title: 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.
authors.affiliation: Departments of Chemistry and Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Bremer
authors.affiliation: Discovery Biology, Takeda San Diego, Inc., 10410 Science Center Drive, San Diego, CA 92121, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hixon
authors.affiliation: Departments of Chemistry and Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Electronic address: kdjanda@scripps.edu.
authors.forename: Kim D
authors.initials: KD
authors.lastname: Janda
mesh_terms: D016227:Benzoquinones / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015195:Drug Design; D007700:Kinetics; D062105:Molecular Docking Simulation; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D016227:Benzoquinones; D011480:Protease Inhibitors; C004532:quinone; D019274:Botulinum Toxins, Type A
keywords: Benzoquinone; Botulinum neurotoxin; Computational docking; Covalent inhibitor; Cysteine targeting; Fragment-based design; Irreversible inhibitor; Juglone; Naphthazarin; Naphthoquinone; Rational design