Abstract:The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M−1 min−1) towards VX and a preferential hydrolysis of the more toxic P(−) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/… Show more
“…However, we were able to identify some critical amino acid positions for expression yield and folding efficiency or thermal stability (306, 303, 233, 259 and 260, 262, 267, 257). This knowledge may prove useful during future biopharmaceutical development as a catalytic bioscavenger for the treatment of acute intoxications by nerve agents 15 as well as OP pesticides, for example when combined with other promising mutants to build a heterodimeric PTE with a broader substrate profile. 56 ■ ASSOCIATED CONTENT…”
Section: ■ Conclusionmentioning
confidence: 99%
“…13 Moreover, we found that this mutant exhibits high hydrolytic activities toward several other nerve agents, including GD, 14 and a PASylated version of this BdPTE variant with an extended plasma half-life showed promising results in vivo in a rat treatment study after VX poisoning. 15 To better understand the role of some enzyme active site residues for the phosphorothioester bond cleavage in VX and related OP substrates, we set out to examine the crystal structure of this mutant BdPTE and, based on this information, we constructed 30 new mutants and analyzed their hydrolytic activities toward a representative set of four nerve agents: VX, VR, GB, and GF (Scheme 1).…”
Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)� stabilized against oxidation by substitution of two unpaired Cys residues�in complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253−276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.
“…However, we were able to identify some critical amino acid positions for expression yield and folding efficiency or thermal stability (306, 303, 233, 259 and 260, 262, 267, 257). This knowledge may prove useful during future biopharmaceutical development as a catalytic bioscavenger for the treatment of acute intoxications by nerve agents 15 as well as OP pesticides, for example when combined with other promising mutants to build a heterodimeric PTE with a broader substrate profile. 56 ■ ASSOCIATED CONTENT…”
Section: ■ Conclusionmentioning
confidence: 99%
“…13 Moreover, we found that this mutant exhibits high hydrolytic activities toward several other nerve agents, including GD, 14 and a PASylated version of this BdPTE variant with an extended plasma half-life showed promising results in vivo in a rat treatment study after VX poisoning. 15 To better understand the role of some enzyme active site residues for the phosphorothioester bond cleavage in VX and related OP substrates, we set out to examine the crystal structure of this mutant BdPTE and, based on this information, we constructed 30 new mutants and analyzed their hydrolytic activities toward a representative set of four nerve agents: VX, VR, GB, and GF (Scheme 1).…”
Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)� stabilized against oxidation by substitution of two unpaired Cys residues�in complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253−276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.
“…The µLC-ESI MS/HR MS (PIS) method (method A) for biomarker detection was applied to heparin plasma obtained from an in vivo study with male Wistar rats. Details on the scope, conditions and results of the study are described by Stigler et al (Stigler et al 2022 ). In brief, male Wistar rats were anaesthetized under continuous surveillance of anaesthesia depth and exposed to VX subcutaneously (s.c.) using a dose of 25 µg/kg (approximately 2 × LD 50 for rats i.m.)…”
Section: Methodsmentioning
confidence: 99%
“…P02770) in plasma is similar to that in humans (40 mg/mL) (Metz und Schütze 1975 ) and RSA also contains a reactive Tyr 411 and a Cys 34 residue exhibiting a free thiol group (Chen et al 2013 ) as well as a LeuProCys 448 sequence as an analogue to the human MetProCys 448 (Kranawetvogl et al 2018a ). Accordingly, we developed and validated a µLC-ESI MS/HR MS method for the qualitative detection of adducted biomarkers and applied it to an in vivo study of VX-exposed rats (Stigler et al 2022 ). Fig.…”
VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.
“…Since 2018, in the toxicological literature, the following lines of investigation can be identified, which are reflected by key contributions to Archives of Toxicology. Clinical reports (Novichok) and improvement of therapy of intoxications by organophosphorus compounds (Hulse et al 2019 ; Steindl et al 2021 ; Vale et al 2018 ; Worek et al 2020 ; Zandona et al 2020 , 2021 ): a special focus is development of novel antidotes, e.g., novel oximes (Machamer et al 2020 ; Zandona et al 2020 , 2021 ) or engineered phoshotriesterases (Stigler et al 2021 ). Given the world-wide incidents attributed to chemical weapons, such as Novichok agents, intensive-care clinicians should know how to rapidly recognize symptoms of acute poisoning and to administer life-saving antidotal therapy, when indicated (Chai et al 2018 ).…”
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