Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Mande Holford

Committee Members

Jeroen Kool

Nicholas Tatonetti

Rein Ulijn

Guillermo Gerona-Navarro

Subject Categories

Biochemistry

Keywords

Venom, high-throughput, peptide synthesis, teretoxins, drug discovery, proteomics, peptide synthesis

Abstract

Bioactive venom peptides are increasingly being explored as drug leads in pharmaceutical research because of their immense therapeutic potential, however there is a need to more efficiently screen and characterize these peptides to discover medicinal leads more quickly.1,2 The Holford lab has leveraged recent technological achievements in genomics, transcriptomics, and proteomics to produce a leap in the discovery of venom peptides from previously hard to characterize small and neglected venomous animals such as Terebridae, an understudied lineage of the Conoidea Superfamily.4, 5–7 With the rise of -omic technologies the floodgates have opened and venom researchers are awash with large datasets of novel, uncharacterized venom peptides. However, the key barrier of progress for the future of venom drug discovery is determining how to efficiently screen animal venom arsenals to identify bioactive peptides with pharmaceutical potential. The long-term goal of this research is to efficiently characterize and screen marine snail peptides for therapeutic targets. The specific objectives of this proposal are to establish a robust high-throughput method for screening purified venom peptides using a variety of approaches that will provide structural and functional characterization. In preliminary findings and published results from the Holford lab, we have shown how to characterize terebrid venom peptides, teretoxins, using proteomic and transcriptomic methods and developed strategies for synthesizing pure venom peptides from our datasets.8,9 Specifically, we first developed a modular plasmid that can encode for any disulfide-rich venomous peptide in its variable region, with a singular purification protocol for the recombinantly expressed peptide. The modular plasmid can be modified to encode an affinity tag, such as HaloTag protein or a TurboID protein, while having the same purification protocol. These teretoxins where then studied for phenotypic and functional peptide screens. However, the peptide-fusions (HaloTag and TurboID) in vivo experiments are still being optimized to test for Protein-Protein interactions (PPIs). Secondly, parameters for characterizing crude Terebridae venom on several instrument platforms, including nano-liquid chromatography mass spectrometry (nano-LCMS) and capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS), were determined. This prompted us to develop a computational program, Toxins-to-Toxins (T2T) that matches proteomic and transcriptomic data to identify novel venom peptide toxins, while using pure teretoxins as controls. Lastly, we used VenomSeq, which is a computational method to predict teretoxin’s biomolecular targets. We exposed IMR-32 cells to nine pure teretoxins and two control peptides using two computational approaches, one without restrictions on modes of action and in which we restricted action to membrane interactions. From this analysis terertoxin Mki 8.7, from the venom Myurella kiliburni was identified as a potential SERCA or serotonin antagonist. This discovery highlights the use of VenomSeq to screen the large reservoir of venom extracts and pure peptides for therapeutic applications.

Our integrated approach develops a system that can rapidly synthesize venomous peptides, screen, and characterize crude venom by various proteomic approaches to strengthen our inhouse Terebridae transcriptomic database, and characterize the function of selected pure venom peptides in bioactive assays. In this manner we created a partially validated teretoxin functional activity pipeline to identify potential therapeutic candidates in a high throughput manner.

This work is embargoed and will be available for download on Saturday, September 30, 2023

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