A mixture of three engineered phosphotriesterases enables rapid detoxification of the entire spectrum of known threat nerve agents

Abstract

Nerve agents are organophosphates (OPs) that potently inhibit acetylcholinesterase, and their enzymatic detoxification has been a long-standing goal. Nerve agents vary widely in size, charge, hydrophobicity and the cleavable ester bond. A single enzyme is therefore unlikely to efficiently hydrolyze all agents. Here, we describe a mixture of three previously developed variants of the bacterial phosphotriesterase (Bd-PTE) that are highly stable and nearly sequence identical. This mixture enables effective detoxification of a broad spectrum of known threat agents—GA (tabun), GB (sarin), GD (soman), GF (cyclosarin), VX and Russian-VX. The potential for dimer dissociation and exchange that could inactivate Bd-PTE has minimal impact, and the three enzyme variants are as active in a mixture as they are individually. To our knowledge, this engineered enzyme ‘cocktail’ comprises the first solution for enzymatic detoxification of the entire range of threat nerve agents.

Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition

“…58 Moreover, it was speculated that PTE heterodimers that could spontaneously form in such mixtures might lead to lower hydrolytic activity. 25 However, our findings suggest that the two different active centers in a PTE heterodimer function independently of each other, retaining high catalytic efficiency, while stabilizing mutations at the interface, if properly designed, have no detrimental impact on activity. Thus, our bispecific enzyme engineering strategy offers a fundamental solution and a viable approach for the broad-band degradation of OPs by employing a single, well-defined biological entity.…”

“…12,56 However, during protein evolution, an increase in enzyme activity often leads to specialization on fewer substrates, thereby narrowing the substrate range and limiting broader application. 57 The previously proposed use of enzyme mixtures as bioscavengers for the efficient inactivation of different OP nerve agents 25 would be very costly because each component has to be manufactured separately under GMP guidelines and validated in clinical studies. This is particularly the case for enzymes that have substantially different substrate specificities as they usually show significant deviations in their amino acid sequences and exhibit distinct physicochemical properties.…”

“…Furthermore, this is a caveat for biopharmaceutical development, where biomanufacturing and clinical trials according to regulatory guidelines require a substantial financial investment for each individual biological drug candidate. Thus, the administration of enzyme mixtures, as proposed, 25 would involve elevated development and manufacturing effort and will most likely not offer a viable solution.…”

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions

“…with N-acyl homoserine lactones [108,109]. Thus, they can be rationally combined to obtain universal enzyme preparations [110] or even hybrid molecules with other enzymes [111] or with 'guide' DNA [112]. This opens up new promising perspectives for their practical application.…”

Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P–S Bonded Organophosphorus as Monitored by Spectrofluorimetry

“…OP hydrolases are of great biotechnological and medical interest. Phosphotriesterases (PTE) of various origins, in particular, can be used for detection of OPs [13], decontamination and remediation [8,[14][15][16][17][18] and for therapy of OP poisoning as catalytic bioscavengers [7,[19][20][21][22]. Because OPs are hemi-substrates of ChEs, research of novel human ChE mutants capable of hydrolyzing OPs is of great interest for improving catalytic bioscavenger-based medical countermeasures of OP poisoning [23][24][25] and implementation of pseudocatalytic bioscavenger systems composed of ChE-reactivator [26][27][28].…”