Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

Abstract

Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins’ proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin’s enzymatic components, to antagonize multiple BoNT serotypes in motor neurons.

Newly Designed Quinolinol Inhibitors Mitigate the Effects of Botulinum Neurotoxin A in Enzymatic, Cell-Based, and ex Vivo Assays

“…Over the past 20 years, a variety of BoNT/A inhibitors have been developed, which target LC endosomal translocation 18–22 or the LC protease directly. 23–31 Zinc-chelating molecules have been shown to be privileged inhibitor scaffolds against zinc metalloproteases e.g.…”

Gαs proteins activate p72Syk and p60-c-Src tyrosine kinases to mediate sickle red blood cell adhesion to endothelium via LW-αvβ3 and CD44–CD44 interactions

“…1B). The selective Src-family kinase inhibitors, PP1 and PP2 (Kiris et al, 2015) (Fig. 1A), and the non-receptor tyrosine kinase p72 Syk inhibitor, piceatannol (Buharalioglu et al, 2011) (Fig.…”

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

“…For BoNT/A holotoxin (Metabiologics) experiments with pre-intoxication conditions ( Figures 1 , 3 ), mouse ES cell-derived motor neurons were cultured in 24-well plate formats and pre-treated with compounds and cultured for 30 min at 37°C with 5% CO 2 prior to intoxication , similar to previous studies ( Kiris et al, 2015a ; Kiris et al, 2015b ). Neurons were then intoxicated with the holotoxin for 4 h in a humidified incubator with 5% CO 2 at 37°C.…”