Implications of unique conformations of botulinum neurotoxin serotype A on drug discovery and structure-guided drug design
Embargo Date
2027-11-24
OA Version
Citation
Abstract
Botulinum neurotoxin (BoNT), the most potent toxin known to man, is the causative agent of botulism, history’s most infamous foodborne illness. Clostridium botulinum, the bacterial that produces BoNT, is classified as a Category A bioterrorism threat by the CDC, alongside agents like Anthrax, Plague, and Smallpox. It represents one of the most sinister potential weapons in the arsenal of global bad actors, was part of Iraq's weapons of mass destruction program under Saddam Hussein, and was employed, thankfully without success, in terror attacks by the Japanese cult, Aum Shinrikyo. Despite its deadly potential, BoNT is also the active ingredient in BOTOX, used in over 10 million cosmetic procedures annually. The widespread use of BOTOX raises concerns about iatrogenic botulism, as many patients and providers may be unaware of the toxin’s inherent risks. To date, there are no clinically viable, post-cellular-intoxication antidotes to cure botulism. While many successful in vitro inhibitors of BoNT have been identified, none of them have translated into effective in vivo inhibitors in mouse bioassays of botulism. This dissertation investigates the structural dynamics of the BoNT serotype A light chain (BoNT/A-LC) and the challenges of developing effective small-molecule inhibitors (SMIs) against this zinc metalloprotease. Initially aimed at obtaining novel inhibitor-bound crystal structures of BoNT/A, the focus shifted to understanding two distinct conformations of the enzyme, termed the alpha and beta forms, which are hypothesized to be the catalytically competent and inactive conformations, respectively. A comprehensive analysis of all reported BoNT/A structures to date revealed that SMIs predominantly bind to the beta form, while peptides and peptidomimetics, ligands more like the substrate, target the alpha form. The difficulty in obtaining SMI-bound structures using a construct that crystallized exclusively in the alpha form, despite extensive troubleshooting efforts, supports this conclusion. Given this new perspective and framework for structure-guided drug design efforts, it is possible that past small-molecule drug design efforts may have targeted the wrong enzyme conformation, reducing inhibitor efficacy in physiological settings, and possibly explaining the in vitro/in vivo disconnect. Orthogonal biochemical evaluation of BoNT/A-LC conformations, by proxy of its susceptibility to proteolytic degradation support the hypothesis that the enzyme’s conformational equilibrium shifts toward the alpha form upon substrate binding. These findings highlight critical considerations for future BoNT/A inhibitor design, with implications for both therapeutic and bioterror countermeasures.
Description
2025