Background
Peptides represent a growing class of therapeutics with potential to treat different conditions such as diabetes and various forms of cancer. However, therapeutic peptides have two intrinsic drawbacks: poor cell permeability and susceptibility to proteolytic degradation, which represent major stumbling blocks for peptide drug development. Macrocyclic peptides are a new class of peptides that have emerged as a valuable class of molecules for the investigation and modulation of protein-protein interactions (PPIs). Compared to linear peptides, macrocyclic peptides often exhibit increased adsorption, greater proteolytic resistance and improved cell permeability. Coupled with their conformational rigidity, cyclic peptides have the potential to interact with large, shallow protein surfaces with high affinity and selectivity, which contribute to their value and utility as chemical probes and potential leads for therapeutic development. Given the increasing importance of macrocyclic peptides, methods for generating and exploring combinatorial libraries of these molecules have become particularly valuable. An inherent limitation in this endeavor remains the restricted pool of building blocks available for library construction, despite notable recent advances to address this issue. Undoubtedly, synthetic libraries can draw upon a much broader spectrum of structures. Combining the advantages of biological and synthetic combinatorial methods would open new opportunities for molecular discovery.
Technology Overview
Researchers at the University of Rochester have developed a methodology for embedding non-proteogenic, synthetic moieties into genetically encoded peptidic frameworks. This strategy enables the modular assembly of macrocyclic organo-peptide hybrids (MOrPHs), whose structure can be readily diversified by varying the nature of the synthetic and biosynthetic moieties. This method for generating and screening cyclic peptides couples a randomly generated peptide between a non-canonical amino acid and an intein in a biosynthetic precursor. The precursor spontaneously forms into a lariat containing the desired peptide and the intein in a loop attached to a non-cannonical amino acid. Notably the linkage can contain non-proteogenic amino acids allowing another level of optimization.
The library is created with a 20n peptide sequence library which is combined with different linkage elements leading to a macrocylic peptide library of up to 109-1010 compounds. Notably the synthesis technique is compatible with a bacterial phage display and the compounds can be generated within the bacterial cell. Our method has been validated against streptavidin (20 nM), Keap1/Nrf2 interaction (31 nM), CTLA4-CD80/86 interaction (327 nM), and the Hedgehog/Patched interaction (160 nM) targets.
Benefits
This technology allows for incorporation of synthetic components such as unnatural amino acids, peptoids, - and -peptides, peptidomimetics, or amino acid unrelated structures into resulting peptides. This special feature makes it possible to create peptides with novel or improved properties such as conformational, binding, or chemical/enzymatic stabilities properties. Freedom in the linkage increases the size of the library. Unique to our method is an ability to generate the library in cells allowing for enhanced screening abilities.
Applications
Identification of new cyclic peptide therapeutics.