Tirbanibulin (KX2‐391) analog KX2‐361 inhibits botulinum neurotoxin serotype A mediated SNAP‐25 cleavage in pre‐ and post‐intoxication models in cells

Abstract Botulinum neurotoxins (BoNT) inhibit neuroexocytosis, leading to the potentially lethal disease botulism. BoNT serotype A is responsible for most human botulism cases, and there are no approved therapeutics to treat already intoxicated patients. A growing body of research has demonstrated that BoNT/A can escape into the central nervous system, and therefore, identification of BoNT/A inhibitors that can penetrate BBB and neutralize the toxin within intoxicated neurons would be important. We previously identified an FDA‐approved, orally bioavailable compound, KX2‐391 (Tirbanibulin) that inhibits BoNT/A in motor neuron assays. Recently, a structural analog of KX2‐391, KX2‐361, has been shown to exhibit good oral bioavailability and cross BBB with high efficiency in mouse experiments. Therefore, in this work, we evaluated the inhibitory effects of KX2‐361 against BoNT/A. Toward this goal, we first evaluated the compound for its effects on cell viability in PC12 cells, via MTT assay, and in mouse embryonic stem cell (mESC)‐derived motor neurons, with imaging‐based assays. Following, we tested KX2‐361 in mESC‐derived motor neurons intoxicated with BoNT/A holotoxin, and the compound exhibited activity against the toxin in both pre‐ and post‐intoxication conditions. Excitingly, KX2‐361 also inhibited BoNT/A enzymatic component (light chain; LC) in PC12 cells transfected with BoNT/A LC. Furthermore, our molecular docking analyses suggested that KX2‐361 can directly bind to BoNT/A LC. Medicinal chemistry approaches to develop structural analogs of KX2‐361 to increase its efficacy against BoNT/A may provide a critical lead compound with BBB penetration capacity for drug development efforts against BoNT/A intoxication.