New Engineered-Botulinum Toxins Inhibit the Release of Pain-Related Mediators.

Minhong Tang,Jianghui Meng,Jiafu Wang

doi:10.3390/ijms21010262

Minhong Tang, Jianghui Meng + Show 1 more

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https://doi.org/10.3390/ijms21010262

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Abstract

Targeted delivery of potent inhibitor of cytokine/pain-mediator into inflammatory or pain-sensing cells is a promising avenue for treating chronic pain, a world-wide major healthcare burden. An unmet need exists for a specific and effective delivery strategy. Herein, we describe a new approach using sortase to site-specifically ligate a non-toxic botulinum neurotoxin D (BoNT/D) core-therapeutic (synaptobrevin-cleaving protease and translocation domains) to cell-specific targeting ligands. An engineered core-therapeutic was efficiently ligated to IL-1β ligand within minutes. The resultant conjugate specifically entered into cultured murine primary macrophages, cleaved synaptobrevin 3 and inhibited LPS/IFN-γ evoked IL-6 release. Likewise, a CGRP receptor antagonist ligand delivered BoNT/D protease into sensory neurons and inhibited K+-evoked substance P release. As cytokines and neuropeptides are major regulators of inflammation and pain, blocking their release by novel engineered inhibitors highlights their therapeutic potential. Our report describes a new and widely-applicable strategy for the production of targeted bio-therapeutics for numerous chronic diseases.

Highlights

Chronic pain, including rheumatoid arthritis (RA), is still a major medical challenge, with a high number of people suffering from persistent pain worldwide
A synthetic gene fragment encoding light chain domain (LC).HN of botulinum neurotoxin D (BoNT/D), with a codon optimized for E. coli expression, was inserted into the pET29a vector
Our strategy is dependent on removal of the botulinum neurotoxins (BoNTs)/D neuronal receptor binding domain, followed by sortase A-mediated conjugation of the targeting ligand

Summary

Introduction

Chronic pain, including rheumatoid arthritis (RA), is still a major medical challenge, with a high number of people suffering from persistent pain worldwide. Three main types of medicine are widely used to treat chronic pain, including nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids with adjuvants such as antidepressants and anticonvulsants These medications are largely ineffective or unsuitable for many sufferers of chronic pain, either due to their short half-lives or various unacceptable adverse side effects, including addiction and overdose [1]. Once BoNTs get internalised into the neurons, the N-terminal half of HC (HN) forms a channel on the synaptic vesicle and/or endosomal membrane, allowing for translocation of the attached LC to the cytosol [11] This active LC protease can cleave and inactivate soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). To avoid the unwanted muscle paralysis side effect associated with the treatment of chronic pain, improvement of BoNT is desired so that it will selectively and target BoNT SNARE-cleaving protease into inflammatory cells and/or sensory neurons rather than motor neurons

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