Presentation Title

Amino Acid Profiling and Peptidomimetic Inhibitors against the Botulinum Neurotoxin

Faculty Mentor

Nicholas T. Salzameda

Start Date

18-11-2017 11:15 AM

End Date

18-11-2017 11:30 AM

Location

9-273

Session

Bio Sciences 3

Type of Presentation

Oral Talk

Subject Area

interdisciplinary

Abstract

The Botulinum neurotoxin serotype A (BoNT/A), a byproduct of the Clostridium botulinum bacteria, is responsible for causing the paralytic disease, Botulism. BoNT/A has a lethal dose (LD50) of 1.3 ng/kg of body weight in humans, making it the world’s most lethal toxin. Its potency, ease of extraction, and reproducibility raises concern the toxin may be weaponized and used in bioterrorism. The therapeutics currently available to treat the disease are not viable for large scale infections, resulting in mass casualties in the case of an attack. Therefore, efforts to develop alternative therapeutics are essential.

The toxin is a dimer composed of a heavy chain (HC) and a light chain (LC). The HC binds to neuronal cells and aids the LC into the cytosol of the cell. Once in the cytosol the LC, a zinc metalloprotease, terminates neurotransmission by cleaving SNARE proteins.

Our laboratory focuses on the development and synthesis of small molecules as a therapeutic countermeasure against BoNT/A LC. Previously, our laboratory discovered a promising small molecule inhibitor consisting of four major components: (1) Hydroxamic acid, (2) Isoleucine, (3) Sulfonamide, and (4) a 4-Chlorobiphenyl ring system. We hypothesized that increasing the number of amino acids in the scaffold would increase the inhibitor’s affinity to the enzyme. The purpose of our research is to optimize lead molecules capable of inhibiting BoNT/A LC. Using a solid phase synthetic strategy, various amino acid sequences were coupled onto a solid support to create a diverse library of diamino-peptides. A 4-chlorobiphenyl sulfonyl chloride was coupled to the peptides to yield the final compound. The molecules were cleaved from the solid support with trifluoroacetic acid to yield the hydroxamic acid on the C-terminus. An enzymatic assay was employed to evaluate the new small molecules. Structure activity relationship studies were used to optimize enzyme inhibition.

Summary of research results to be presented

In our efforts, we discovered five small molecules that displayed 84% or greater inhibition at 15µM. We have successfully improved one of our lead molecules to 75% inhibition at a concentration of 0.5 µM. The molecule contained a hydroxamic acid, isoleucine, phenylalanine, and a 3,5-Dichlorobenzene sulfonamide instead of the biphenyl ring system. These results will shift our focus to optimize the ring system in future compound libraries.

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Nov 18th, 11:15 AM Nov 18th, 11:30 AM

Amino Acid Profiling and Peptidomimetic Inhibitors against the Botulinum Neurotoxin

9-273

The Botulinum neurotoxin serotype A (BoNT/A), a byproduct of the Clostridium botulinum bacteria, is responsible for causing the paralytic disease, Botulism. BoNT/A has a lethal dose (LD50) of 1.3 ng/kg of body weight in humans, making it the world’s most lethal toxin. Its potency, ease of extraction, and reproducibility raises concern the toxin may be weaponized and used in bioterrorism. The therapeutics currently available to treat the disease are not viable for large scale infections, resulting in mass casualties in the case of an attack. Therefore, efforts to develop alternative therapeutics are essential.

The toxin is a dimer composed of a heavy chain (HC) and a light chain (LC). The HC binds to neuronal cells and aids the LC into the cytosol of the cell. Once in the cytosol the LC, a zinc metalloprotease, terminates neurotransmission by cleaving SNARE proteins.

Our laboratory focuses on the development and synthesis of small molecules as a therapeutic countermeasure against BoNT/A LC. Previously, our laboratory discovered a promising small molecule inhibitor consisting of four major components: (1) Hydroxamic acid, (2) Isoleucine, (3) Sulfonamide, and (4) a 4-Chlorobiphenyl ring system. We hypothesized that increasing the number of amino acids in the scaffold would increase the inhibitor’s affinity to the enzyme. The purpose of our research is to optimize lead molecules capable of inhibiting BoNT/A LC. Using a solid phase synthetic strategy, various amino acid sequences were coupled onto a solid support to create a diverse library of diamino-peptides. A 4-chlorobiphenyl sulfonyl chloride was coupled to the peptides to yield the final compound. The molecules were cleaved from the solid support with trifluoroacetic acid to yield the hydroxamic acid on the C-terminus. An enzymatic assay was employed to evaluate the new small molecules. Structure activity relationship studies were used to optimize enzyme inhibition.