Permeability Analysis and Conformational Investigation of Cyclic Peptide Scaffolds Cyclized via Backbone Thioether or 1,4-Disubstituted-1,2,3-Triazole

The effort to modulate challenging, undruggable protein targets has stimulated interest in ligands that are larger and more complex than typical small molecule drugs. While combinatorial techniques such as macrocyclic peptide mRNA display and DNA encoded libraries routinely produce high-affinity lead compounds against classically undruggable targets, poor membrane permeability has limited their use toward primarily extracellular targets. Understanding the passive membrane permeability of macrocyclic peptides would, in principle, improve our ability to design libraries whose leads can be more readily optimized against intracellular targets.Chapter 1 seeks to investigate the permeabilities of over 200 cyclic 10-mer scaffolds using the thioether cyclization motif commonly found in mRNA display macrocycle libraries. We identified the optimal lipophilicity range for achieving permeability in three 10-mer cyclic peptide-peptoid hybrid scaffolds inspired by permeable, non-thioether scaffolds and showed that permeability could be maintained even when multiple stereocenters were inverted. The low-dielectric (membrane-associated) conformation of one of these scaffolds, as revealed by circular dichroism, NMR spectroscopy, and molecular dynamics, revealed a unique, saddle-shaped fold in which all four backbone NH groups were sequestered from solvent. This work provides an example by which pre-existing physicochemical knowledge of a scaffold can benefit the design of macrocyclic peptide mRNA display libraries, pointing toward an approach for biasing libraries toward permeability by design. In Chapter 2, the same principles are used to investigate the permeability landscape of 1,4-disubstituted-1,2,3-triazole cyclized 10mers. We confirmed that passive permeability is still achieved in the three model scaffolds we identified, although the interplay with lipophilicity is distinctly different from their parent and thioether derivatives. Additionally, cassette analysis of hydrocarbon-water partition coefficients supports the hypothesis that the triazole scaffolds are also able to sequester all hydrogen-bond donors in low-dielectric media, similar to their parent congeners. Overall, the compounds described herein are a further demonstration that many diverse and permeable scaffolds exist well beyond conventional drug-like chemical space