Lewis D Turner1, Alexander L Nielsen1,2, Lucy Lin1, Sabine Pellett3, Takashi Sugane1, Margaret E Olson1, Kim D Janda1. 1. Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, Worm Institute of Research and Medicine (WIRM), Scripps Research, 10550 N Torrey Pines Road, La Jolla, CA 92037, United States. 2. Center for Biopharmaceuticals & Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark. 3. Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, United States.
Abstract
INTRODUCTION AND OBJECTIVES: Botulinum neurotoxin A (BoNT/A) is extremely toxic possessing an estimated intravenous LD50 of 1-2 ng/kg and as such has been designated a category A bioterrorism agent.1, 2 BoNT/A also possesses an extremely long half-life and persists within muscle neurons for months to >1 year.3 Because of BoNT/A longevity, we have utilized covalent inhibition as a means to abrogate BoNT/A's toxicity. To this end, we describe an approach to designing inhibitors that possess both electrophilic warheads and metal-binding groups for the bifunctional inhibition of BoNT/A. METHODS: Small molecule inhibitors that possessed electrophilic moieties were designed, using X-ray crystallography as guidance, to target both the zinc metal-binding region and Cys165 within the active site of BoNT/A. Synthesized compounds were evaluated for covalent inhibition using a continuous SNAPtide FRET assay4 and exhaustive dialysis. Compounds were also evaluated against a C165A variant. Compound reactivity, stability, MMP selectivity and cellular efficacy/toxicity was also evaluated. RESULTS: Several electrophilic warhead types were confirmed to inhibit BoNT/A LC covalently with substantial differences in time-dependent inhibition between the WT and C165A variant. A trend in warhead reactivity was reflected in inhibitor stability and toxicity. Compounds exhibited moderate potency in a BoNT/A neuronal cellular assay but were not further explored due to undesirable therapeutic potential. CONCLUSIONS: A fundamental framework for the bifunctional covalent inhibition of BoNT/A LC has been established. This approach has potential to be translated to other small molecule metal-binding inhibitors of BoNT/A LC with the vision that different pharmacophores, possessing improved physicochemical properties, will address BoNT/As toxicity and longevity within cells.
INTRODUCTION AND OBJECTIVES: Botulinum neurotoxin A (BoNT/A) is extremely toxic possessing an estimated intravenous LD50 of 1-2 ng/kg and as such has been designated a category A bioterrorism agent.1, 2 BoNT/A also possesses an extremely long half-life and persists within muscle neurons for months to >1 year.3 Because of BoNT/A longevity, we have utilized covalent inhibition as a means to abrogate BoNT/A's toxicity. To this end, we describe an approach to designing inhibitors that possess both electrophilic warheads and metal-binding groups for the bifunctional inhibition of BoNT/A. METHODS: Small molecule inhibitors that possessed electrophilic moieties were designed, using X-ray crystallography as guidance, to target both the zinc metal-binding region and Cys165 within the active site of BoNT/A. Synthesized compounds were evaluated for covalent inhibition using a continuous SNAPtide FRET assay4 and exhaustive dialysis. Compounds were also evaluated against a C165A variant. Compound reactivity, stability, MMP selectivity and cellular efficacy/toxicity was also evaluated. RESULTS: Several electrophilic warhead types were confirmed to inhibit BoNT/A LC covalently with substantial differences in time-dependent inhibition between the WT and C165A variant. A trend in warhead reactivity was reflected in inhibitor stability and toxicity. Compounds exhibited moderate potency in a BoNT/A neuronal cellular assay but were not further explored due to undesirable therapeutic potential. CONCLUSIONS: A fundamental framework for the bifunctional covalent inhibition of BoNT/A LC has been established. This approach has potential to be translated to other small molecule metal-binding inhibitors of BoNT/A LC with the vision that different pharmacophores, possessing improved physicochemical properties, will address BoNT/As toxicity and longevity within cells.
Authors: S S Arnon; R Schechter; T V Inglesby; D A Henderson; J G Bartlett; M S Ascher; E Eitzen; A D Fine; J Hauer; M Layton; S Lillibridge; M T Osterholm; T O'Toole; G Parker; T M Perl; P K Russell; D L Swerdlow; K Tonat Journal: JAMA Date: 2001-02-28 Impact factor: 56.272
Authors: Lucy Lin; Margaret E Olson; Takashi Sugane; Lewis D Turner; Margarita A Tararina; Alexander L Nielsen; Elbek K Kurbanov; Sabine Pellett; Eric A Johnson; Seth M Cohen; Karen N Allen; Kim D Janda Journal: J Med Chem Date: 2020-09-18 Impact factor: 7.446
Authors: Patrick G Foran; Nadiem Mohammed; Godfrey O Lisk; Sharuna Nagwaney; Gary W Lawrence; Eric Johnson; Leonard Smith; K Roger Aoki; J Oliver Dolly Journal: J Biol Chem Date: 2002-10-14 Impact factor: 5.157