Non-quaternary oximes detoxify nerve agents and reactivate nerve agent-inhibited human butyrylcholinesterase

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Sodium phosphate salts, acetylthiocholine, DTNB, Bovine serum albumin, 2-PAM, and HI-6 were purchased from Merck (Sigma, Israel). O-ethyl methyl phosphonate methyl cyano-coumarin (EMP-MeCyC) was synthesized as described previously21. Resorufin pivalate was synthesized according to ref. 22. The HTS hit 3-hydroxy 2-amidoxime pyridine (PCM-0212399, Fig. 1a) was synthesized according to ref. 17. A nucleophile-focused library of 1348 nucleophilic compounds was composed of 1298 aldoximes, hydroxamates, alkylhydroxamtes, ketoximes, and amidoximes purchased from Enamine, Kiev. Ukraine. The following 18 quaternary aryl-imidazolium aldoximes (ImOx’s), ProImOxmCl, ProImOxpCl, ButImOxmCl, ButImOxpCl, BeImOxmCl, BeImOxpCl, pClpClImOx, pClmClImOx, mClmClImOx. pCH3pClImOx, pCH3mClImOx, mCH3pClImOx, mCH3mClImOx, BeImOx, pClImOx, mClImOx, pCH3ImOx and mCH3ImOx, together with 3 Cinchona-derived quinuclidinium-quinoline oximes Ox(C1), OxCH3I (C2) and OxBnBr (C3)—were kindly provided by Z. Kovarik, IMI, Zagreb, Croatia. The following 7 substituted benzohydroxamic acids (BHA), HA-1, HA-2, HA-2, MeBHA-2, MeBHA-3, BuBHA-4, and BuBHA as well as 13 Pyridinium aldoxime N-substituted by alkyl amides and alkyl amino acids, IL-1, IL-2, IL-3, IL-Ala-1, IL-Ala-2, IL-Ala-3, IL-Phe-1, IL-Phe-2, IL-Phe-3, IL-PheO-1, IL-PheO-2, IL-PheO-3, and IL-Phe-acid, were kindly provided by Y. Karpichev, Tallinn University of Technology, Tallinn, Estonia. The following nine bis-quarternary pyridinium oximes: K027, K048, K203, K865, K866, K867, K868, K869, and K870 were kindly provided by Kamil Musilek, University of Hradec Kralove, Hradec Kralove, Czech Republic. The following alkyne analogs of the hit oximes were custom synthesized at Enamine, Kiev, Ukraine. PCM-0214580, PCM-0214579, PCM-0214535, PCM-0214534, PCM-0214533 and PCM-0214518 (Supplementary Scheme S6).

VX and sarin were synthesized at IIBR according to previously published procedures with utmost safety precautions. Purified human serum BChE was kindly provided by O. Lockridge, Omaha Medical Center, Omaha, Nebraska, USA. Purified recombinant Human AChE was kindly provided by the Department of Biotechnology, IIBR, Israel.

Screening libraries

The HTS screening campaigns for detoxification and reactivation were conducted with the small molecule compound library of the Israel National Center for Personalized Medicine at the Weizmann Institute of Science. The library is composed of commercial sets from ChemDiv (Diversity), Chembridge (DiversetCl), Enamine (Drug-like Set, and Nucleophiles), Maybridge (Hitfinder), Microsource (Spectrum), Selleck (Bioactives), Prestwick (Known Drugs), and Sigma (LOPAC). Additional contributions from academic collections (listed above) were registered and integrated into the screening deck prior to conducting the screens.

HTS equipment

High-throughput screens, counter screens, orthogonal screens and hit validation experiments were performed using the HTS equipment: Echo555 Acoustic transfer system (Labcyte, Germany), MultiDrop 384 (Thermo Scientific), Washer Dispenser II (GNF, San Diego, CA, USA), EL406 Microplate Washer Dispenser (BioTek, Winooski, VT, USA), Bravo Automated Liquid Handling (Agilent, Santa Clara, CA, USA). Fluorescence/absorbance signals were measured by luminescence module of PheraStar FS plate reader (BMG Labtech, Ortenberg, Germany). 1536-well plates were from Nunc (#264711), and 384-well plates from Greiner (#781162).


The synthetic pathway for preparing the O-propargyl alkyne benzamido oxime analogs PCM-0214528 (4a) PCM-0214517 (4b), PCM-0214522 (4c) are shown in the Supplementary Information (Supplementary Scheme 2).

HTS fluorescent detoxification assay

The assay buffer (AB) was 10 mM phosphate buffer, pH = 7.9. 1536-well plates were pre-plated with compounds and controls (480 nl/well, final 600 µM) using the Echo transfer system. The nucleophilic compound Benzhydroxamic acid (BHA) was used as a standard. 10 mM DMSO stocks of either EMP- MeCyC (working solution, neutral control) or MeCyC-OH (positive control) were diluted 2,000-fold in AB to 5 µM (final concentration of 2.5 µM). 8 µl of the solutions were added to appropriate wells of 1536-well plates and 380/430 fluorescence emission was read twice: immediately (time 0 h) and after 3 h incubation using a PheraStar plate reader. Data analysis was as follows: calculation of average – standard deviation (SD) of fluorescence at time 0 h; calculation of average fluorescence at time 3 h; calculation of the average ratio of fluorescence at 3 h/0 h. Compounds that were able to increase fluorescence signal at least 70% above BHA-induced fluorescence (standard) were selected for validation. In order to filter out false-positive compounds, hits were also subjected to a counter assay, where MeCyC-OH was used instead of EMP-MeCyC.

HTS fluorescent reactivation assay

The assay buffers were: Enzyme buffer (EB), 10 mM Na Phosphate pH 7.6; Substrate buffer (SB), 10 mM Na phosphate pH 6.7, 0.01% BSA. 1536-well plates were pre-plated with compounds and controls (40 nl/well, final 100 µM in 4 µl) using an Echo transfer system. 2-PAM was used as a reference oxime. Either BChE (non-inhibited enzyme, positive control) or a mixture of BChE + EMP-MeCyC (inhibited enzyme, optimized to ~85% enzyme inhibition; working solution, neutral control) was diluted in EB under sterile conditions (BChE stock 25 µM in EB, EMP-MeCyC 10 mM stock in DMSO; final BChE concentration of 1.6 µM). These solutions were incubated for 30 min at room temperature and then diluted 50-fold in EB to 32 nM enzyme solution. 4 µl of each solution were added to appropriate wells of 1536-well plates and incubated in the presence of tested compounds for a further one hour at room temperature (reactivation reaction). Resorufin pivalate, 10 mM stock, was diluted 2000-fold in SB to 5 µM, and 4 µl were added to each assay well (final concentration of the enzyme and EMP-MeCyC was 16 nM; RP 2.5 µM). After additional incubation for one hour at room temperature in the dark, the 540/590 fluorescence signal was read. Compounds that yielded an equivalent or larger increase in fluorescence signal to that obtained for 2-PAM were selected for follow-up validation in a double dilution assay. Using 2-PAM as a selection gate minimized false hits from direct oximolysis of the substrate by the nucleophiles. BChE and BChE + EMP-MeCyC were diluted to 6.4 µM, incubated for 30 min, then diluted 50-fold to a concentration of 128 nM. Next, the solutions were added to 384-well plates and diluted again 4-fold to 32 nM BChE. All following steps were as above.

The suitability and reliability of the detoxification and reactivation HTS assays were evaluated by determining the screening window coefficient parameter Z-prime (Z′). Z′ is reflective of both the assay signal dynamic range and the data variation associated with signal readouts. We measured Z′ value as an assay quality assessment and to demonstrate that the fluorescent readout in our HTS assays was large enough to warrant reliability and reproducibility23. Z′ was calculated by the following equation:

$$Z^{prime}=1{-}3({sigma }_{{rm{p}}}+{sigma }_{{rm{n}}})/|{mu }_{{rm{p}}}{-}{mu }_{{rm{n}}}|$$

where σp and σn are the standard deviations of the mean values µp and µn of the positive (p) and negative (n) control signals. The mean and standard deviation of the positive and negative controls were based on 32 repeated readouts of these controls in 1536-well plate. Usually, Z′ values of 0.5–0.9 reflect highly reliable HTS assays.

HTS absorbance-based Ellman assay

The assay buffers EB and SB were as described above. 384-well plates were pre-plated with potential hit compounds discovered in the fluorescent HTS described above. BChE and BChE + EMP-MeCyC were diluted in EB to 1.6 µM and incubated for 30 min at room temperature. Then, the solutions were diluted 200-fold in EB to 8 nM, dispensed to appropriate wells, and incubated for a further one hour at room temperature. Then, the recovered activity of BChE was measured by the Ellman absorbance assay16 as follows: 60 µl of SB were added to each well, following by the addition of 15 µl of DNTB (stock 5 mM, final 0.75 mM and substrate acetylthiocholine (ATC), stock 7.27 mM, final 1.1 mM). Absorbance was measured continually at 412 nm (OD412) over time. In parallel, the absorbance at 412 nm in wells containing all Ellman reagents, without the enzyme, was measured in order to evaluate the rate of direct oximolysis of ATC.

Detoxification kinetics using an “enzyme rescue assay”

Prior to starting the OPNA degradation reaction, a 96-well plate was filled with 120 µl of 50 mM sodium phosphate buffer at pH 7.4, 30 µl of DTNB (final 0.75 mM), and 10 µl of BChE (final 1 × 10−9 M). The OPNA degradation reaction (1 µM of either VX or sarin) was incubated with the screened oximes (0.5 mM) at pH 8 at room temperature.

At specified time intervals, samples of 10 µl of the degradation reaction mixture were drawn and added to the 96-well plate (diluted 20-fold in 200 µl), that contained BChE (10−9 M) in 50 mM phosphate buffer pH 7.4 with DTNB, and incubated for 3 min at room temperature. The 3-min inhibition period was determined independently for obtaining 90–95% inhibition of BChE (10-9 M) by intact OPNA (5 × 10−8M sarin). Finally, 10 µl of the substrate acetylthiocholine (ATC) solution was added (final ATC concentration was 1.1 mM in 200 µl total volume) to start the Ellman enzymatic activity reaction (increase in OD405nm 1 min readout time). The Ellman assay for BChE activity was performed at pH 7.4 that keeps ATC stable and reactive. It should be emphasized that in the OP degradation assay, BChE serves only for monitoring the residual potency of the OPNA as enzyme inhibitor during its degradation by the oximes at pH 8 (using pH 8 for the reason described above) and the Ellman assay at which ATC is more stable and reactive, was performed at pH 7.4. The decrease in BChE inhibition caused by progressive degradation of VX and sarin with time is presented in Fig. 1b, c, respectively. Absorbance was read at 405 nm using a Tecan, Infinity F200 M spectrophotometer.

Reactivation of VX-BChE and sarin-BChE with HTS-selected Oximes

The reactivation reaction was performed in two stages. First, BChE at a concentrated solution (2.5 × 10−6 M) was inhibited by the OPNA (5 × 10−6 M sarin) in 20 mM phosphate buffer pH 8 containing 0.01% BSA—to provide about 90–95% steady-state inhibition within 30–60 min. Secondly, the OP-BChE conjugate was diluted 4-fold into 20 mM phosphate buffer pH 8 containing 0.01% BSA and then further diluted 25× fold into either the same buffer (pH 8) or buffer containing the screened oxime (0.5 mM). In parallel, free BChE was diluted 4-fold and then 25-fold in pH 8 to serve as a control uninhibited enzyme (E0). The total dilution of the concentrated OP-BChE conjugates was 100-fold in buffer or oxime-containing buffer. The 4-fold and 25-fold consecutive dilutions were done to handle solutions volume scale in a more operationally convenient manner. The reactivation reaction mixture, as well as the E0 solution, were sampled (10 µl) at the same specified time intervals and diluted into 160 µl of 50 mM phosphate pH 7.4 that contained DTNB. Finally, 30 µl of ATC was added to start the Ellman activity assay for a 1 min readout time. It should be noted that the activity of free BChE (E0) was decreased by about 20–25% after 24 h at room temperature in 20 mM phosphate 0.01% BSA pH 8, however, the regained BChE activity during reactivation was compared to the corresponding E0 value measured at the same specified time point.


Adult male Hsd:ICR mice (Envigo, Rehovot, Israel), weighing 25–30 g, were housed 10 per plastic cage with bedding, under controlled environment at 21 °C ± 2 °C and a 12-h light/dark cycle with lights on at 7 am. The mice were acclimatized for 1 week before the experiment. Food and water were available ad libitum.

All the procedures involving mice were approved by and conducted in accordance with the institutional Animal Care and Use Committee at the Israel Institute for Biological Research (protocol-M-21-20, Israel Institute for Biological Research), and are also in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press, Washington DC, 2011).

All oximes were dissolved in DMSO at 45 mg/ml. A group of four mice weighing 25–30 g was initially treated with each oxime at a dose of 150 mg/kg by injecting ~100 µl of the oxime solution intraperitoneally (i.p.). Toxic signs were monitored for 8 days, and the mice were weighed each day. Two of the oximes PCM-0211955 and PCM-0211088 that displayed toxicity at 150 mg/kg (i.p.) were estimated for their LD50 using the up and down toxicity method24.

Statistics and reproducibility

HTS assays

The suitability and reliability of the detoxification and reactivation HTS assays were evaluated by determining the screening window coefficient parameter Z-prime (Z′) as defined above in the HTS methods.

Detoxification and reactivations kinetics

All kinetics of Sarin and VX detoxification as well as reactivation of VX- and Sarin-inhibited BChE were performed by three repeats of each experiment (n = 3). The symbols  represent individual values (n = 3). The kinetic parameters of detoxification in Fig. 1b, c (kobs and t1/2) were calculated from a linear transformation of the data to Ln[%BChE Inhibition] vs. time. The kinetic parameters of reactivation (kobs, t1/2) in Fig. 2a, b were calculated from a non-linear fit to the mean values at each time point.

Toxicity in vivo

The time-course of change in mice body weight over 8 days after treatment with four oximes are presented in Fig. 3 as individual points (n = 4). Ordinary one-way ANOVA comparing body weight of oxime-treated to DMSO treated mice (control) was performed over a period of 8 days. All statistical analyses were performed with GraphPad Prism software.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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