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Issues in therapeutic development against botulinum neurotoxins (BONT)
ARTICLE IN PRESS 8
Abstracts Toxins 2008 / Toxicon 51 (2008) 1–54
21. Relevance of the crystal vs. solution structure of botulinum neurotoxin for understanding its mechanism of action and development of inhibitors Bal Ram Singh, Shuowei Cai, Roshan Kukreja Botulinum Research Center, and Department of Chemistry and Biochemistry, University of Massachusetts, Dartmouth, USA
Botulinum neurotoxins are multifaceted molecules, which are truly unique not only in their mode of action, but also in their utility as a drug carrier either across the gut wall or to the nerve terminals. The molecule is divided into clear functional domains that can operate independently. This feature can be used to employ them as cargo carrier by linking other drugs or vaccines with the binding and translocation domains of BoNT. While the domain structures are largely independent of each other, the dynamic structure of these domains, especially that of the enzymatic domain (L chain), is quite different from the reported crystal structures for several BoNT serotypes and their enzymatic domain. This presentation will discuss the comparative structures of BoNT in crystal and solution for their relevance to the molecular mechanism of BoNT action. We have recently discovered that the enzymatically active structure of the BoNT exists as a molten-globule and that of the endopeptidase domain as a novel PRIME conformation, which may be relevant to the mechanistics of BoNT endopeptidase activity against SNAP-25 and to the development of inhibitors against it.
modified LC. Investigation of the properties of these modified species is therefore also very important. We have investigated several factors that lead to physical and catalytic instability of the LC. They include an autocatalytic reaction whereby the LC is fragmented into two major peptides with largely diminished activity that was prevented by adding polyols. We also found that inadvertent, gentle agitation rapidly precipitated the LC that is catalytically inactive, but did not affect BSA, ovalbumin, and thermolysin. Because the precipitation of BoNT domains must preclude some partly denaturation step(s), the resulting denatured species may display varied activity, stability, and solubility. Protection from precipitation and inactivation was attained by including low concentrations of nonionic detergents. The research was supported by JSTO-CBD Project# 3.10011_06_RD_B. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Keywords: LC; Stability; Precipitation 10.1016/j.toxicon.2008.04.024
23. Human botulism immune globulin for the treatment of infant botulism: The first four years postlicensure
This work was supported by DTRA under a DoD/Army Contract No. W911NF-06-1-0095.
Stephen S. Arnon, J.R. Payne, Y. Drummond, J.R. Barash
Keywords: Botulinum; Enzyme; Inhibitors; Mechanism; SNAP-25; Structure 10.1016/j.toxicon.2008.04.023
Infant botulism is the intestinal (infectious) toxemia in which swallowed spores of Clostridium botulinum activate in the lumen of the colon and thereby produce botulinum toxin, which after absorption causes flaccid paralysis. Human Botulism Immune Globulin (BIG-IV) neutralizes botulinum toxin types A and B. The US FDA licensed BIG-IV to the California Department of Public Health, its creator and developer, as the public service orphan drug BabyBIGs in October 2003 (N. Engl. J. Med. 2006;354:462–471). International distribution began on a trial basis in July 2005. This report summarizes the 2003–2007 distribution, efficacy and safety data for BIGIV. In the US, a total of 333 eventually laboratoryconfirmed patients residing in 35 states were treated within 1 week of hospital admission: 142 type A, 188 type B, 2 type Ba and 1 type Bf. Mean length of hospital stay was 2.271.5 vs. 5.775.1 wks for untreated patients in the 1992–97 BIG-IV randomized clinical trial (Po0.001) and did not differ significantly by toxin type of illness. Aggregate hospital stay avoided through use of BIG-IV totaled 22.4 years; aggregate hospital costs avoided totaled at least $28 million (2007 dollars). Four other infant botulism patients (2 type E, 2 type F) were also treated with BIG-IV. Internationally, 10 laboratory-confirmed cases in 4 countries were treated, 4 type A, 5 type B and 1 type Ba. No serious adverse events associated with BIG-IV treatment were reported. In the 4 years since licensure of human BIG-IV (BabyBIGs), its use globally to
3. Pharmacological properties and factors that influence duration of action 22. Issues in therapeutic development against botulinum neurotoxins (BONT) S. Ashraf S. Ahmed, Leonard Smith, Stephen Toth USAMRIID, Fort Detrick, USA
Studies of the BoNT holotoxin are limited by its intrinsic toxicity. The N-terminal light chain (LC) catalytic domain is a target for therapeutic drug development. But investigators have experienced large variations in its solubility, stability, and specific activity. Efforts to develop inhibitors are likely to be adversely affected by such variations. Thus, a thorough understanding of the factors behind these variations, and finding ways in obtaining a consistently soluble, stable, and active preparation of the LC are very important. In addition, LC has been reported to undergo post-translocation modification in neurons. The most logical targets for drug development should be such
California Department of Public Health, Richmond, USA
Abstracts Toxins 2008 / Toxicon 51 (2008) 1–54
21. Relevance of the crystal vs. solution structure of botulinum neurotoxin for understanding its mechanism of action and development of inhibitors Bal Ram Singh, Shuowei Cai, Roshan Kukreja Botulinum Research Center, and Department of Chemistry and Biochemistry, University of Massachusetts, Dartmouth, USA
Botulinum neurotoxins are multifaceted molecules, which are truly unique not only in their mode of action, but also in their utility as a drug carrier either across the gut wall or to the nerve terminals. The molecule is divided into clear functional domains that can operate independently. This feature can be used to employ them as cargo carrier by linking other drugs or vaccines with the binding and translocation domains of BoNT. While the domain structures are largely independent of each other, the dynamic structure of these domains, especially that of the enzymatic domain (L chain), is quite different from the reported crystal structures for several BoNT serotypes and their enzymatic domain. This presentation will discuss the comparative structures of BoNT in crystal and solution for their relevance to the molecular mechanism of BoNT action. We have recently discovered that the enzymatically active structure of the BoNT exists as a molten-globule and that of the endopeptidase domain as a novel PRIME conformation, which may be relevant to the mechanistics of BoNT endopeptidase activity against SNAP-25 and to the development of inhibitors against it.
modified LC. Investigation of the properties of these modified species is therefore also very important. We have investigated several factors that lead to physical and catalytic instability of the LC. They include an autocatalytic reaction whereby the LC is fragmented into two major peptides with largely diminished activity that was prevented by adding polyols. We also found that inadvertent, gentle agitation rapidly precipitated the LC that is catalytically inactive, but did not affect BSA, ovalbumin, and thermolysin. Because the precipitation of BoNT domains must preclude some partly denaturation step(s), the resulting denatured species may display varied activity, stability, and solubility. Protection from precipitation and inactivation was attained by including low concentrations of nonionic detergents. The research was supported by JSTO-CBD Project# 3.10011_06_RD_B. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. Keywords: LC; Stability; Precipitation 10.1016/j.toxicon.2008.04.024
23. Human botulism immune globulin for the treatment of infant botulism: The first four years postlicensure
This work was supported by DTRA under a DoD/Army Contract No. W911NF-06-1-0095.
Stephen S. Arnon, J.R. Payne, Y. Drummond, J.R. Barash
Keywords: Botulinum; Enzyme; Inhibitors; Mechanism; SNAP-25; Structure 10.1016/j.toxicon.2008.04.023
Infant botulism is the intestinal (infectious) toxemia in which swallowed spores of Clostridium botulinum activate in the lumen of the colon and thereby produce botulinum toxin, which after absorption causes flaccid paralysis. Human Botulism Immune Globulin (BIG-IV) neutralizes botulinum toxin types A and B. The US FDA licensed BIG-IV to the California Department of Public Health, its creator and developer, as the public service orphan drug BabyBIGs in October 2003 (N. Engl. J. Med. 2006;354:462–471). International distribution began on a trial basis in July 2005. This report summarizes the 2003–2007 distribution, efficacy and safety data for BIGIV. In the US, a total of 333 eventually laboratoryconfirmed patients residing in 35 states were treated within 1 week of hospital admission: 142 type A, 188 type B, 2 type Ba and 1 type Bf. Mean length of hospital stay was 2.271.5 vs. 5.775.1 wks for untreated patients in the 1992–97 BIG-IV randomized clinical trial (Po0.001) and did not differ significantly by toxin type of illness. Aggregate hospital stay avoided through use of BIG-IV totaled 22.4 years; aggregate hospital costs avoided totaled at least $28 million (2007 dollars). Four other infant botulism patients (2 type E, 2 type F) were also treated with BIG-IV. Internationally, 10 laboratory-confirmed cases in 4 countries were treated, 4 type A, 5 type B and 1 type Ba. No serious adverse events associated with BIG-IV treatment were reported. In the 4 years since licensure of human BIG-IV (BabyBIGs), its use globally to
3. Pharmacological properties and factors that influence duration of action 22. Issues in therapeutic development against botulinum neurotoxins (BONT) S. Ashraf S. Ahmed, Leonard Smith, Stephen Toth USAMRIID, Fort Detrick, USA
Studies of the BoNT holotoxin are limited by its intrinsic toxicity. The N-terminal light chain (LC) catalytic domain is a target for therapeutic drug development. But investigators have experienced large variations in its solubility, stability, and specific activity. Efforts to develop inhibitors are likely to be adversely affected by such variations. Thus, a thorough understanding of the factors behind these variations, and finding ways in obtaining a consistently soluble, stable, and active preparation of the LC are very important. In addition, LC has been reported to undergo post-translocation modification in neurons. The most logical targets for drug development should be such
California Department of Public Health, Richmond, USA