Benzoquinones as inhibitors of botulinum neurotoxin serotype A

Abstract

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low μM IC50 values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low μM IC50 competitive inhibitors that depend on the BQ moiety for activity.

Radicals: Reactive Intermediates with Translational Potential

“…In fact, many of the quinone adducts surveyed during the course of the reaction development represented new structural entities. 89 Despite their semblance of Michael acceptors, quinones rarely undergo smooth conjugate additions with organometallic reagents; 90 their inertia toward transition metal catalysis is evidenced by their roles as ligands or oxidants in such reactions. 91 This simple chemical avenue, through a radical process, tames quinones’ unique electronic properties.…”

Cellular Protection of SNAP-25 against Botulinum Neurotoxin/A: Inhibition of Thioredoxin Reductase through a Suicide Substrate Mechanism

“…(15) As an alternative means to enhance the potency of LC inhibitors, we turned our attention to Cys165, which is embedded within the active site of the BoNT/LC (highlighted with yellow in the crystal structure, Figure 1). (16) Moreover, Dive and co-workers reported that when this Cys residue was covalently modified, catalysis was stopped and the enzyme was inactivated. (17) This observation prompted us to prepare a series of compounds based upon our inhibitor 1 that could extend into the space that Cys165 occupies.…”

Impact of an Upper Respiratory Tract Infection on Botulinum Toxin Efficacy in Spasmodic Dysphonia Patients

“…The first of these is a black tea extract thearubigin, which in mouse models is protective against BoNT types A, B and E by directly binding with the toxins . Benzoquinone, which is produced naturally by certain plant species as well as commercially as quinone anti‐cancer medications, also inhibits BoNT type A . This agent acts as an irreversible inhibitor by modifying cysteine 165 of the BoNT type A light chain complex.…”