Peptide Ligands Stabilized by Small Molecules

Abstract

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100-fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needlefree application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X-ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide-protein complexes and contribute to the high binding affinity.

Highly Constrained Bicyclic Scaffolds for the Discovery of Protease-Stable Peptides via mRNA Display

“…The use of 1,3,5-trisbromomethylbenzene (TBMB) 37 to generate bicyclic phage displayed libraries has been used to develop high affinity ligands specific for human plasma kallikrein 38 and human urokinase-type plasminogen activator 18, 39 . Similar strategies involving 1,3,5-triacryloyl-1,3,5-triazinane (TATA) and N , N ′, N ″-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB) have also been reported 40 . While the size of each macrocycle can be varied using these linkers, conformations are limited to two independent loops because of the reliance on a central, trisubstituted linker.…”

Encoded libraries of chemically modified peptides

“…The inhibitors of PK and uPA even did not inhibit the murine orthologs [14,38 ]. X-ray structures of bicyclic peptides with their targets showed a high shape complementarity of peptide and target, and explained the good target selectivity (Figure 4b) [14,42].…”

“…Like TBMB, TATA and TBAB were shown to efficiently and selectively cyclize peptides containing three cysteines [46]. Panning of peptide phage libraries cyclized with either TBMB, TATA or TBAB against the target uPA yielded entirely different families of ligands [42]. Most of the isolated peptides bound the target only when cyclized with the linker that was used in the selection but not when cyclized with one of the other two linkers.…”

“…Most of the isolated peptides bound the target only when cyclized with the linker that was used in the selection but not when cyclized with one of the other two linkers. X-ray crystallographic analysis of the bicyclic peptides in complex with uPA showed that some of the linkers played important structural roles; in one peptide, the linker even formed Hbonds with the peptide and the target protein [42].…”

A phage display approach to identify highly selective covalent binders

“…We describe here a general, unbiased approach to directly screen pools of covalent probes with exceptional diversity using the power of phage display. Inspired by the work of others in which phage are chemically modified to produce cyclic and bicyclic peptides directly on the phage surface [21][22][23][24], we developed a small molecule linker that could be used to both form cyclic peptides and also introduce a weak electrophile 'warhead' directly on the cyclized phage coat protein segment ( Fig. 1).…”