William H. Tepp

University of Wisconsin–Madison

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Eric A. Johnson

Johns Hopkins University

104

Sabine Pellett

University of Wisconsin–Madison

Marite Bradshaw

University of Wisconsin–Madison

Joseph Barbieri

Medical College of Wisconsin

Christina L. Pier

University of Wisconsin–Madison

Guangyun Lin

University of Wisconsin–Madison

Min Dong

Boston Children's Hospital

Kim D. Janda

Scripps Research Institute

Edwin R. Chapman

University of Wisconsin–Madison

Regina C. M. Whitemarsh

University of Wisconsin–Madison

Cooperative Institutions

University of Wisconsin–Madison

Scripps Research Institute

Harvard University

Medical College of Wisconsin

Stanford University

University of California, San Francisco

Centers for Disease Control and Prevention

Howard Hughes Medical Institute

Johns Hopkins University

University of Illinois Urbana-Champaign

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Potency and duration of action of BoNT/FA in cell models and in mice

Toxicon (2016)

Sabine Pellett William H. Tepp Marite Bradshaw Eric A. Johnson

10.1016/j.toxicon.2016.11.188

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Citations (0)

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells

Biochemical and Biophysical Research Communications (2010)

Sabine Pellett Zhongwei Du Christina L. Pier William H. Tepp Su‐Chun Zhang

Neurotoxin

Botulinum neurotoxin

Clostridium botulinum

10.1016/j.bbrc.2010.11.128

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Citations (33)

Crystal structure of botulinum neurotoxin type A and implications for toxicity

Nature Structural & Molecular Biology (1998)

D. Borden Lacy William H. Tepp A Cohen Bibhuti R. DasGupta Raymond C. Stevens

Neurotoxin

Botulinum neurotoxin

Clostridium botulinum

10.1038/2338

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Citations (745)

Transformation of a Metal Chelate into a “Catch and Anchor” Inhibitor of Botulinum A Protease

International Journal of Molecular Sciences (2023)

Lucy Lin Ealin N. Patel Alexander L. Nielsen Lewis D. Turner William H. Tepp

Targeting the botulinum neurotoxin light chain (LC) metalloprotease using small-molecule metal chelate inhibitors is a promising approach to counter the effects of the lethal toxin. However, to overcome the pitfalls associated with simple reversible metal chelate inhibitors, it is crucial to investigate alternative scaffolds/strategies. In conjunction with Atomwise Inc., in silico and in vitro screenings were conducted, yielding a number of leads, including a novel 9-hydroxy-4H-pyrido [1,2-a]pyrimidin-4-one (PPO) scaffold. From this structure, an additional series of 43 derivatives were synthesized and tested, resulting in a lead candidate with a Ki of 150 nM in a BoNT/A LC enzyme assay and 17 µM in a motor neuron cell-based assay. These data combined with structure-activity relationship (SAR) analysis and docking led to a bifunctional design strategy, which we termed “catch and anchor” for the covalent inhibition of BoNT/A LC. Kinetic evaluation was conducted on structures prepared from this catch and anchor campaign, providing kinact/Ki values, and rationale for inhibition seen. Covalent modification was validated through additional assays, including an FRET endpoint assay, mass spectrometry, and exhaustive enzyme dialysis. The data presented support the PPO scaffold as a novel candidate for targeted covalent inhibition of BoNT/A LC.

Docking (animal)

Clostridium botulinum

10.3390/ijms24054303

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Citations (3)

156. Differences in mode of action of the different toxins: distinct characteristics of botulinum neurotoxin A subtypes

Toxicon (2014)

Sabine Pellett Regina C. M. Whitemarsh William H. Tepp Marite Bradshaw Guangyun Lin

Botulinum neurotoxin

Neurotoxin

Mode of Action

10.1016/j.toxicon.2014.11.159

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Citations (0)

A Microscale Neuron and Schwann Cell Coculture Model for Increasing Detection Sensitivity of Botulinum Neurotoxin Type A

Toxicological Sciences (2013)

Won S. Hong Edmond W. K. Young William H. Tepp Eric A. Johnson David J. Beebe

Botulinum neurotoxin (BoNT) is a potent and specific biomolecule that is both implicated as a potential threat in bioterrorism and used in therapeutics. Highly sensitive and robust assays that measure BoNT activity are needed to manage outbreak or controlled distribution of BoNT. Current in vivo and in vitro assays have limitations, including high costs and variability for mouse bioassays, extensive preparations for primary and stem cell–derived neurons, and inherent low sensitivity for cell lines. Sensitivity of cell lines can be increased by direct differentiation and with their physiological relevance (compared with cell-free strategies) and robustness (compared with primary cell strategies); adopting cell lines is an attractive alternative to in vivo assays. Here, we present two distinct strategies that improved sensitivity of a cell line to BoNT serotype A (BoNT/A) without direct differentiation. We developed a cell-based BoNT assay using microscale culture and coculture of neuronal and Schwann cell lines, NG108-15 and S16, respectively, to improve both sensitivity and physiological relevance. Results showed that NG108-15 and S16 coculture decreased EC50 from 12.5 to 0.8ng/µl (p < 0.001) in macroscale and from 2.6 to 1.1ng/µl (p = 0.006) in microscale. In addition, NG108-15 monoculture at microscale decreased EC50 from 12.5 to 2.6ng/µl (p < 0.001) compared with macroscale. Finally, controlling the spatial arrangement of microscale coculture revealed that S16-derived soluble factors can increase sensitivity. Thus, our study demonstrates two distinct strategies for increasing the sensitivity of a cell line to BoNT using coculture and microscale culture, thereby advancing assay technology for BoNT detection.

Neurotoxin

Microscale chemistry

Schwann cell

10.1093/toxsci/kft082

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Citations (21)

Cultured Human-Induced Pluripotent Stem Cell–Derived Motor Neurons as a Sensitive Model for BoNT Detection

Toxicon (2022)

Molly M. Sneller William H. Tepp Michael Hendrickson Sabine Pellett

10.1016/j.toxicon.2022.05.034

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Citations (0)

Status of Cys Residues in the Covalent Structure of Botulinum Neurotoxin Types A, B, and E

Journal of Protein Chemistry (1998)

Babu S. Antharavally William H. Tepp Bibhuti R. DasGupta

DTNB

Guanidine

Iodoacetamide

Clostridium botulinum

Residue (chemistry)

Ammonium sulfate precipitation

10.1023/a:1022572332150

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Citations (14)

Characterization of Botulinum Neurotoxin A Subtypes 1 Through 5 by Investigation of Activities in Mice, in Neuronal Cell Cultures, and In Vitro

Infection and Immunity (2013)

Regina C. M. Whitemarsh William H. Tepp Marite Bradshaw Guangyun Lin Christina L. Pier

Botulinum neurotoxins (BoNTs) are synthesized by Clostridium botulinum and exist as seven immunologically distinct serotypes designated A through G. For most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. BoNT/A1 is the most well-characterized subtype of the BoNT/A serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. In contrast, much remains unknown of the other BoNT/A subtypes. In this study, BoNT/A subtype 1 (BoNT/A1) to BoNT/A5 were characterized utilizing a mouse bioassay, an in vitro cleavage assay, and several neuronal cell-based assays. The data indicate that BoNT/A1 to -5 have distinct in vitro and in vivo toxicological properties and that, unlike those for BoNT/A1, the neuronal and mouse results for BoNT/A2 to -5 do not correlate with their enzymatic activity. These results indicate that BoNT/A1 to -5 have distinct characteristics, which are of importance for a greater understanding of botulism and for pharmaceutical applications.

Botulism

Clostridium botulinum

Botulinum neurotoxin

Neurotoxin

In vitro toxicology

10.1128/iai.00536-13

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Citations (103)

Investigations of Thiosemicarbazides as Botulinum Toxin Active-Site Inhibitors: Enzyme, Cellular, and Rodent Intoxication Studies

ACS Infectious Diseases (2024)

Ealin N. Patel Lucy Lin Hyeri Park Molly M. Sneller Lisa M. Eubanks

Botulinum neurotoxin type A (BoNT/A) is an exceptionally potent neurotoxin of great therapeutic value; however, it is also considered a weapon of mass destruction, as it is one of the most poisonous biological substances known to man. The etiology behind BoNT/A is its action as a zinc-dependent protease, which can cause extended paralysis through the cleavage of SNARE proteins. Thiosemicarbazones, known zinc chelators, provide a privileged scaffold that can be leveraged for the development of BoNT/A LC inhibitors. Through a combination of biochemical and kinetic assays, it was demonstrated that the thiosemicarbazone ZMC1, an antitumor agent, is an effective competitive inhibitor of the BoNT/A LC. Based on these results, a series of thiosemicarbazones were designed/synthesized using structure-based analysis and examined in enzyme activity and cell-based assays. From this screen, two analogues presented noteworthy cellular activity. The most potent inhibitors were then tested in a BoNT/A mouse lethality assay, providing statistically significant prolonged survival.

Clostridium botulinum

10.1021/acsinfecdis.4c00750

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Citations (1)