Phosphatase Inhibitors Function as Novel, Broad Spectrum Botulinum Neurotoxin Antagonists in Mouse and Human Embryonic Stem Cell-Derived Motor Neuron-Based Assays

Abstract

There is an urgent need to develop novel treatments to counter Botulinum neurotoxin (BoNT) poisoning. Currently, the majority of BoNT drug development efforts focus on directly inhibiting the proteolytic components of BoNT, i.e. light chains (LC). Although this is a rational approach, previous research has shown that LCs are extremely difficult drug targets and that inhibiting multi-serotype BoNTs with a single LC inhibitor may not be feasible. An alternative approach would target neuronal pathways involved in intoxication/recovery, rather than the LC itself. Phosphorylation-related mechanisms have been implicated in the intoxication pathway(s) of BoNTs. However, the effects of phosphatase inhibitors upon BoNT activity in the physiological target of BoNTs, i.e. motor neurons, have not been investigated. In this study, a small library of phosphatase inhibitors was screened for BoNT antagonism in the context of mouse embryonic stem cell-derived motor neurons (ES-MNs). Four inhibitors were found to function as BoNT/A antagonists. Subsequently, we confirmed that these inhibitors protect against BoNT/A in a dose-dependent manner in human ES-MNs. Additionally, these compounds provide protection when administered in post-intoxication scenario. Importantly, the inhibitors were also effective against BoNT serotypes B and E. To the best of our knowledge, this is the first study showing phosphatase inhibitors as broad-spectrum BoNT antagonists.

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.…”

New Steroidal 4-Aminoquinolines Antagonize Botulinum Neurotoxin Serotype A in Mouse Embryonic Stem Cell Derived Motor Neurons in Postintoxication Model

“…The extent of SNAP-25 cleavage was quantified using standard immunoblotting procedures with SNAP-25 antibodies that detect both the full length and the BoNT/A cleaved large fragment, as described previously. 7,10 Briefly, the cell lysates were processed, run on 12% Tris Glycine gels (Invitrogen, #XP00125), and transferred to nitrocellulose membranes. Membranes were blocked in 5% non-fat milk for 1hr, and then incubated with primary antibodies against GAPDH (Millipore, #MAB374), and SNAP-25 (BioLegend, SMI-81, #836304) in TBST buffer containing 5% milk overnight at 4 °C.…”

“…The cells were then washed with PBS thoroughly and lysed in NP-40 cell lysis buffer. The extent of SNAP-25 cleavage was quantified using standard immunoblotting procedures with SNAP-25 antibodies that detect both the full length and the BoNT/A cleaved large fragment, as described previously. , Briefly, the cell lysates were processed, run on 12% Tris glycine gels (Invitrogen, no. XP00125), and transferred to nitrocellulose membranes.…”

“…5,6 Very recently, several approaches using SMNPIs have been explored by targeting the host intoxication pathways not by directly inhibiting the toxins’ proteolytic activity but focusing on Src family kinase signaling, 7 thioredoxin reductase system 8,9 and by the development of phosphatase inhibitors that would have an impact on BoNT intoxication in motor neurons. 10 The alternative approach was also developed, the inhibitors that interact with BoNT/A LC; among them the hydroxamates 11 and aminoquinolines showed high inhibitory activities against BoNT/A LC in cell free assays. 12–16 Diverse cell-based assays have been used as more relevant methods for evaluation of new drugs capable of protecting SNAP-25 from BoNT/A LC cleavage.…”

“…There are at least seven distinct serotypes (A–G); however, three of them (A, B, and E) are considered the most noxious in humans. , The majority of efforts have been focused on identification of BoNT/A inhibitors with intracellular activity because antibody-based treatments were found successful only before toxin enters a neuron . Recent reviews have covered the in vitro–in vivo gap in this field, focusing on small molecule non-peptide inhibitors (SMNPI), potential drug targets, and the mechanism of action (MOA) of these SMNPIs. , Very recently, several approaches using SMNPIs have been explored by targeting the host intoxication pathways, not by directly inhibiting the toxins’ proteolytic activity but focusing on Src family kinase signaling, thioredoxin reductase system , and by the development of phosphatase inhibitors that would have an impact on BoNT intoxication in motor neurons . The alternative approach was also developed, the inhibitors that interact with BoNT/A LC; among them the hydroxamates and aminoquinolines showed high inhibitory activities against BoNT/A LC in cell-free assays. − Diverse cell-based assays have been used as more relevant methods for evaluation of new drugs capable of protecting SNAP-25 from BoNT/A LC cleavage. − More importantly, these methods simulate all key steps in intoxication process, starting from holotoxin binding to the cell surface to cleavage of the SNARE proteins. , Since there is a continuous need for discovering new therapeutics for treatment of BoNT/A intoxication, this method is considered to be a very helpful tool for narrowing the spectrum of compounds for testing in animal models.…”