Targeting the Enzymatic Site of Botulinum Neurotoxin Type E With 8‐Hydroxyquinolinol–Based Inhibitors: In Silico, In Vitro, and In Vivo Evaluation

Botulinum neurotoxins are the most potent toxins responsible for causing flaccid paralysis of muscles by blocking the release of acetylcholine at the neuromuscular junction. There are no postexposure therapeutics and effective active/passive prophylaxis available for the treatment. Therefore, it is highly desirable to develop a potential antidote to counter botulinum neurotoxicity. In this study, ~800 molecules were mined by a structure similarity search from open databases and docked into the pocket of the catalytic domain of botulinum toxin type E using AutoDock 4.2. Twenty-four small molecules with the best scoring function were selected and evaluated using in vitro and in vivo assays. Among these, two molecules, NSC1011 and NSC1012, were identified as inhibiting the catalytic activity of BoNT/E, with IC₅₀ values of 31.25 ± 1.0 μM and 55.45 ± 5.2 μM and K_D of 5.54E-07 and 6.51E-06 M, respectively. To find inhibitors that can reverse the neurotoxicity more effectively, we have derived and synthesized 12 analogs of NSC1011. These compounds showed higher inhibition than the parent molecules, with IC₅₀ and K_D values of 4.375 ± 2.3 µM and 1.61E-08 M (C25.12) and 10.25 ± 3.0 µM and 4.70E-08 M (C25.9). Compounds C25.9 and C25.12 completely protected mice in premixed doses and led to significant extension in survival of up to 60 h with therapeutic treatment. This study showed that these 8-HQ derivatives had the potency to inhibit BoNT/E by interacting with the active site. Further studies could lead to the development of undiscovered postexposure therapeutics against this deadly neurotoxin.