Date

2022-8-25

Author

Koç, Dilara

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Botulinum neurotoxins (BoNTs) are the most poisonous neurotoxins known, and intoxication by this toxin leads to botulism, a potentially lethal disease. BoNT/A, one of the 7 serotypes of BoNTs, is the most common serotype that causes human botulism. BoNT/A primarily targets the peripheral cholinergic nerves, especially motor neurons, and causes inhibition of acetylcholine release by cleaving SNAP-25 protein. Once BoNTs are internalized into motor neurons, there are no therapeutic options to neutralize them within the cytoplasm. Our previous studies evaluated libraries of small molecules for their inhibitory activities against BoNT/A. Such work led to the discovery of an Src inhibitor, KX2-391 (Tirbanibulin), as a BoNT/A inhibitor in motor neuron assays. Despite the promising features of the compound, unfortunately, KX2-391 failed to protect significantly against BoNT/A intoxication in mice. One possible reason for results obtained from in vivo studies may be the insufficient penetration capacity of KX2-391 to cross the Blood-Brain Barrier (BBB). Recently, a structural analog of KX2-391, KX2-361, specifically designed to cross BBB, has been developed and published. Therefore, in this thesis work, our primary goal was to evaluate the inhibitory effects of KX2-361 against BoNT/A. Towards this goal, we evaluated both KX2-391 and KX2-361 molecules for their potential effects on cell viability in PC12 cells via MTT assay, and our findings show no significant cell viability differences, especially with lower doses. We then generated motor neurons from HBG3 mouse embryonic stem cells and tested the possible effects on cell viability of KX2-361 on motor neurons with imaging-based assays. Following this, we tested the compound's inhibitory effects against BoNT/A in mESC-derived motor neurons in pre-intoxication conditions, and excitingly, KX2-361 provided dose-dependent protection against the toxin. We then evaluated the compound in post-intoxication conditions, which exhibited activity against the toxin. Furthermore, KX2-361 inhibited BoNT/A LC enzymatic components in PC12 cells transfected with BoNT/A LC. The results of this work may open new avenues to develop structural analogs of KX2-361 to increase its efficacy against BoNT/A, which may provide a critical lead compound for drug development efforts against BoNT intoxication.

Subject Keywords

Mouse embryonic stem cells, Motor neuron, KX2-361, KX2-391, Botulinum neurotoxin

URI

https://hdl.handle.net/11511/98794

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Graduate School of Natural and Applied Sciences, Thesis