PhD Dissertation Defense/BMEBT Seminar by Wonsup Choi
When: Tuesday,
December 8, 2015
4:00 PM
-
5:00 PM
Description: TOPIC: DEVELOPMENT OF A SPECIFIC PEPTIDE LIGAND FOR TARGETING THE LIGHT CHAIN OF BOTULINUM NEUROTOXIN A
LOCATION: VIOLETTE BLDG - 201
ABSTRACT: The overall goal of this research aims at identification of peptide molecules that can inhibit Botulinum neurotoxins (BoNTs) to address preparedness for bio-terrorism attacks using BoNTs. The light chain (LC) of BoNTs inhibits neurotransmitter release by destroying exocytotic fusion. LC acts as an endopeptidase against specific intracellular protein targets present either on the plasma membrane or on the synaptic vesicle. Since LC cleaves proteins of the SNARE inside the cytosol and this is a key step to the development of botulism, it is a valid target for design of inhibitors agains BoNTs.
Phage display has been widely used for identification of receptor ligands, drug discovery, studying protein-protein interactions, and epitope mapping of antibodies. In this study, phage display was used to identify peptides that specifically bind and inhibit the light chain of BoNT type A (LC BoNT/A). After several rounds of panning, clones were sequenced, binding selectivity evaluated by enzyme-linked immunosorbant assay (ELISA), and inhibition of LCA endopeptidase activity investigated with the standard SNAPtide® assay. Two phage clones were chosen as the best candidates, then the sequences corresponding to free peptides were synthesized for detailed characterizations by ITC and SNAPtide® assay; the data showed 2.7 to 11.5 μM of IC50 and 0.45 to 0.51 μM of Kd value, which are significantly improved than those of a known peptide inhibitor for BoNT/A.
Following characterizations of the free peptide, multivalent forms (Polyamidoamine dendrimers and Cyclodextrins) of the peptide were used to mimic the original multivalent phage display and investigate impact of such on binding and inhibition of the toxin. In the results, multivalency increased not only the potency of the peptide inhibitor by > 100 fold in LC BoNT/A inhibition, but also peptide resistance to proteolytic degradation. Furthermore, to elucidate the capability of the peptide inhibitors as unique inhibitors for the action of LC BoNT/A in the body, neuronal cell based inhibition assay was conducted using the human neuroblastoma cell line. This study demonstrates that the peptides developed from phage display technique are promising lead inhibitors, presenting the foundation for the generation of a more dominant derivative that can translate into rapid detection technology and therapeutic treatments.
LOCATION: VIOLETTE BLDG - 201
ABSTRACT: The overall goal of this research aims at identification of peptide molecules that can inhibit Botulinum neurotoxins (BoNTs) to address preparedness for bio-terrorism attacks using BoNTs. The light chain (LC) of BoNTs inhibits neurotransmitter release by destroying exocytotic fusion. LC acts as an endopeptidase against specific intracellular protein targets present either on the plasma membrane or on the synaptic vesicle. Since LC cleaves proteins of the SNARE inside the cytosol and this is a key step to the development of botulism, it is a valid target for design of inhibitors agains BoNTs.
Phage display has been widely used for identification of receptor ligands, drug discovery, studying protein-protein interactions, and epitope mapping of antibodies. In this study, phage display was used to identify peptides that specifically bind and inhibit the light chain of BoNT type A (LC BoNT/A). After several rounds of panning, clones were sequenced, binding selectivity evaluated by enzyme-linked immunosorbant assay (ELISA), and inhibition of LCA endopeptidase activity investigated with the standard SNAPtide® assay. Two phage clones were chosen as the best candidates, then the sequences corresponding to free peptides were synthesized for detailed characterizations by ITC and SNAPtide® assay; the data showed 2.7 to 11.5 μM of IC50 and 0.45 to 0.51 μM of Kd value, which are significantly improved than those of a known peptide inhibitor for BoNT/A.
Following characterizations of the free peptide, multivalent forms (Polyamidoamine dendrimers and Cyclodextrins) of the peptide were used to mimic the original multivalent phage display and investigate impact of such on binding and inhibition of the toxin. In the results, multivalency increased not only the potency of the peptide inhibitor by > 100 fold in LC BoNT/A inhibition, but also peptide resistance to proteolytic degradation. Furthermore, to elucidate the capability of the peptide inhibitors as unique inhibitors for the action of LC BoNT/A in the body, neuronal cell based inhibition assay was conducted using the human neuroblastoma cell line. This study demonstrates that the peptides developed from phage display technique are promising lead inhibitors, presenting the foundation for the generation of a more dominant derivative that can translate into rapid detection technology and therapeutic treatments.
Contact: > See Description for contact information
Topical Areas: University Community, Chemistry and Biochemistry, Bioengineering