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Characteristics of Botulinum Neurotoxins that determine potency

决定效力的肉毒杆菌神经毒素的特征

基本信息

批准号：
10539300
负责人：
Joseph T Barbieri
金额：
$ 63.6万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-18 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10539300
关键词：
Affect Amaze Amino Acid Substitution Amino Acids Animals Area Binding Biological Biological Assay Biological Products Botulinum Toxins Botulism C-terminal CCRL2 gene Caring Categories Cell model Cells Cessation of life Characteristics Chimera organism Classification Data Development Disease Dose Elements Engineering Esthetics Gangliosides Human Immunologics In Vitro Individual Intoxication Investigation Kinetics Knowledge Laboratories Light Maps Medical Medicine Membrane Modeling Molecular Mus Mutagenesis Mutate Mutation N-terminal Neuromuscular Diseases Neurons Paralysed Pathogenicity Pathologic Pathology Patients Pharmacologic Substance Property Protein Family Proteins Receptor Cell Recombinants Research Rodent Role Serotyping Severity of illness Structural Models Supportive care System Testing Therapeutic Time Toxic effect Toxin Variant antitoxin base beta-Lactamase bioweapon cost digital experimental study holotoxins improved in vivo insight mouse model nervous system disorder novel novel therapeutics pathogen pharmacologic receptor receptor binding respiratory trafficking uptake weapons

项目摘要

Abstract Botulinum Neurotoxins (BoNTs) are a large family of protein toxins and are of great significance due to their extreme potency and the severity of the disease they cause in humans and animals. Botulism is a neuroparalytic disease of long duration, lasting up to several months. Without proper medical care, naturally occurring botulism is lethal in up to 50% of cases, and even with respiratory and other supportive care and antitoxin administration, up to 5 % of patients die. While naturally occurring botulism is rare, BoNTs are classified as a Tier 1 Category A Select Agent due to their threat as potential bioterrorist weapons. Amazingly, BoNTs are also widely used in medicine to treat more than 100 neuromuscular disorders and for aesthetic purposes. BoNTs are immunologically divided into 7 serotypes, which are further subdivided into subtypes. Today, 100s of BoNT variants have been identified by sequencing efforts, but only few have been investigated at the protein level. It is noteworthy that out of all the BoNT variants only two (BoNT/A1 and /B1) are currently used as therapeutics, and studies examining properties of other variants are infrequent. Our research groups have determined for the first time that subtypes within the BoNT/A serotype have distinct biologic properties, including cell entry kinetics, duration of action, cell and mouse toxicity, and immunological variations. This RO1 project proposes to determine on a molecular and structural level the mechanisms underlying these unique properties of A subtypes and the chimeric BoNT/FA. Specifically, this project will investigate the mechanisms underlying the shorter duration of action of BoNT/A3, the faster and more efficient cell entry by BoNT/A2, /A6, and /FA, the 1,000 fold lower toxicity of BoNT/A4. Our collaborative efforts are unique in this area, as we are able to combine detailed mechanistic studies on subdomains with cell-based and in vivo studies on the holotoxin level. The Barbieri lab will use structural modeling to guide mutagenesis studies on functional domains of BoNTs and investigate mechanisms of receptor binding and cell trafficking using neuronal cell models. The Johnson/Pellett laboratory will utilize these data to create targeted holotoxin constructs in their native hosts and conduct detailed investigations in various rodent and human cell models. Finally, based on the data from these studies, select holotoxin constructs will be investigated in mice to determine pathologic and pharmacologic consequences of structural alterations. Using this approach, we will investigate a large number of amino acid substitutions in functional domain studies and select specific alterations for the more effort- and cost-intensive construction of recombinant holotoxins. By utilizing several cell models before conducting in vivo studies, we are able to reduce the number of required animals and also use human specific models. Finally, the combination of in vitro, cell-based, and in vivo studies will provide novel insight into the mechanisms underlying the observed pathologic and pharmacologic properties of these toxins.

摘要