Reply to Comment on “Cysteine-Targeted Insecticides against A. gambiae Acetylcholinesterase Are Neither Selective nor Reversible Inhibitors”

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 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.
• (2)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.
• (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 article¹ 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 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 findings¹ 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.