Print Email Facebook Twitter Activity based protein profiling leads to identification of novel protein targets for nerve agent VX Title Activity based protein profiling leads to identification of novel protein targets for nerve agent VX Author Carmany, D. Walz, A.J. Hsu, F.L. Benton, B. Burnett, D. Gibbons, J. Noort, D. Glaros, T. Sekowski, J.W. Publication year 2017 Abstract Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics. © 2017 American Chemical Society. Subject Observation, Weapon & Protection SystemsCBRN - CBRN ProtectionTS - Technical SciencesAcylglycerol lipaseFatty acid amidaseIsocitrate dehydrogenaseIsocitrate dehydrogenase 2Isocitrate dehydrogenase 3Malate dehydrogenaseMethylphosphonothioic acid s (2 diisopropylaminoethyl) o ethyl esterStreptavidinSuccinate coenzyme A ligaseUnclassified drugAnimal tissueBrain tissueCell lysateCholinergic systemControlled studyCovalent bondEiaphragmEnergy metabolismEnzyme inhibitionHeart tissueKidney tissueLiquid chromatography-mass spectrometryLiver tissueNonhumanProtein interactionProtein targetingQuantitative analysisRatAcylglycerol lipase, 9040-75-9Fatty acid amidase, 153301-19-0Isocitrate dehydrogenase, 9001-58-5Malate dehydrogenase, 9001-64-3Methylphosphonothioic acid s (2 diisopropylaminoethyl) o ethyl ester, 50782-69-9Streptavidin, 9013-20-1Succinate coenzyme A ligase, 37341-57-4, 9014-36-2, 9080-33-5 To reference this document use: http://resolver.tudelft.nl/uuid:03bdf64e-30f1-459e-adcc-765e8ba244fd DOI https://doi.org/10.1021/acs.chemrestox.6b00438 TNO identifier 762766 Publisher American Chemical Society ISSN 0893-228X Source Chemical Research in Toxicology, 30 (4), 1076-1084 Article number 28267914 Bibliographical note Funding details: CB3810, DTRA, Defense Threat Reduction Agency Funding text: This project received support from the Defense Threat Reduction Agency − Joint Science and Technology Office for Chemical and Biological Defense (Project CB3810) to J.W.S. Document type article Files To receive the publication files, please send an e-mail request to TNO Library.