Prof. Pang made a comment on our recent article.(1) We take the opportunity to address six critical points that were raised.

(1)	The enzyme was in native form which was determined by its specific activity and assessing KM (SI, 6.1, p 43). No evidence of required enzyme pretreatment was formerly stated.(2) Moreover, the possible absence of active cysteine for conjugation with Cys-targeted compounds corroborates our results of the nonselective agAP-AChE inhibition by these molecules.
(2)	Although there is no listed kcat, our enzyme was characterized by a standard Michaelis–Menten procedure.(1) The specific enzyme activity, IC50 values, and calculation of KM value were considered as sufficient to obtain reproducible results. However, based on the SDS-PAGE analysis, confirmation of the enzyme purity, and determination of protein concentration by linearized Bradford assay, the kcat can be calculated as 6.8 × 104 min–1, which correlates with published data.(3,4) The produced agAP-AChE was fully comparable with similar proteins.
(3)	Maleimides are nonselective agents that have never been used as insecticides. But in ref (2), entitled “Novel selective and irreversible mosquito acetylcholinesterase inhibitors for controlling malaria···” they are intended to be mosquito acetylcholinesterase inhibitors (potential insecticides). Besides the title, the authors wrote: “insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides”.(2) As such, title/text could be considered as misleading,(2) and thus, our article(1) only revisited the published statements.
(4)	In our study,(1) all compounds were analyzed under constant conditions (i.e., temperature, protein concentration, controls) and were tested at ∼IC50 value. As such, the used concentrations can hardly be described as inappropriate in contrast to ref (2), where PM20 was analyzed in the concentration ∼100-times lower compared to its IC501 and the paraoxon was tested at the concentration ∼10-times higher compared to its IC50.(3) Thus, there was a dramatic difference in the used concentrations.(2) Moreover, all assays in ref (2) were performed by using 1.7% DMSO, which can significantly affect the enzyme activity.(5) The determination of enzyme activity for only 2 min is not the best practice since it brings a high error. Thus, the presented data raise serious questions about the experimental design.(2)
(5)	The specific enzyme activity was demonstrated.(1) In contrast to the ref (2) dilution experiment, the enzyme-specific activity was unchanged during dialysis. Moreover, the possible contamination caused by nonspecific binding of the inhibitor was eliminated, because the enzyme was transferred to a clean tube after dialysis and the final dilution factor was greater than 15 × 109. The use of 1 nM PM202 for determining the reversibility of inhibition seems to be irrelevant since it is ∼800 times lower than its IC50 value. Thus, it could not be possible to achieve the required level of inhibition (>95%). The validity of our protocol was confirmed on the series of standard compounds which proved that enzyme can be reversibly (bendiocarb, carbofuran) or irreversibly inhibited (paraoxon) when results with Cys-targeted molecules are in contrast with Dou et al.(2)
(6)	Although the determination by kinact/Ki parameters would yield more accurate results, this was not the intention of the present publication. All experiments were performed under standard conditions with necessary controls. The inhibition time was adjusted to the inhibition rate, which was constant at the time of determination. We have serious doubts about the results in ref (2). The conclusions are based namely on kinact/Ki results, which are incomplete. The curves in Figure 5 are not sufficient for calculation of nonlinear regression, and the error had to be enormous.

The enzyme was in native form which was determined by its specific activity and assessing K_M (SI, 6.1, p 43). No evidence of required enzyme pretreatment was formerly stated.(2) Moreover, the possible absence of active cysteine for conjugation with Cys-targeted compounds corroborates our results of the nonselective agAP-AChE inhibition by these molecules. Although there is no listed k_cat, our enzyme was characterized by a standard Michaelis–Menten procedure.(1) The specific enzyme activity, IC₅₀ values, and calculation of K_M value were considered as sufficient to obtain reproducible results. However, based on the SDS-PAGE analysis, confirmation of the enzyme purity, and determination of protein concentration by linearized Bradford assay, the k_cat can be calculated as 6.8 × 10⁴ min^–1, which correlates with published data.(3,4) The produced agAP-AChE was fully comparable with similar proteins. Maleimides are nonselective agents that have never been used as insecticides. But in ref (2), entitled “Novel selective and irreversible mosquito acetylcholinesterase inhibitors for controlling malaria···” they are intended to be mosquito acetylcholinesterase inhibitors (potential insecticides). Besides the title, the authors wrote: “insect-specific AChE cysteine is a unique and unexplored target to develop new insecticides”.(2) As such, title/text could be considered as misleading,(2) and thus, our article(1) only revisited the published statements. In our study,(1) all compounds were analyzed under constant conditions (i.e., temperature, protein concentration, controls) and were tested at ∼IC₅₀ value. As such, the used concentrations can hardly be described as inappropriate in contrast to ref (2), where PM20 was analyzed in the concentration ∼100-times lower compared to its IC₅₀¹ and the paraoxon was tested at the concentration ∼10-times higher compared to its IC₅₀.(3) Thus, there was a dramatic difference in the used concentrations.(2) Moreover, all assays in ref (2) were performed by using 1.7% DMSO, which can significantly affect the enzyme activity.(5) The determination of enzyme activity for only 2 min is not the best practice since it brings a high error. Thus, the presented data raise serious questions about the experimental design.(2) The specific enzyme activity was demonstrated.(1) In contrast to the ref (2) dilution experiment, the enzyme-specific activity was unchanged during dialysis. Moreover, the possible contamination caused by nonspecific binding of the inhibitor was eliminated, because the enzyme was transferred to a clean tube after dialysis and the final dilution factor was greater than 15 × 10⁹. The use of 1 nM PM20² for determining the reversibility of inhibition seems to be irrelevant since it is ∼800 times lower than its IC₅₀ value. Thus, it could not be possible to achieve the required level of inhibition (>95%). The validity of our protocol was confirmed on the series of standard compounds which proved that enzyme can be reversibly (bendiocarb, carbofuran) or irreversibly inhibited (paraoxon) when results with Cys-targeted molecules are in contrast with Dou et al.(2) Although the determination by k_inact/K_i parameters would yield more accurate results, this was not the intention of the present publication. All experiments were performed under standard conditions with necessary controls. The inhibition time was adjusted to the inhibition rate, which was constant at the time of determination. We have serious doubts about the results in ref (2). The conclusions are based namely on k_inact/K_i results, which are incomplete. The curves in Figure 5 are not sufficient for calculation of nonlinear regression, and the error had to be enormous. In summary, our findings(1) are different from ref (2), proposing cysteine-targeted molecules for selective mosquito acetylcholinesterase inhibition. This concept has to be carefully revisited based on the new data. The work was supported by Ministry of Health of the Czech Republic (no. NV16-34390A), University of Hradec Kralove (no. SV2115-2018, no. VT2019-2021 and postdoctoral job positions at UHK), and University of Defence (Faculty of Military Health Sciences, Long-term development plan and SV/FVZ2019/01). This article references 5 other publications.

中文翻译：

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对“针对冈比亚曲霉乙酰胆碱酯酶的半胱氨酸类杀虫剂既不是选择性抑制剂也不是可逆抑制剂”的评论答复。

庞教授对我们最近的文章发表了评论。（1）我们借此机会解决提出的六个关键点。

（1）	该酶为天然形式，通过其比活性和评估K M来确定（SI，6.1，第43页）。以前没有证据表明需要进行酶预处理。（2）此外，可能没有活性半胱氨酸与Cys靶向化合物结合，证实了我们对这些分子非选择性agAP-AChE抑制的结果。
（2）	尽管没有列出k cat，但我们的酶已通过标准的Michaelis-Menten程序进行了表征。（1）比酶活性，IC 50值和K M值的计算被认为足以获得可重复的结果。但是，基于SDS-PAGE分析，酶纯度确认和通过线性化Bradford分析确定蛋白质浓度，k cat可以计算为6.8×10 4 min –1，与公开的数据相关。（3， 4）产生的agAP-AChE与相似的蛋白质完全可比。
（3）	马来酰亚胺是从未用作杀虫剂的非选择性试剂。但是在参考文献（2）中，标题为“用于控制疟疾的新型选择性和不可逆的蚊虫乙酰胆碱酯酶抑制剂···”意在将它们用作蚊虫乙酰胆碱酯酶抑制剂（潜在的杀虫剂）。除了标题外，作者还写道：“昆虫特有的AChE半胱氨酸是开发新杀虫剂的独特且未开发的目标。”（2）因此，标题/文字可能被认为具有误导性，（2），因此，我们的文章（ 1）仅重新审视已发布的声明。
（4）	在我们的研究中，（1）所有化合物均在恒定条件下（即温度，蛋白质浓度，对照）进行分析，并在〜IC 50值下进行测试。因此，与参考文献（2）相比，所使用的浓度很难被描述为不合适的，参考文献（2）中分析的PM20浓度比其IC 50 1低约100倍，而对氧磷的浓度约为10倍。高于其IC 50。（3）因此，所用浓度存在显着差异。（2）此外，参考文献（2）中的所有测定均使用1.7％DMSO进行，这会显着影响酶的活性。（5）测定仅2分钟的酶活性不是最佳实践，因为它会带来很高的误差。因此，提出的数据对实验设计提出了严重的问题。（2]
（5）	证明了酶的比活性。（1）与参考（2）稀释实验相反，在透析过程中酶的比活性没有变化。此外，消除了由于抑制剂非特异性结合而引起的可能的污染，因为透析后酶被转移到了干净的试管中，并且最终的稀释系数大于15×10 9。使用1 nM PM20 2来确定抑制作用的可逆性似乎无关紧要，因为它比其IC 50低约800倍。值。因此，不可能达到所需的抑制水平（> 95％）。我们的方案的有效性在一系列标准化合物上得到了证实，这些标准化合物证明当与Cys靶向的分子结果相反时，酶可以被可逆地（苯二威，呋喃丹）或不可逆地抑制（对氧磷）抑制（Dou等人[2]）。
（6）	尽管通过k inact / K i参数确定会得到更准确的结果，但这不是本出版物的目的。所有实验均在标准条件下进行必要的对照。将抑制时间调节至抑制率，该抑制率在测定时是恒定的。我们对参考文献（2）中的结果有严重怀疑。该结论是基于即上ķ INACT / ķ我的结果，这是不完整的。图5中的曲线不足以计算非线性回归，并且误差必须很大。

该酶为天然形式，通过其比活性和评估K _M来确定（SI，6.1，第43页）。以前没有证据表明需要进行酶预处理。（2）此外，可能没有活性半胱氨酸与Cys靶向化合物结合，证实了我们对这些分子非选择性agAP-AChE抑制的结果。尽管没有列出k _cat，但我们的酶的特征在于采用标准的Michaelis–Menten程序。（1）比酶活性，IC ₅₀值和K _M的计算值被认为足以获得可重复的结果。但是，基于SDS-PAGE分析，酶纯度确认和通过线性化Bradford分析确定蛋白质浓度，k _cat可以计算为6.8×10 ⁴ min ^–1（3,4）产生的agAP-AChE与相似的蛋白质完全可比。马来酰亚胺是从未用作杀虫剂的非选择性试剂。但是在参考文献（2）中，标题为“用于控制疟疾的新型选择性和不可逆的蚊虫乙酰胆碱酯酶抑制剂···”意在将它们用作蚊虫乙酰胆碱酯酶抑制剂（潜在的杀虫剂）。除了标题外，作者还写道：“昆虫特有的AChE半胱氨酸是开发新杀虫剂的独特且未开发的目标。”（2）因此，标题/文字可能被认为具有误导性，（2），因此，我们的文章（ 1）仅重新审视已发布的声明。在我们的研究中，（1）在恒定条件下（即温度，蛋白质浓度，对照）对所有化合物进行了分析，并在〜IC _50下进行了测试。值。因此，与参考文献（2）相比，所使用的浓度很难被描述为不合适的，参考文献（2）中分析的PM20浓度比其IC ₅₀ ¹低约100倍，而对氧磷的浓度约为10倍。高于其IC ₅₀。（3）因此，所用浓度存在显着差异。（2）此外，参考文献（2）中的所有测定均使用1.7％DMSO进行，这会显着影响酶的活性。（5）测定仅2分钟的酶活性不是最佳实践，因为它会带来很高的误差。因此，提出的数据对实验设计提出了严重的问题。（2）证明了酶的比活性。（1）与ref（2）稀释实验相反，在透析过程中酶的比活性没有变化。此外，消除了由于抑制剂非特异性结合而引起的可能的污染，因为透析后酶被转移到了干净的试管中，并且最终的稀释系数大于15×10 ⁹。使用1 nM PM20 ²确定抑制作用的可逆性似乎无关紧要，因为它比其IC ₅₀值低约800倍。因此，不可能达到所需的抑制水平（> 95％）。我们的方案的有效性在一系列标准化合物上得到了证实，这些标准化合物证明当与Cys靶向的分子结果相反时，酶可以被可逆地（苯二威，呋喃丹）或不可逆地抑制（对氧磷）抑制（Dou等人）[2]。由k _inact / K _i确定参数将产生更准确的结果，这不是本出版物的目的。所有实验均在标准条件下进行必要的对照。将抑制时间调节至抑制率，该抑制率在测定时是恒定的。我们对参考文献（2）中的结果有严重怀疑。结论是基于k _inact / K _i结果，这是不完整的。图5中的曲线不足以计算非线性回归，并且误差必须很大。总而言之，我们的发现（1）与参考文献（2）有所不同，提出了以半胱氨酸为靶点的分子可选择性抑制蚊虫乙酰胆碱酯酶。必须根据新数据仔细重新考虑此概念。这项工作得到了捷克共和国卫生部（编号NV16-34390A），赫拉德茨·克拉洛韦大学（编号SV2115-2018，编号VT2019-2021和UHK的博士后职位）和国防大学（学院）的支持。军事卫生科学，长期发展计划和SV / FVZ2019 / 01）。本文引用了其他5个出版物。