PhotoRaPID: Development of a trillion-member discovery platform to identify de novo light-responsive cyclic peptides
File(s)
Author(s)
Jackson, Thomas
Type
Thesis
Abstract
Cyclic peptides are an emerging class of exciting chemical tools, bridging the gap between small molecule therapeutics and larger biologics. They have recently gained traction probing classically undruggable protein targets of interest. Many powerful methods to identify novel cyclic peptides have been developed, including mRNA display-based RaPID (Random Non-standard Peptide Integrated Discovery). Such platforms enable the screening of >1 trillion-member cyclic peptide libraries incorporating non-proteogenic amino acids, against any protein of interest.
Despite the development of cyclic peptides as novel tools to answer questions in Chemical Biology, the fine-tuned spatiotemporal druggability of complex biological pathways remains elusive. Light is an incredibly powerful non-invasive stimulus to introduce temporal control within a system. Photoswitches, which harness light as a stimulus, are small molecules that can undergo isomerisation when irradiated at a specific wavelength of light. This project focuses on the integration of light controllable ‘photoswitches’ within every macrocycle of the trillion-member library, via genetic code re-programming, inducing global conformational changes within the macrocyclic peptides structure to give switchable binding.
As a proof-of-concept, methodology for the identification of photoswitchable macrocyclic peptides has been developed against human Protein Arginine Deiminase II. Identified peptides show nanomolar ZKDs and up to 29-fold differential binding and inhibition of PADI2 between each of the two light-responsive (E/Z) macrocyclic isomers. Further work to streamline the methodology was also undertaken. Preliminary data suggests a general correlation between differential sequence enrichment of the two light-responsive isomers binding to PADI2 during the selection and their respective binding affinities determined via SPR. This will potentially streamline the transition from light-responsive candidate peptides to validated hits, a bottleneck of high-throughput screening platforms.
In summary, this work has developed a methodology to allow for the robust identification of light-responsive photoswitchable macrocyclic peptides against any protein target of interest and offers the potential to interrogate complex biological systems in novel and exciting ways.
Despite the development of cyclic peptides as novel tools to answer questions in Chemical Biology, the fine-tuned spatiotemporal druggability of complex biological pathways remains elusive. Light is an incredibly powerful non-invasive stimulus to introduce temporal control within a system. Photoswitches, which harness light as a stimulus, are small molecules that can undergo isomerisation when irradiated at a specific wavelength of light. This project focuses on the integration of light controllable ‘photoswitches’ within every macrocycle of the trillion-member library, via genetic code re-programming, inducing global conformational changes within the macrocyclic peptides structure to give switchable binding.
As a proof-of-concept, methodology for the identification of photoswitchable macrocyclic peptides has been developed against human Protein Arginine Deiminase II. Identified peptides show nanomolar ZKDs and up to 29-fold differential binding and inhibition of PADI2 between each of the two light-responsive (E/Z) macrocyclic isomers. Further work to streamline the methodology was also undertaken. Preliminary data suggests a general correlation between differential sequence enrichment of the two light-responsive isomers binding to PADI2 during the selection and their respective binding affinities determined via SPR. This will potentially streamline the transition from light-responsive candidate peptides to validated hits, a bottleneck of high-throughput screening platforms.
In summary, this work has developed a methodology to allow for the robust identification of light-responsive photoswitchable macrocyclic peptides against any protein target of interest and offers the potential to interrogate complex biological systems in novel and exciting ways.
Version
Open Access
Date Issued
2023-11-01
Date Awarded
2024-02-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Walport, Louise
Fuchter, Matthew
Tate, Edward
Sponsor
Engineering and Physical Sciences Research Council
Centre for Doctoral Training
Grant Number
CHTP.G98215
Publisher Department
Department of Chemistry and The Francis Crick Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)