title: Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX.
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.
authors.affiliation: Excet, Inc. , 6225 Brandon Avenue, Suite 360, Springfield, Virginia 22150, United States.
authors.forename: Dan
authors.initials: D
authors.lastname: Carmany
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Walz
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: Fu-Lian
authors.initials: FL
authors.lastname: Hsu
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: Bernard
authors.initials: B
authors.lastname: Benton
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: David
authors.initials: D
authors.lastname: Burnett
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Gibbons
authors.affiliation: TNO Defense, Security, and Safety, P.O. Box 45, 2280 AA Rijswijk, The Netherlands.
authors.forename: Daan
authors.initials: D
authors.lastname: Noort
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: Trevor
authors.initials: T
authors.lastname: Glaros
authors.affiliation: Research and Technology Division, US Army Edgewood Chemical Biological Center , Aberdeen Proving Ground, Maryland 21010, United States.
authors.forename: Jennifer W
authors.identifier: 0000-0003-4561-640X
authors.initials: JW
authors.lastname: Sekowski
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006321:Heart / Q000187:drug effects; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D008297:Male; D000067397:Nerve Agents / Q000737:chemistry* / Q000633:toxicity; D009946:Organothiophosphorus Compounds / Q000737:chemistry* / Q000633:toxicity; D010455:Peptides / Q000032:analysis; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D053719:Tandem Mass Spectrometry; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D007521:Isocitrate Dehydrogenase; D008291:Malate Dehydrogenase; D000067397:Nerve Agents; D009946:Organothiophosphorus Compounds; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D000099252:Fatty Acid Amide Hydrolases; C009680:VX-agent