- The inhibition, reactivation and mechanism of VX-, sarin-, fluoro-VX and fluoro-sarin surrogates following their interaction with HuAChE and HuBuChE
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In this study, the mechanisms of HuAChE and HuBChE inhibition by Me-P(O) (OPNP) (OR) [PNP = p-nitrophenyl; R = CH2CH3, CH2CH2F, OCH(CH3)2, OCH(CH3) (CH2F)] representing surrogates and fluoro-surrogates of VX and sarin were studied by in vitro kinetics and mass spectrometry. The in vitro measures showed that the VX- and fluoro-VX surrogates were relatively strong inhibitors of HuAChE and HuBChE (ki ~ 105-106 M?1min?1) and underwent spontaneous and 2-PAM-mediated reactivation within 30 min. The sarin surrogates were weaker inhibitors of HuAChE and HuBChE (ki ~ 104-105 M?1min?1), and in general did not undergo spontaneous reactivation, although HuAChE adducts were partially reactivatable at 18 h using 2-PAM. The mechanism of HuAChE and HuBChE inhibition by the surrogates was determined by Q-TOF and MALDI-TOF mass spectral analyses. The surrogate-adducted proteins were trypsin digested and the active site-containing peptide bearing the OP-modified serine identified by Q-TOF as triply- and quadruply-charged ions representing the respective increase in mass of the attached OP moiety. Correspondingly, monoisotopic ions of the tryptic peptides representing the mass increase of the OP-adducted peptide was identified by MALDI-TOF. The mass spectrometry analyses validated the identity of the OP moiety attached to HuAChE or HuBChE as MeP(O) (OR)-O-serine peptides (loss of the PNP leaving group) via mechanisms consistent with those found with chemical warfare agents. MALDI-TOF MS analyses of the VX-modified peptides versus time showed a steady reduction in adduct versus parent peptide (reactivation), whereas the sarin-surrogate-modified peptides remained largely intact over the course of the experiment (24 h). Overall, the presence of a fluorine atom on the surrogate modestly altered the rate constants of inhibition and reactivation, however, the mechanism of inhibition (ejection of PNP group) did not change.
- Chao, Chih-Kai,Balasubramanian, Narayanaganesh,Gerdes, John M.,Thompson, Charles M.
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- An improved radiosynthesis of O-(2-[18F]fluoroethyl)-O-(p-nitrophenyl)methylphosphonate: A first-in-class cholinesterase PET tracer
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O-(2-Fluoroethyl)-O-(p-nitrophenyl) methylphosphonate 1 is an organophosphate cholinesterase inhibitor that creates a phosphonyl-serine covalent adduct at the enzyme active site blocking cholinesterase activity in vivo. The corresponding radiolabeled O-(2-[18F]fluoroethyl)-O-(p-nitrophenyl) methylphosphonate, [18F]1, has been previously prepared and found to be an excellent positron emission tomography imaging tracer for assessment of cholinesterases in live brain, peripheral tissues, and blood. However, the previously reported [18F]1 tracer synthesis was slow even with microwave acceleration, required high-performance liquid chromatography separation of the tracer from impurities, and gave less optimal radiochemical yields. In this paper, we report a new synthetic approach to circumvent these shortcomings that is reliant on the facile reactivity of bis-(O,O-p-nitrophenyl) methylphosphonate, 2, with 2-fluoroethanol in the presence of DBU. The cold synthesis was successfully translated to provide a more robust radiosynthesis. Using this new strategy, the desired tracer, [18F]1, was obtained in a non-decay–corrected radiochemical yield of 8?±?2% (n?=?7) in >99% radiochemical and >95% chemical purity with a specific activity of 3174?±?345 Ci/mmol (EOS). This new facile radiosynthesis routinely affords highly pure quantities of [18F]1, which will further enable tracer development of OP cholinesterase inhibitors and their evaluation in vivo.
- Neumann, Kiel D.,Thompson, Charles M.,Blecha, Joseph E.,Gerdes, John M.,VanBrocklin, Henry F.
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- Novel Organophosphate Ligand O-(2-Fluoroethyl)-O-(p-Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability and Analysis of the Inhibition and Reactivation of Cholinesterases
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The organophosphate O-(2-fluoroethyl)-O-(p-nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-18 radiolabeled organophosphate inhibitor ([18F]1) of acetylcholinesterase (AChE). In rats, [18F]1 localizes in AChE rich regions of the brain and other tissues where it likely exists as the (CH3)(18FCH2CH2O)P(O)-AChE adduct (ChE-1). Characterization of this adduct would define the inhibition mechanism and subsequent postinhibitory pathways and reactivation rates. To validate this adduct, the stability (hydrolysis) of 1 and ChE-1 reactivation rates were determined. Base hydrolysis of 1 yields p-nitrophenol and (CH3) (FCH2CH2O)P(O)OH with pseudo first order rate constants (kobsd) at pH 7.4 (PBS) of 3.25 × 10-4 min-1 (t1/2 = 35.5 h) at 25 °C and 8.70 × 10-4 min-1 (t1/2 = 13.3 h) at 37 °C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; ki = 7.5 × 105 M-1 min-1), electric eel acetylcholinesterase (EEAChE) (ki = 3.0 × 106 M-1 min-1), and human serum butyrylcholinesterase (HuBChE; 1.95 × 105 M-1 min-1). Spontaneous and oxime-mediated reactivation rates for the (CH3) (FCH2CH2O)P(O)-serine ChE adducts using 2-PAM (10 μM) were (a) HuAChE 8.8 × 10-5 min-1 (t1/2 = 131.2 h) and 2.41 × 10-2 min-1 (t1/2 = 0.48 h), (b) EEAChE 9.32 × 10-3 min-1 (t1/2 = 1.24 h) and 3.33 × 10-2 min-1 (t1/2 = 0.35 h), and (c) HuBChE 1.16 × 10-4 min-1 (t1/2 = 99.6 h) and 4.19 × 10-2 min-1 (t1/2 = 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo is the formation of (CH3)(FCH2CH2O)P(O)-serine adducts and monoacid (CH3)(FCH2CH2O)P(O)OH from hydrolysis and reactivation.
- Chao, Chih-Kai,Ahmed, S. Kaleem,Gerdes, John M.,Thompson, Charles M.
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p. 1810 - 1817
(2017/04/21)
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- Synthesis and anti-acetylcholinesterase properties of novel β- And γ-substituted alkoxy organophosphonates
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Activated organophosphate (OP) insecticides and chemical agents inhibit acetylcholinesterase (AChE) to form OP-AChE adducts. Whereas the structure of the OP correlates with the rate of inhibition, the structure of the OP-AChE adduct influences the rate at
- Kaleem Ahmed,Belabassi, Yamina,Sankaranarayanan, Lakshmi,Chao, Chih-Kai,Gerdes, John M.,Thompson, Charles M.
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p. 2048 - 2051
(2013/04/23)
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- CHOLINESTERASE INHIBITORS
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The invention provides compounds that inhibit cholinesterases, such as acetylcholinesterase and butyrylcholinesterase. Such compounds are useful to prevent or treat exposure of a patient (e.g., a human) to an organophosphoric nerve agent (e.g., sarin and
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