in the electrostatic properties and solvent penetration of the two active sites and, most critically, the role of residues that make no direct contact with either substrate in acting as “specificity switches” between the two enzymes. Specifically, we demonstrate that key residues that are structurally and functionally critical for the paraoxonase activity of PON1 prevent it from being able to hydrolyze DFP with its fluoride leaving group. These insights expand our understanding of the drivers of the evolution of divergent substrate specificity in enzymes with identical active sites and guide the future design of organophosphate hydrolases that hydrolyze compounds with challenging leaving groups.

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In vitro and in vivo efficacy of PEGylated diisopropyl fluorophosphatase (DFPase)

Cited by 21 publications

References 39 publications

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes

Catalytic Soman Scavenging by the Y337A/F338A Acetylcholinesterase Mutant Assisted with Novel Site-Directed Aldoximes

Enzymatic Bioremediation of Organophosphate Compounds—Progress and Remaining Challenges

Similar Active Sites and Mechanisms Do Not Lead to Cross-Promiscuity in Organophosphate Hydrolysis: Implications for Biotherapeutic Engineering

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