Nerve Agent Sarin Research Articles

Detection of Chemical Warfare Agents with a Miniaturized High-Performance Drift Tube Ion Mobility Spectrometer Using High-Energetic Photons for Ionization.

A growing demand for low-cost gas sensors capable of detecting the smallest amounts of highly toxic substances in air, including chemical warfare agents (CWAs) and toxic industrial chemicals (TICs), has emerged in recent years. Ion mobility spectrometers (IMS) are particularly suitable for this application due to their high sensitivity and fast response times. In view of the preferred mobile use of such devices, miniaturized ion drift tubes are required as the core of IMS-based lightweight, low-cost, hand-held gas detectors. Thus, we evaluate the suitability of a miniaturized ion mobility spectrometer featuring an ion drift tube length of just 40 mm and a high resolving power of Rp = 60 for the detection of various CWAs, such as nerve agents sarin (GB), tabun (GA), soman (GD), and cyclosarin (GF), as well as the blister agent sulfur mustard (HD), the blood agent hydrogen cyanide (AC) and the choking agent chlorine (CL). We report on the limits of detection reaching minimum concentration levels of, for instance, 29 pptv for sarin (GB) within an averaging time of only 1 s. Furthermore, we investigate the effects of precursors, simulants, and other common interfering substances on false positive alarms.

Polyaniline impregnated activated carbon fabric: Adsorption and protection studies against nerve agent sarin

The synergistic effect of polyaniline (PANI) was explored in detail by conducting experiments with liquid and vapour sarin (GB) exposure tests. For that, different samples were prepared and breakthrough behaviour, liquid sarin elution profile and dynamic adsorption of vapour were studied at a different doping level of PANI and activated carbon fabric (PANI-ACF) was optimized to achieve optimum breakthrough behaviour. The results reveal that PANI-ACF of a surface density of 1.2–1.27 g/cm2 has a high surface area (914–980 m2/g) and micropore volume (0.20–0.22 (mL/g). Specific breakthrough volume (BTV), breakthrough time (BTT) and average breakthrough concentration rate were determined and found to be 243 L/g, 220–350 min and 0.004–0.0014 μg h−1cm−2 respectively which is much better than only ACF. Hence, PANI reinforced ACF applies to selective adsorption of nerve agent sarin and consecutively in chemical nerve agent protection.

Forensic analysis of the deuterium/hydrogen isotopic ratios of the nerve agent sarin, its reaction by-product diisopropyl methylphosphonate and their precursors by 2H SNIF-NMR

The Deuterium/Hydrogen (D/H) isotope ratios of sarin (5), diisopropyl methylphosphonate (3) and their precursors Isopropanol (1), methylphosphonic acid dichloride (2) and methylphosphonic acid difluoride (4) were measured by the 2H SNIF-NMR technique. The D/H isotope ratios of 1 show a large variation. It is shown, that the formation of 3 by reaction of 1 with 2, the fluorination of 2 to form 4 and the reaction of 4 with 1 to form 5, the D/H isotope ratios of the methyl and isopropyl moieties in 3, 4 and 5 are not significantly changed compared to 1 and 2.

High-performance p-hexafluoroisopropanol phenyl functionalized multi-walled carbon nanotube film on surface acoustic wave device for organophosphorus vapor detection

A delay line-type surface acoustic wave (SAW) gas sensor based on p-hexafluoroisopropanol phenyl (HFIPPH) functionalized multi-walled carbon nanotube (MWCNT) film is developed to detect organophosphorus dimethyl methylphosphonate (DMMP) vapor (a simulant of chemical nerve agent sarin). Inspired by the transfer process of Cu-based graphene, a uniform and size-controllable HFIPPH-MWCNT film is successfully prepared on the SAW device via a wet-etching transfer method. For the first time, we use the method of measuring the change of the sensor’s insertion loss to achieve the detection of ultra-low concentration DMMP vapor. The designed sensor exhibits a fast response/recovery time about 3 s/50 s, and a low detection limit of 0.1 ppm. Additionally, the stability and selectivity of the sensor and the influence of humidity on its response are evaluated through experiments. The acoustoelectric effect is proved to be the sensing mechanism of the sensor insertion loss response.

Part per trillion level DMMP gas sensor based on calixarene modified organic thin film transistor

Dimethyl methylphosphonate (DMMP) is a simulant of nerve agent sarin, which still remains a great challenge to detect due to lack of sensitive, selective and stable sensor. In this work, calixarenes are designed to apply on organic thin film transistor (OTFT) to form a high-performance gas sensor for DMMP detection. The theoretical calculation indicates that the binding energy between calixarene (4-tert-Butylcalix[6]arene, denote as TB6) and DMMP is stronger (–25.34 kcal/mol) than that of poly(3-hexylthiophene-2,5-diyl) (semiconductor layer of this OTFT sensor, denote as P3HT) (–10.83 kcal/mol), which could bring significant improvement to the sensitivity of this sensor. The experimental result confirms that the limit of detection (LOD) of TB6-P3HT based sensor can be as high as 10 ppt which is about four orders of magnitude higher than pristine P3HT. The drain current changes after exposure to 50 ppm DMMP, ethyl acetate, toluene, isopropanol and nitrogen dioxide are 39.0%, 2.9%, 2.7%, 2.1% and 1.3%, which also suggests the good selectivity of this sensor. Besides, the TB6-P3HT based sensor exhibits a better stability (14.1% degradation after one month) than pristine P3HT (55.8%) because of the better antioxidant capacity of this bilayer structure. Further, this sensor shows its ability to work in different relative humidity and temperatures, which demonstrates the potential application in complex environment. We anticipate this general approach could offer solutions for high performance gas detection.

Development of the Göttingen Minipig Model: GB and GF Toxicity

The dwindling availability of nonhuman primates (NHP) and increased ethical concerns over their use as experimental animals has driven a search for alternative animal models that can predict the effects of organophosphorus nerve agent (OPNA) in humans and to develop better medical countermeasures (MCM). Göttingen minipigs are physiologically and anatomically similar to humans, and are being considered as a potential alternative species for OPNA and MCM research. In the current study, fifteen sexually mature male Göttingen minipigs were utilized to determine the intramuscular (IM) 24‐hour median lethal dose (LD50) for the nerve agent sarin (GB) and cyclosarin (GF) using an up‐and‐down dose selection method. The initial doses for GB and GF were selected by approximating established lethality data for VX in Göttingen minipigs across IM and subcutaneous (SC) routes of administration. All animals exhibited typical signs of nerve agent poisoning immediately after being exposed to GB/GF. Three unique doses were required to estimate a GB LD50 of 40.00 µg/kg (95% confidence interval 33.25 µg/kg ‐ 49.76 µg/kg) and a GF LD50 of 40.57 µg/kg (95% confidence interval 33.03 µg/kg ‐ 49.73 µg/kg) with maximum likelihood estimation. The progression of toxic signs and the LD50 estimates obtained support the further development and use of Göttingen minipigs as a model to study MCMs and OPNAs.

Influence of Experimental End Point on the Therapeutic Efficacy of Essential and Additional Antidotes in Organophosphorus Nerve Agent-Intoxicated Mice.

The therapeutic efficacy of treatments for acute intoxication with highly toxic organophosphorus compounds, called nerve agents, usually involves determination of LD50 values 24 h after nerve agent challenge without and with a single administration of the treatment. Herein, the LD50 values of four nerve agents (sarin, soman, tabun and cyclosarin) for non-treated and treated intoxication were investigated in mice for experimental end points of 6 and 24 h. The LD50 values of the nerve agents were evaluated by probit-logarithmical analysis of deaths within 6 and 24 h of i.m. challenge of the nerve agent at five different doses, using six mice per dose. The efficiency of atropine alone or atropine in combination with an oxime was practically the same at 6 and 24 h. The therapeutic efficacy of the higher dose of the antinicotinic compound MB327 was slightly higher at the 6 h end point compared to the 24 h end point for soman and tabun intoxication. A higher dose of MB327 increased the therapeutic efficacy of atropine alone for sarin, soman and tabun intoxication, and that of the standard antidotal treatment (atropine and oxime) for sarin and tabun intoxication. The therapeutic efficacy of MB327 was lower than the oxime-based antidotal treatment. To compare the 6 and 24 h end points, the influence of the experimental end point was not observed, with the exception of the higher dose of MB327. In addition, only a negligible beneficial impact of the compound MB327 was observed. Nevertheless, antinicotinics may offer an additional avenue for countering poisoning by nerve agents that are difficult to treat, and synthetic and biological studies towards the development of such novel drugs based on the core bispyridinium structure or other molecular scaffolds should continue.

In Vivo Evaluation of the Acute Systemic Toxicity of (1S,2E,4R,6R,7E,11E)-Cembratriene-4,6-diol (4R) in Sprague Dawley Rats.

The tobacco cembranoid (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (4R) interacts with nicotinic acetylcholine receptors, which results in neuroprotection against organophosphate toxicity, brain ischemia, and Parkinson’s disease. The present study is a continuation of our previous research in which we applied a single dose of 4R 1 h before or 24 h after exposure to diisopropylfluorophosphate (DFP) (analog of the nerve agent sarin). The 4R dose robustly decreased neuroinflammation and neuronal death at both timepoints. Here, we investigated the toxicity of a single dose of 4R in male and female Sprague Dawley (SD) rats after a subcutaneous (s.c.) injection of 6, 24, or 98 mg/kg. Body weight was not affected by 4R during the 7-day observation period. No histopathologic changes in the organs were attributed to 4R. Minor hematological and blood composition variations were detected on Day 3 in the mid- and the high-dose males, but these were resolved by Day 8. At the area of the s.c. injection site, alopecia and dry skin were detected in both the 4R-treated males and females and in the female controls.

Chemical forensic profiling and attribution signature determination of sarin nerve agent using GC-MS, LC-MS and NMR.

Sarin is a highly toxic nerve agent classified by the Chemical Weapon Convention as a Schedule 1 chemical with no use other than to kill or injure. Moreover, in recent times, chemical warfare agents have been deployed against both military and civilian populations. Chemical warfare agents always contain minor impurities that can provide important chemical attribution signatures (CAS) that can aid in forensic investigations. In order to understand the trace molecular composition of sarin, various analytical approaches including GC-MS, LC-MS and NMR were used to determine the chemical markers of a set of sarin samples. Precursor materials were studied and the full characterisation of a synthetic process was undertaken in order to provide new insights into potential chemical attribution signatures for this agent. Several compounds that were identified in the precursor were also found in the sarin samples linking it to its method of preparation. The identification of these CAS contributes critical information about a synthetic route to sarin, and has potential for translation to related nerve agents.

Sensing of Sarin Nerve Agent by BN Nanoclusters: DFT and TDDFT Calculation

Sensing of Sarin Nerve Agent by BN Nanoclusters: DFT and TDDFT Calculation

Synthesis of Novel CuO/ZnFe2O4/NaA Zeolite Nanocomposite Adsorbent for the Decontamination of Nerve Agent Sarin Simulant

The aim of present research is to evaluate the decontamination of nerve agent sarin simulant dimethyl methyl phosphonate (DMMP) in water media using the CuO/ZnFe2 O4 /NaA zeolite as a novel ternary nanocomposite adsorbent. In this regard, the CuO and ZnFe2 O4 nanoparticles were successfully synthesized within the NaA zeolite applying the ultrasound-assisted hydrothermal route and characterized via XRD, FESEM, FTIR, EDAX, VSM and BET analyses. Then, the CuO/ZnFe2 O4 /NaA nanocomposite activity was investigated for the decontamination of DMMP molecule and monitored by a GC-FID and GC-MS analyses. Plus, the impacts of several parameters, including contact time, adsorbent dose and adsorbent type on the decontamination of DMMP were studied. The gained data from GC-FID analysis confirmed the maximum decontamination more than 98.4% for DMMP. The parameters including: adsorbent amount of 50 mg and contact time of 40 min were achieved as the optimized values for the decontamination reaction. In addition, the non-toxic methyl phosphoric acid (MPA) as the DMMP degradation product in the presence of CuO/ZnFe2 O4 /NaA adsorbent was characterized.

Substrate Analogues for the Enzyme-Catalyzed Detoxification of the Organophosphate Nerve Agents-Sarin, Soman, and Cyclosarin.

The G-type nerve agents, sarin (GB), soman (GD), and cyclosarin (GF), are among the most toxic compounds known. Much progress has been made in evolving the enzyme phosphotriesterase (PTE) from Pseudomonas diminuta for the decontamination of the G-agents; however, the extreme toxicity of the G-agents makes the use of substrate analogues necessary. Typical analogues utilize a chromogenic leaving group to facilitate high-throughput screening, and substitution of an O-methyl for the P-methyl group found in the G-agents, in an effort to reduce toxicity. Till date, there has been no systematic evaluation of the effects of these substitutions on catalytic activity, and the presumed reduction in toxicity has not been tested. A series of 21 G-agent analogues, including all combinations of O-methyl, p-nitrophenyl, and thiophosphate substitutions, have been synthesized and evaluated for their ability to unveil the stereoselectivity and catalytic activity of PTE variants against the authentic G-type nerve agents. The potential toxicity of these analogues was evaluated by measuring the rate of inactivation of acetylcholinesterase (AChE). All of the substitutions reduced inactivation of AChE by more than 100-fold, with the most effective being the thiophosphate analogues, which reduced the rate of inactivation by about 4-5 orders of magnitude. The analogues were found to reliably predict changes in catalytic activity and stereoselectivity of the PTE variants and led to the identification of the BHR-30 variant, which has no apparent stereoselectivity against GD and a kcat/Km of 1.4 × 106, making it the most efficient enzyme for GD decontamination reported till date.

UiO-66-NH2 and Zeolite-Templated Carbon Composites for the Degradation and Adsorption of Nerve Agents.

Composites of metal-organic frameworks and carbon materials have been suggested to be effective materials for the decomposition of chemical warfare agents. In this study, we synthesized UiO-66-NH2/zeolite-templated carbon (ZTC) composites for the adsorption and decomposition of the nerve agents sarin and soman. UiO-66-NH2/ZTC composites with good dispersion were prepared via a solvothermal method. Characterization studies showed that the composites had higher specific surface areas than pristine UiO-66-NH2, with broad pore size distributions centered at 1–2 nm. Owing to their porous nature, the UiO-66-NH2/ZTC composites could adsorb more water at 80% relative humidity. Among the UiO-66-NH2/ZTC composites, U0.8Z0.2 showed the best degradation performance. Characterization and gas adsorption studies revealed that beta-ZTC in U0.8Z0.2 provided additional adsorption and degradation sites for nerve agents. Among the investigated materials, including the pristine materials, U0.8Z0.2 also exhibited the best protection performance against the nerve agents. These results demonstrate that U0.8Z0.2 has the optimal composition for exploiting the degradation performance of pristine UiO-66-NH2 and the adsorption performance of pristine beta-ZTC.

Extended retrospective detection of regenerated sarin (GB) in rabbit blood and the IMPA metabolite in urine: a pharmacokinetics study.

Long-term retrospective monitoring of exposure to organophosphorus nerve agents is challenging. We recently developed two highly sensitive analytical methods for regenerated sarin (GB) nerve agent in blood and its primary metabolite, isopropyl-methylphosphonic acid (IMPA), in urine. These methods were implemented in a toxicokinetics study carried out with sarin injected (i.v.) to rabbits at doses corresponding to 0.1, 0.5 or 0.9 LD50. The time frame for monitoring regenerated sarin from blood was 70days for 0.1 LD50 and 0.5 LD50 and 77days for 0.9 LD50, where rapid elimination occurred in the first 8days with an initial average half-life of 1.2days, followed by a second, slower elimination, with a terminal average half-life of 8.4days. The time frame for monitoring IMPA in urine was 7, 15 and 16days for 0.1 LD50, 0.5 LD50 and 0.9 LD50 intoxications, respectively. Rapid elimination of IMPA in urine occurred after exposure, with an average half-life of ~ 0.8days on days 2-6. For the first time, a slower elimination route for IMPA, with an average half-life of ~ 4days from day 6 onwards, was revealed. Both IMPA and regenerated sarin pharmacokinetics exhibit linearity with dose. The overlaid pharmacokinetic profiles of regenerated sarin in blood along with IMPA in urine emphasize the dominance of IMPA with a rapid decay in urine in the first week and the slower long-term decay of protein-bound sarin later in blood. To our knowledge, the two new sensitive methods exhibit the longest monitoring time frame reported in biological samples.

Neuroprotection by delayed triple therapy following sarin nerve agent insult in the rat

The development of refractory status epilepticus (SE) induced by sarin intoxication presents a therapeutic challenge. In our current research we evaluate the efficacy of a delayed combined triple treatment in ending the abnormal epileptiform seizure activity (ESA) and the ensuing of long-term neuronal insult. SE was induced in male Sprague-Dawley rats by exposure to 1.2LD50 sarin insufficiently treated by atropine and TMB4 (TA) 1 min later. Triple treatment of ketamine, midazolam and valproic acid was administered 30 min or 1 h post exposure and was compared to a delayed single treatment with midazolam alone. Toxicity and electrocorticogram activity were monitored during the first week and behavioral evaluation performed 3 weeks post exposure followed by brain biochemical and immunohistopathological analyses. The addition of both single and triple treatments reduced mortality and enhanced weight recovery compared to the TA-only treated group. The triple treatment also significantly minimized the duration of the ESA, reduced the sarin-induced increase in the neuroinflammatory marker PGE2, the brain damage marker TSPO, decreased the gliosis, astrocytosis and neuronal damage compared to the TA+ midazolam or only TA treated groups. Finally, the triple treatment eliminated the sarin exposed increased open field activity, as well as impairing recognition memory as seen in the other experimental groups. The delayed triple treatment may serve as an efficient therapy, which prevents brain insult propagation following sarin-induced refractory SE, even if treatment is postponed for up to 1 h.

Resolving whether inhalation of depleted uranium contributed to Gulf War Illness using high-sensitivity mass spectrometry

Of the hypothesized causes of Gulf War Illness (GWI), a chronic multi-symptom illness afflicting approximately 25% of military personnel deployed to the 1991 Gulf War, exposure to depleted uranium (DU) munitions has attracted international concern. Past research has not tested the potential association of GWI with inhaled DU nor used isotope mass spectrometry of sufficient sensitivity to rigorously assess prior DU exposure. We applied a standard biokinetic model to predict the urinary concentration and uranium isotopic ratios for a range of inhalation exposures. We then applied sensitive mass spectrometry capable of detecting the predicted urinary DU to 154 individuals of a population-representative sample of U.S. veterans in whom GWI had been determined by standard case definitions and DU inhalation exposures obtained by medical history. We found no difference in the 238U/235U ratio in veterans meeting the standard case definitions of GWI versus control veterans, no differences by levels of DU inhalation exposure, and no 236U associated with DU was detected. These findings show that even the highest likely levels of DU inhalation played no role in the development of GWI, leaving exposure to aerosolized organophosphate compounds (pesticides and sarin nerve agent) as the most likely cause(s) of GWI.

Composites based on conductive polymer with carbon nanotubes in DMMP gas sensors – an overview

A number of recent terrorist attacks make it clear that rapid response, high sensitivity and stability are essential in the development of chemical sensors for the detection of chemical warfare agents. Nerve agent sarin [2-(fluoro-methyl-phosphoryl) oxypropane] is an organophosphate (OP) compound that is recognized as one of the most toxic chemical warfare agents. Considering sarin’s high toxicity, being odorless and colorless, dimethyl methylphosphonate (DMMP) is widely used as its simulant in the laboratory because of its similar chemical structure and much lower toxicity. Thus, this review serves to introduce the development of a variety of fabricated chemical sensors as potential sensing materials for the detection of DMMP in recent years. Furthermore, the research and application of carbon anotubes in DMMP polymer sensors, their sensitivity and limitation are highlighted. For sorption-based sensors, active materials play crucial roles in improving the integral performances of sensors. The novel active materials providing hydrogen-bonds between the polymers and carbon nanotubes are the main focus in this review.

The tertiary oxime monoisonitrosoacetone penetrates the brain, reactivates inhibited acetylcholinesterase, and reduces mortality and morbidity following lethal sarin intoxication in guinea pigs

The brain is a critical target for the toxic action of organophosphorus (OP) inhibitors of acetylcholinesterase (AChE) such as the nerve agent sarin. However, the available oxime antidote 2-PAM only reactivates OP-inhibited AChE in peripheral tissues. Monoisonitrosoacetone (MINA), a tertiary oxime, reportedly reactivates AChE in the central nervous system (CNS). The current study investigated whether MINA would be beneficial as a supplemental oxime treatment in preventing lethality and reducing morbidity following lethal sarin exposure, MINA supplement would improve AChE recovery in the body, and MINA would be detectable in the CNS. Guinea pigs were exposed to sarin and treated with atropine sulfate and 2-PAM at one minute. Additional 2-PAM or MINA was administered at 3, 5, 15, or 30 min after sarin exposure. Survival and morbidity were assessed at 2 and 24 h. AChE activity in brain and peripheral tissues was evaluated one hour after MINA and 2-PAM treatment. An in vivo microdialysis technique was used to determine partitioning of MINA into the brain. A liquid chromatography-tandem mass spectrometry method was developed for the analysis of MINA in microdialysates. MINA-treated animals exhibited significantly higher survival and lower morbidity compared to 2-PAM-treated animals. 2-PAM was significantly more effective in reactivating AChE in peripheral tissues, but only MINA reactivated AChE in the CNS. MINA was found in guinea pig brain microdialysate samples beginning at ~10 min after administration in a dose-related manner. The data strongly suggest that a centrally penetrating oxime could provide significant benefit as an adjunct to atropine and 2-PAM therapy for OP intoxication.

Study on the Degradation Effect of Three Organophosphorus Hydrolase Mutant on Sarin

Organophosphorus hydrolase can effectively degrade organic phosphorus compounds such as sarin. In this study, we constructed a recombinant Bacillus subtilis mutant expressing organophosphorus hydrolase, measured the effect of the mutant on the degradation rate of nerve agent sarin, and selected the optimal mutation scheme. Three different hydrolase mutant genes, 257L, 257Y and 303T, were ligated to PMA0911 vector and transferred into Bacillus subtilis WB800 to construct the target recombinant strain successfully. The recombinant bacteria secreted the target protein by fermentation. The effect of enzyme protein on the degradation of sarin was determined by the benzidine method. The optimal mutant was screened, and its enzymatic performance was explored. The effects of three organophosphorus hydrolase mutants on the hydrolysis rate of sarin were detected. The results showed that the 257Y mutant accelerated the hydrolysis of sarin significantly. Point mutation can improve the enzyme activity of wild-type organophosphorus hydrolase to a certain extent, laying the foundation for subsequent in-depth research.

QM/MM study of the enzymatic catalysis

Enzyme catalysis generally includes multiple physical and chemical processes, such as the substrate transportion and binding to the active site, the chemical reaction in the protein confined environment, and the product release, where any chemical or nonchemical step is probably to dominate the activity of enzyme. In order to understand the enzymatic efficiency, the classical molecular dynamics (MM MD) and combined quantum and classical molecular dynamics (QM/MM MD) simulations, in combination with the umbrella sampling, have been used to explore mechanisms for the enzymatic catalysis and the substrate transportion in several biological systems, including the detoxification of the G-type nerve agent sarin by phosphotriesterase (PTE), the hydrolysis of uridine by the pyrimidine-specific nucleoside hydrolase Yeik (CU-NH), and the transportation of naphthalimide-polyamine compounds into the drug sites by the bovine serum albumin (BSA). Extensive QM/MM MD and MM MD simulations show that the wild-type PTE enzyme is capable of hydrolyzing sarin but with a low catalytic efficiency. Dissociation of the degraded product from the binuclear zinc center is predicted to be the rate-determining step with the free-energy barrier of 21.0 kcal/mol, and the multiple chemical steps are involved in the dissociation process. The P-F cleavage of the substrate sarin follows a two-step mechanism, in which the nucleophilic attack of the bridging hydroxide leads to the P-Oµ formation with the free-energy barrier of 9.8 kcal/mol, and the subsequent P-F cleavage and the proton transfer from the µ-OH to the residue Asp301 yields the degraded product with the free-energy span of 12.3 kcal/mol. The relatively strong hydrogen-bond interactions between the substrate uridine and key residues in the active site of CU-NH play an important role in the substrate specificity. The enzymatic N -glycosidic bond cleavage of uridine by CU-NH is the rate-limiting step with the free energy barrier of about 20 kcal/mol, and the substrate protonation less influences the activity of CU-NH, completely different from other nucleoside hydrolases. The combined random acceleration molecular dynamics and MD simulations (RAMD MD) have been used to explore possible transportation channels and the most favorable pathways for the delivery of the naphthalimide-polyamine complex to two drug sites of BSA, as well as their thermodynamic and dynamic properties. MM MD and RAMD MD simulations show that the selective specificity of these two drug sites strongly depends on the electron-withdrawing and electron-donating substituents of the substrate, which can be used for the design of new naphthalimide drugs. Such site-selectivity-dependence design strategy may open up an avenue in drug discovery. With the fast development of low-scaling QM methodologies and the application of artificial intelligence neural network approaches to theoretical and computational chemistry, it can be expected to reduce the computational costs of QM treatment and improve the efficiency of QM/MM, which makes the direct QM/MM study of larger and more complex systems become possible. In addition, the improvement and development of coarse-grained models can surmount the spatial and temporal limitation of multiscale calculations and simulations. Accordingly, global simulations of extremely large and complicated biosystems can be realized in the future, which may provide a theoretical basis for further expanding the application of enzyme engineering.