Small Molecules Showing Significant Protection of Mice against Botulinum Neurotoxin Serotype A

Yuan-Ping Pang,Jewn Giew Park,James J Schmidt,Charles B Millard,Madhusoodana P Nambiar,Shaohua Wang,Jon Davis

doi:10.1371/journal.pone.0010129

Abstract

Botulinum neurotoxin serotype A (BoNTA) causes a life-threatening neuroparalytic disease known as botulism that could afflict large, unprotected populations if the toxin were employed in an act of bioterrorism. Current post-exposure therapy is limited to symptomatic treatment or passive immunization that is effective for treating infant botulism at a cost of US $45,300 per treatment regimen. Antibodies can neutralize the extracellular but not the intracellular BoNTA. Moreover, antibody production, storage, and administration in a mass casualty scenario pose logistical challenges. Alternatively, small-molecule inhibitors of BoNTA endopeptidase (BoNTAe) are sought to antagonize the extracellular or intracellular toxin. While several such molecules reportedly demonstrated efficacy in protecting cells against BoNTA, there is scant information to show that small molecules can significantly protect mammals against BoNTA. Herein we report the development of effective small-molecules BoNTAe inhibitors with promising in vivo pharmacokinetics. One such molecule has an in vivo half-life of 6.5 hours and is devoid of obvious sign of toxicity. Pre-treatment with this molecule at 2 mg/kg protected 100% and 70% of treated mice against BoNTA at 5 times of its median-lethal dose during the periods of 2 and 4 half-lives of the inhibitor, respectively. In contrast, 40% and 0% of untreated mice survived during the respective periods. Similar levels of protection were also observed with two other small molecules. These results demonstrate that small molecules can significantly protect mice against BoNTA and support the pursuit of small-molecule antagonists as a cost-effective alternative or as an adjunct to passive immunity for treating botulism.

Highlights

Seven distinct serotypes (A to G) of the spore-forming Clostridium botulinum have been characterized based upon production of structurally and functionally unique botulinum neurotoxins (BoNTs) [1]
In optimizing H3H for water solubility and higher potency in inhibiting BoNTA endopeptidase (BoNTAe), we encountered problems in derivatizing H3H caused by chemical instability under acidic conditions that was presumably due to the proton at position 3 of the indole ring
Recognizing the synthesis step as the rate-determining step of the optimization, we set out to first establish a facile synthetic scheme that can lead to a group of inhibitor analogues and use computer simulations of the inhibitor-bound BoNTAe complexes to prioritize the syntheses of the analogues

Summary

Introduction

Seven distinct serotypes (A to G) of the spore-forming Clostridium botulinum have been characterized based upon production of structurally and functionally unique botulinum neurotoxins (BoNTs) [1]. Such toxins can cause a life-threatening neuroparalytic disease known as botulism [1] by inhibiting normal release of the neurotransmitter acetylcholine at peripheral neuromuscular junctions and thereby causing prolonged flaccid paralysis, serious medical sequelae, or death [1]. Even in carefully controlled clinical scenarios, overdoses of BoNTA can occur and result in systemic botulism [4]; such incidents may rise as the number of therapeutic indications increases [5]. There is an urgent need for small-molecule BoNTA inhibitors as effective and safe post-exposure treatment for BoNTA intoxication to respond to food poisoning, accidental clinical overdoses, and mass-casualty situations

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Results

Conclusion