DOI
https://doi.org/10.25772/VD0Q-BA64
Author ORCID Identifier
0000-0001-6894-2027
Defense Date
2023
Document Type
Thesis
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Dr. Matthew C. T Hartman
Second Advisor
Dr. Brian Fuglestad
Third Advisor
Dr. Ashton Cropp
Fourth Advisor
Dr. Xuwei Wang
Abstract
Peptide therapeutics have shown unique abilities in targeting protein-protein interactions involved in disease states in contrast to small molecules. The composition and size of peptides have great advantages especially when large interfaces with undefined structures are involved. However, peptides aren’t largely bioavailable, making them an unpopular choice for drug discovery. In the past few decades, the stability and affinity of therapeutic peptides have seen huge improvements using noncanonical amino acids (ncAAs) and cyclization which promotes their drug-like properties by imparting more stable and rigid conformations. The ribosomal incorporation of such ncAAs is challenging as they can be poor substrates in in vitro translation compared to canonical amino acids (cAAs) thus limiting the amino acid palette. I have investigated the use of a hyperaccurate ribosome that efficiently incorporates ncAAs by eliminating competition with the canonical aminoacyl tRNA substrates in in vitro translation system. Here, I demonstrate that this application can be extended to different Genetic code expansion platforms where competing cAA-tRNA can be misincorporated and result in poor ncAAs incorporation. ncAAs play an important role in discovery of peptide therapeutics using platforms like mRNA display, where trillions of unique peptides can be screened together for their affinity towards a target. The mRNA sequence that codes for peptides are covalently attached thus forming a direct genotype-phenotype link, making mRNA display compatible for directed evolution-like screening process. Using efficiently incorporated ncAAs that are involved in cyclization (CuAAC and DBX), I demonstrate a library of bicyclic peptides that will be screened against a target involved in Neurofibromatosis type 1 disease using mRNA display. The two cyclic regions of our peptide library assist in i) cell penetration along with cargo and ii) affinity binding to the target. This library is strategically designed to offer diversity in structure of cyclization and cycle length to find high affinity binders to the target protein which may have inherent cell penetrating capabilities.
My work presents an accessible method for improving the fidelity of in vitro translation systems using hyperaccurate ribosomes, characterizes its strengths in Genetic code expansion platforms. It shows that the hyperaccurate ribosomes are superior to wildtype for the translation of peptides bearing non-canonical amino acids. The enhanced in vitro translation for ncAAs is a crucial aspect of peptide therapeutics screening platforms, where it can assure predictable fates of peptide libraries being screened against therapeutic targets for affinity.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
6-29-2023
Included in
Biochemistry Commons, Biotechnology Commons, Cancer Biology Commons, Molecular Biology Commons, Organic Chemistry Commons