V-type Agents Research Articles

Comprehensive insight on protein modification by V-type agent: A chemical proteomic approach employing bioorthogonal reaction.

Organophosphorus compounds (OPs) such as chemical agents and pesticides are posing critical threats to civilians due to their irreversible phosphonylation of diverse amino acids residues forming different protein adducts. However, traditional analytical approaches are quite limited in capturing the myriad of post-translational events that affect protein functions, especially in identifying the low-abundance OP adducts. Herein a systematic proteomic strategy based on a typical click-enrich-release-identify bioorthogonal operation was firstly developed by employing an alkynyl-tagged V-type agent probe (AVP) and a biotin-based azido-enrichment linker (BTP-N3 ). AVP targeting peptides from human serum albumin (HSA) or plasma were captured by BTP-N3 via CuAAC click reaction, enriched by streptavidin beads, released by selective alkaline hydrolysis of phenacyl ester bond, and subsequently sequenced by LC-MS/MS. This strategy has helped identifying 1115 unique OP adduction sites on 163 proteins in human plasma, and covers lots of OP adducts that cannot be achieved by traditional detection methods. The comprehensive coverage of novel OP substrates provided a general and sensitive approach to retrospective verification and/or dose assessment of toxic OPs.

Software-Assisted Automated Detection and Identification of "Unknown" Analogues: Implementation on V-Type Nerve Agents.

V-type nerve agents are among the most toxic organophosphorus chemical warfare agents, and they are under strict regulation and supervision by the OPCW (Organization for the Prohibition of Chemical Weapons). The V-type class of materials refers to a potentially large number of analogues and isomers. In order to expose instances of unfulfillment of the OPCW treaty, it is essential to have the ability to detect and identify "unknown" analogues of this family, even in the absence of an analytical standard. This work demonstrates a new automated tool for the detection and identification of V-type analogues, using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" software data processing. This software, originally developed for metabolism and metabolomics screening, is used here to automatically detect various V-type analogues by picking peaks and comparing them to "in-silico" calculated modifications made on a predefined basic V-backbone structure (according to the OPCW definitions for V-type agents). Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component, for both the V-type analogue (using ESI(+) analysis) as well as its hydrolysis product (using ESI(-) analysis) for a better elucidation of the phosphonate "head" structure. This method was found to be useful for the detection and identification of several "unknown" analogues, at low ng/mL levels in soil extracts.

Retrospective detection for V-type OPNAs exposure via phosphonylation and disulfide adducts in albumin

Organophosphorus nerve agents (OPNAs) that damage the central nervous system by inhibiting acetylcholinesterase activity, pose severe threats to human health and life security. Reliable biomarkers that quickly and accurately detect OPNAs exposure are urgently needed to help diagnose quickly and treat in time. Albumins that covalently bind to OPNAs could serve as important targets for retrospective verification of OPNAs exposure. The goal of this study is to explore the potential biomarkers in albumins with high reactivity and good stability and expand the group of potential biomarkers in different species for detecting the exposure of V-type OPNAs including O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). Taking human serum albumin (HSA), bovine serum albumin (BSA) and rabbit serum albumin (RSA) as the research objectives, multiple active sites including phosphonylation and disulfide adduct sites were observed in albumins from different species. Numerous phosphonylation sites labeled by all agents in one type of albumin were found. Among the different species, four shared phosphonylation sites with high reactivity include K499, K549, K249, and Y108. In addition, Y108 on ETY*GEMADCCAK, Y287 on Y*ICENQDSISSK, Y377 on TY*ETTLEK and Y164 on YLY*EIAR in HSA were stably phosphonylated by all agents in gradient concentration, making them stable and suitable potential biomarkers for V-type OPNAs exposure. Notably, Y108 on ETY*GEMADCCAK in HSA, on DTY*GDVADCCEK in RSA, and on ETY*GDMADCCEK in BSA were highly reactive to all V-type agents, regardless of species. It was also successfully labeled in HSA exposed to class V agents in gradient concentration. Y108 is expected to be used to screen and identify the exposure of V-type agents in the retrospective research. Disulfide adducts sites, consisted of four sites in HSA and two sites in BSA were also successfully labeled by V-type agents, and characteristic ion fragments from these disulfide adducts were also identified by secondary mass spectrometry. Molecular simulation of the stably modified sites were conducted to discover the promoting factors of covalent adduct formation, which help further clarify formation mechanism of albumin adducts at active sites.

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M−1 min−1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC–MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(−) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(−) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

Tracing and attribution of V-type nerve agents in human exposure by strategy of assessing the phosphonylated and disulfide adducts on ceruloplasmin

V-type agents are highly toxic organophosphorus nerve agents that inhibit acetylcholinesterase in the nervous system, causing a series of poison symptoms. Trace analytical methods are essential for the specific verification of exposure to these agents, especially for human exposure. This paper investigates the phosphonylated and disulfide adducts between human ceruloplasmin and O-ethyl S-(2-(diisopropylamino)ethyl) methylphosphonothioate (VX), O-isobutyl S-(2-(diethylamino)ethyl) methylphosphonothioate (VR), and O-butyl S-(2-(diethylamino)ethyl) methylphosphonothioate (Vs). After being digested by trypsin, the mixture of peptides was separated by a nano-liquid chromatography (nano-LC) and analyzed using quadrupole-orbitrap mass spectrometry (Q-Orbitrap-MS). The sensitive LC-MS/MS-assisted proteomics approach was developed to achieve the identification of human exposure to V-type agents based on these modified sites; results revealed that potential biomarkers could be derived from adducts based on the sulfur- and phosphorus-containing groups of V-type agents. This work offered a novel insight into the mechanism of disulfide-containing adducts resulting from the replacement of disulfide bridges by the thiolate groups from the V-type agents. Moreover, four disulfide adducts on human ceruloplasmin were also discovered during this research, specifically confirming exposure to the V-type agents. Furthermore, molecular simulation testified to the reactivity of the modified sites. Collectively, our findings suggest that the eleven binding sites on human ceruloplasmin have the potential use as a selective marker for prediction the V-type agent exposure in humans.

Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(−) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5×106M−1min−1). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg−1 i.v.) 5min after s.c. challenge with VX (∼2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg−1) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg−1 C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(−)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.

Rational design of organophosphorus hydrolase with high catalytic efficiency for detoxifying a V-type nerve agent

V-type nerve agents, known as VX, are organophosphate (OP) compounds, and show extremely toxic effects on human and animals by causing cholinergic overstimulation of synapses. The bacterial organophosphorus hydrolase (OPH) has attracted much attention for detoxifying V-type agents through hydrolysis of the P–S bond. However, low catalytic efficiency of OPH has limited the practical use of the enzyme. Here we present rational design of OPH with high catalytic efficiency for a V-type nerve agent. Based on the model structure of the enzyme and substrate docking simulation, we predicted the key residues that appear to enhance the access of the substrate to the active site of the enzyme, and constructed numerous OPH mutants. Of them, double mutant, L271/Y309A, was shown to exhibit a 150-fold higher catalytic efficiency for VX than the wild-type.

Changes in extracellular striatal acetylcholine and brain seizure activity following acute exposure to nerve agents in freely moving guinea pigs

Organophosphorus nerve agents irreversibly inhibit acetylcholinesterase (AChE) in the peripheral and central nervous systems, causing an increase in the concentration of acetylcholine (ACh) in the synapse or neuromuscular junction and subsequent adverse effects. In this study, in vivo microdialysis was utilized to collect samples from the striatum for monitoring changes in extracellular ACh levels along with cortical electroencephalographic (EEG) recordings for identifying seizure activity after acute subcutaneous (s.c.) exposure to 1.0 × LD50 of the nerve agents sarin, soman, or one of two V-type agents (VX, or a Russian V-agent, designated VR) in unanesthetized freely moving guinea pigs. Based on EEG recordings, these animals were subsequently divided into groups that developed seizures (S) and those that did not develop seizures (NS). Maximum ACh levels in the striatum were observed at 60–70 min for sarin and soman S groups and 105 min for VX and VR S groups. In all NS groups the greatest increase in extracellular ACh occurred within 30 min after exposure, although in the sarin NS group a few sporadic increases of ACh from control occurred. Animals that developed seizures, regardless of the nerve agent, had significantly higher extracellular striatal ACh levels compared to the controls or those animals that did not develop seizures, yet both S and NS groups displayed similar levels of blood AChE inhibition. Regardless of the agent, all animals in the non-seizure groups survived 24 h, while lethality (25–42%) was observed only in animals that experienced seizure activity.