ABSTRACT
The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.
Subject(s)
Acetylcholinesterase/metabolism , Cholinesterase Inhibitors/pharmacology , Drug Design , Organophosphorus Compounds/pharmacology , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/chemistry , Structure-Activity RelationshipABSTRACT
Dust was investigated for its ability to retain source attribution profiles (SAPs) after chemical exposure. Three distinct sources of the organophosphate pesticide acephate were investigated as a proof-of-concept model. In addition, attribution profiles were created and tested using compounds related to chemical warfare agents (CWAs), specifically VX and G-series agents: O-ethyl methylphosphonothioate (EMPTA), N,N-diisopropylmethylamine (DIPMA), N,N-diisopropylethylamine (DIEA), diisopropylamine (DIPA), diethyl aniline (DEA), diethyl ethyl phosphonate (DEEP), trimethyl phosphite (TMP), dimethyl hydrogen phosphite (DMHP), diethyl hydrogen phosphite (DEHP), triethyl phosphate (TEP), ethyl methylphosphonate (EMPA), and diisopropyl methylphosphonate (DIMP). Dust was collected from a storage shed, aliquots deposited on carpet and loaded with distinct chemical profiles using an exposure chamber and aerosolizer. After a given period of time (1h, 24h, or 72 h), the dust was extracted and its SAP analyzed by gas chromatography-mass spectrometry (GC-MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Principal components analysis (PCA) was used to determine the association of dust exposed to the same and different chemical sources. PCA results demonstrate that dust samples exposed to distinct chemical sources are clearly differentiated from one another across all collection times. Furthermore, dust aliquots exposed to the same source can be clearly associated with one another across all collection times. When the CWA-related compounds were subjected to elevated temperature (90°C) conditions, it was found that the signature was stable at the 1h and 24h collections. At 72 h and elevated temperature, larger deviations from the control were observed for some compounds. Elevated pH (10) affected the profile to a lesser degree than elevated temperature. Overall, dust is found to be an effective media for the in situ collection of source attribution profiles.
ABSTRACT
A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.
