Endopeptidase activity of anthrax lethal factor (aLF) prevents the destroy of anthracis spore intracellularly by host macrophages, meanwhile disables the signaling pathways extracellularly that leads to host lethality. Hence, inhibitory of this activity is expected to be an alternative option to cure anthrax infection. Herein, we fabricated a nanopore platform via transmembrane pore construction in vitro, which allows precise mimics, monitoring of intercellular proteinic transport and enables the quantitative detection of aLF endopeptidase activity towards MAPKK signaling protein at single molecule level. Next, we inhibited the aLF activity via screening approaches of protein-metal ion acquisition and other condition controlment (proton/hydroxide strength, adapted temperature, ionizing irradiation), which were identified by nanopore electrokinetic study. Upon the results, we found that Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺ collaborating with Zn²⁺ promote aLF activity efficiently. In contrary, Cd²⁺, Co²⁺, Cu²⁺ have great inhibitory effect. Result further revealed that, the speed of aLF endopeptidase activity with different ions functions as the nanopore signal frequency in linear manner, which enables evident distinction of those divalent ions using this proteinase assay. We also found the higher strength of the proton or hydroxide, the higher the inhibitory to aLF activity. Besides, adapted temperature and γ-ray also play integral roles in inhibiting this activity. Our results lay experimental basis for accurate detection of aLF activity, meanwhile provide new direction to screening novel stimuli-responsive inhibitors specific to aLF.

炭疽致死因子（aLF）的内肽酶活性可防止宿主巨噬细胞对细胞内炭疽孢子的破坏，同时禁用导致宿主死亡的细胞外信号通路。因此，抑制这种活性有望成为治愈炭疽感染的替代选择。在此，我们通过体外跨膜孔构建构建了一个纳米孔平台，该平台可以精确模拟、监测细胞间蛋白质运输，并能够在单分子水平上定量检测 aLF 内肽酶对 MAPKK 信号蛋白的活性。接下来，我们通过筛选蛋白质-金属离子捕获方法和其他条件控制（质子/氢氧化物强度、适应温度、电离辐射）来抑制 aLF 活性，这些方法通过纳米孔电动研究确定。根据结果​​，我们发现Ca ²⁺、Mg ²⁺、Mn ²⁺、Ni ²⁺与Zn ²⁺协同有效地促进aLF活性。相反，Cd ²⁺、Co ²⁺、Cu ²⁺则有较大的抑制作用。结果进一步表明，不同离子的 aLF 内肽酶活性速度与纳米孔信号频率呈线性关系，这使得使用该蛋白酶测定能够明显地区分那些二价离子。我们还发现质子或氢氧根的强度越高，对 aLF 活性的抑制作用就越高。此外，适应的温度和γ射线也在抑制这种活性中发挥着重要作用。我们的研究结果为准确检测aLF活性奠定了实验基础，同时为筛选新型aLF特异性刺激响应抑制剂提供了新方向。