In the article by Gorecki et al. ( ACS Med. Chem. Lett. 2020, 11, 65−71), the authors reported that maleimides irreversibly inhibited both the African malaria mosquito (Anopheles gambiae) Ace-paralogous acetylcholinesterase (aka agAP-AChE(1)) bearing a free Cys and the human counterpart devoid of the Cys and concluded that “Cys-targeted insecticides against A. gambiae acetylcholinesterase are neither selective nor reversible inhibitors”. This conclusion is problematic for at least six reasons.

(1)	A prerequisite to claim that maleimide PM20(2) does not conjugate with the agAP-AChE Cys is to confirm that the Cys is not oxidized. However, in the Gorecki article, no evidence was provided to show that the Cys in their agAP-AChE was active for conjugation.
(2)	In the Gorecki article, their agAP-AChE was claimed to be “in very good correlation” with the reported agAP-AChE(3) that was used in ref (2). However, only KM was reported for their agAP-AChE. Their omission of kcat, which is also needed for enzyme comparison, raises concerns with their specific enzyme activity. Their enzyme could be reasonably active (like crude extracts) and suitable for certain inhibitor characterizations but unsuitable for the kinetic characterization of PM20 described in ref (2).
(3)	In the Gorecki article, 10 μM PM20 and 10 μM paraoxon (rather than 1 nM PM20 and 200 nM paraoxon in ref (2)) showed no agAP-AChE activity recovery after dialysis. Using higher inhibitor concentrations here would prompt for determining the specific enzyme activity at the time when the assay was performed. However, no specific enzyme activity was reported in the Gorecki article. This substantiates the activity concerns above.
(4)	Potential irreversible inhibitors can be identified by their progressive inhibition from reversible inhibitors that exhibit constant inhibition in a time-course experiment. This requires controls at proper concentrations to define a period of time over which the progressive inhibition can be observed. Using high inhibitor concentrations can make the progressive inhibition period too short to practically measure the enzyme activity over the period. In ref (2), 6 nM PM20, 300 nM PMS20, and 700 nM paraoxon showed 20–30% agAP-AChE inhibition after 3 min, and then PM20 and paraoxon showed progressive agAP-AChE inhibition over the 3–15 min period, whereas PMS20 showed constant 20% agAP-AChE inhibition over the same period, wherein PMS20 (a maleimide-free analog of PM20) and paraoxon (known to conjugate with the catalytic Ser of cholinesterases) were controls to show respective constant and progressive inhibitions. In the Gorecki article, 800 nM PM20 exhibited nearly constant agAP-AChE inhibition over the 3–15 min period in their experiment without using known inhibitors (e.g., PMS20 and paraoxon) to show that their experimental conditions are suitable to observe constant and progressive inhibitions. The use of a high PM20 concentration and the lack of controls raise concerns about their study design.
(5)	The conclusion of the Gorecki article regarding inhibitor selectivity is IC50-based and contradicts the textbook(4) on irreversible inhibitor evaluation using kinact/KI: “An important point to realize here is that attempts to quantify the relative potency of irreversible enzyme inactivators by more traditional parameters, such as IC50 values, are entirely inappropriate because these values will vary with time, in different ways for different compounds. Hence the SAR derived from IC50 values, determined at a fixed time point in the reaction progress curve, is meaningless and can be misleading in terms of compound optimization.”
(6)	Maleimides are nonselective alkylating agents that have never been used as insecticides. PM20 was intended only for mass spectrometric evidence for Cys conjugation with agAP-AChE.(2) Equating maleimides with “Cys-targeted insecticides” in the Gorecki article is improper and misleading.

A prerequisite to claim that maleimide PM20(2) does not conjugate with the agAP-AChE Cys is to confirm that the Cys is not oxidized. However, in the Gorecki article, no evidence was provided to show that the Cys in their agAP-AChE was active for conjugation. In the Gorecki article, their agAP-AChE was claimed to be “in very good correlation” with the reported agAP-AChE(3) that was used in ref (2). However, only K_M was reported for their agAP-AChE. Their omission of k_cat, which is also needed for enzyme comparison, raises concerns with their specific enzyme activity. Their enzyme could be reasonably active (like crude extracts) and suitable for certain inhibitor characterizations but unsuitable for the kinetic characterization of PM20 described in ref (2). In the Gorecki article, 10 μM PM20 and 10 μM paraoxon (rather than 1 nM PM20 and 200 nM paraoxon in ref (2)) showed no agAP-AChE activity recovery after dialysis. Using higher inhibitor concentrations here would prompt for determining the specific enzyme activity at the time when the assay was performed. However, no specific enzyme activity was reported in the Gorecki article. This substantiates the activity concerns above. Potential irreversible inhibitors can be identified by their progressive inhibition from reversible inhibitors that exhibit constant inhibition in a time-course experiment. This requires controls at proper concentrations to define a period of time over which the progressive inhibition can be observed. Using high inhibitor concentrations can make the progressive inhibition period too short to practically measure the enzyme activity over the period. In ref (2), 6 nM PM20, 300 nM PMS20, and 700 nM paraoxon showed 20–30% agAP-AChE inhibition after 3 min, and then PM20 and paraoxon showed progressive agAP-AChE inhibition over the 3–15 min period, whereas PMS20 showed constant 20% agAP-AChE inhibition over the same period, wherein PMS20 (a maleimide-free analog of PM20) and paraoxon (known to conjugate with the catalytic Ser of cholinesterases) were controls to show respective constant and progressive inhibitions. In the Gorecki article, 800 nM PM20 exhibited nearly constant agAP-AChE inhibition over the 3–15 min period in their experiment without using known inhibitors (e.g., PMS20 and paraoxon) to show that their experimental conditions are suitable to observe constant and progressive inhibitions. The use of a high PM20 concentration and the lack of controls raise concerns about their study design. The conclusion of the Gorecki article regarding inhibitor selectivity is IC₅₀-based and contradicts the textbook(4) on irreversible inhibitor evaluation using k_inact/K_I: “An important point to realize here is that attempts to quantify the relative potency of irreversible enzyme inactivators by more traditional parameters, such as IC₅₀ values, are entirely inappropriate because these values will vary with time, in different ways for different compounds. Hence the SAR derived from IC₅₀ values, determined at a fixed time point in the reaction progress curve, is meaningless and can be misleading in terms of compound optimization.” Maleimides are nonselective alkylating agents that have never been used as insecticides. PM20 was intended only for mass spectrometric evidence for Cys conjugation with agAP-AChE.(2) Equating maleimides with “Cys-targeted insecticides” in the Gorecki article is improper and misleading. This article references 4 other publications.

中文翻译：

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

在Gorecki等人的文章中。（ACS医药化学快报。 2020，11，65-71）中，作者报道了马来酰亚胺不可逆地抑制两者非洲疟蚊（冈比亚按蚊）ACE-旁系同源乙酰胆碱酯酶（又名AGAP-AChE的（1））带有游离Cys和没有Cys的人类对应物得出结论，“针对冈比亚曲霉乙酰胆碱酯酶的Cys靶向杀虫剂既不是选择性抑制剂也不是可逆抑制剂”。该结论至少有六个原因是有问题的。

（1）	声称马来酰亚胺PM20（2）不与agAP-AChE Cys结合的前提是确认Cys没有被氧化。但是，在Gorecki文章中，没有提供证据表明其agAP-AChE中的Cys具有共轭活性。
（2）	在Gorecki的文章中，他们的agAP-AChE被声称与参考文献（2）中使用的报道的agAP-AChE（3）具有“很好的相关性”。然而，只有ķ中号报道了他们的AGAP乙酰胆碱酯酶。它们省略了k cat，这也是酶比较所必需的，这引起了对它们特定酶活性的关注。它们的酶可能具有适当的活性（如粗提物），适合某些抑制剂的表征，但不适合参考文献（2）中所述的PM20的动力学表征。
（3）	在Gorecki文章，10μM PM20和10μM对氧磷（而不是1nM的PM20和200nM对氧磷在参考文献（2））显示透析后没有AGAP-AChE活性的恢复。在此处使用较高的抑制剂浓度将提示您在进行测定时确定比酶活性。但是，Gorecki文章中没有报道特定的酶活性。这证实了上面的活动关注。
（4）	潜在的不可逆抑制剂可以通过与在时间过程实验中表现出恒定抑制作用的可逆抑制剂的逐步抑制作用来鉴定。这要求对照以适当的浓度来限定可以观察到进行性抑制的时间段。使用高浓度的抑制剂会使逐步抑制的时间太短而无法实际测量该时间段内的酶活性。在参考文献（2），6纳米PM20，300nM的PMS20，和700nm处显示出对氧磷20-30％AGAP-AChE的抑制3分钟后，再PM20和对氧磷呈渐进agAP-AChE在3至15分钟的时间内受到抑制，而PMS20在同一时期内显示20％的agAP-AChE恒定抑制作用，其中PMS20（PM20不含马来酰亚胺的类似物）和对氧磷（已知与胆碱酯酶的催化Ser结合） ）是分别显示恒定和渐进抑制的对照。在Gorecki文章中，在不使用已知抑制剂（例如PMS20和对氧磷）的情况下，实验中3到15分钟内，800 nM PM20表现出几乎恒定的agAP-AChE抑制作用，表明它们的实验条件适合观察恒定的和进行性抑制。高浓度PM20的使用和缺乏对照物引起了对其研究设计的关注。
（5）	Gorecki文章关于抑制剂选择性的结论是基于IC 50的，与使用k inact / K I对不可逆抑制剂评估的教科书（4）背道而驰：“此处重要的一点是，试图量化不可逆酶的相对效力用更传统的参数（例如IC 50值）制成的灭活剂是完全不合适的，因为这些值会随时间变化，对于不同的化合物会以不同的方式变化。因此，从IC 50值得出的SAR在反应进程曲线的固定时间点确定，是没有意义的，并且在化合物优化方面可能会产生误导。”
（6）	马来酰亚胺是从未用作杀虫剂的非选择性烷基化剂。PM20仅用于与agAP-AChE进行Cys偶联的质谱证据。（2）在Gorecki文章中将马来酰亚胺与“靶向Cys的杀虫剂”等同是不恰当和误导的。

声称马来酰亚胺PM20（2）不与agAP-AChE Cys结合的前提是确认Cys没有被氧化。但是，在Gorecki文章中，没有提供证据表明其agAP-AChE中的Cys具有共轭活性。在Gorecki的文章中，他们的agAP-AChE被声称与参考文献（2）中使用的报道的agAP-AChE（3）具有“很好的相关性”。然而，只有ķ_中号报道了他们的AGAP乙酰胆碱酯酶。他们省略了k _cat进行酶比较也需要用到它的特定酶活性。它们的酶可能具有适当的活性（如粗提物），适合某些抑制剂的表征，但不适合参考文献（2）中所述的PM20的动力学表征。在Gorecki文章，10μM PM20和10μM对氧磷（而不是1nM的PM20和200nM对氧磷在参考文献（2））显示透析后没有AGAP-AChE活性的恢复。在此使用较高的抑制剂浓度将提示您在进行测定时确定比酶的活性。然而，在Gorecki的文章中没有报道特定的酶活性。这证实了上面的活动关注。潜在的不可逆抑制剂可通过其识别在时程实验中表现出持续抑制作用的可逆抑制剂的渐进抑制作用。这要求对照以适当的浓度来限定可以观察到进行性抑制的时间段。使用高浓度的抑制剂会使逐步抑制的时间太短而无法实际测量该时间段内的酶活性。在参考文献（2），6纳米PM20，300nM的PMS20，和700nm处显示出对氧磷20-30％AGAP-AChE的抑制3分钟后，再PM20和对氧磷呈渐进AGAP乙酰胆碱酯酶抑制在3-15分钟内，而PMS20显示在同一时间段内恒定的20％agAP-AChE抑制作用，其中PMS20（不含PM20的顺丁烯二酰亚胺的类似物）和对氧磷（已知与胆碱酯酶的催化Ser缀合）是对照，分别显示出恒定的和逐步的抑制作用。在Gorecki文章中，在不使用已知抑制剂（例如PMS20和对氧磷）的情况下，实验中3到15分钟内，800 nM PM20表现出几乎恒定的agAP-AChE抑制作用，表明它们的实验条件适合观察恒定和渐进的抑制作用。使用高PM20注意力集中和缺乏控制引起对他们的研究设计的担忧。Gorecki文章关于抑制剂选择性的结论是基于IC ₅₀的，与使用k _inact / K _I对不可逆抑制剂评估的教科书（4）_背道而驰：“此处重要的一点是，试图量化不可逆酶的相对效力由更传统的参数（例如IC ₅₀值）制成的灭活剂是完全不合适的，因为这些值会随时间变化，对于不同的化合物会以不同的方式变化。因此，SAR源自IC ₅₀在反应进程曲线的固定时间点确定的值是没有意义的，并且在化合物优化方面可能会产生误导。” 马来酰亚胺是从未用作杀虫剂的非选择性烷基化剂。PM20仅用于与agAP-AChE进行Cys偶联的质谱证据。（2）在Gorecki文章中将马来酰亚胺与“靶向Cys的杀虫剂”等同是不恰当和误导的。本文引用了其他4个出版物。