10 min Rapid Fire International Peptide Symposium 2023

Assessing the cellular permeability of peptidic macrocycles in high-throughput   (#36)

Christian Bech-Bartling 1 , Alexander L. Nielsen 2 , Kristian Strømgaard 1 , Christian Heinis 2
  1. University of Copenhagen, Copenhagen, CAPITAL REGION, Denmark
  2. Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

Cyclic peptides provide attractive drug development modalities due to their ability to bind challenging targets. Compared to linear peptides, the cyclic scaffolds convey high proteolytic stability and the potential to cross cell membranes to reaching intracellular proteins. Advances in the synthesis of large libraries of small cyclic peptides facilitated the discovery of ligands to several disease targets. Specifically, “m” short linear peptides containing thiol groups at both ends can combinatorically be cyclized with "n" bis-electrophilic linker reagents to obtain m × n cyclic peptides, which can be tested as crude products. While the approach can yield promising leads, not all of the hits are membrane permeable. Hence, a full picture of membrane permeability for peptidic macrocycles is lacking. In this work in progress, we take advantage of the chloroalkane penetration assay (CAPA), that has recently emerged as a robust method to determine exclusively cytosolic permeability of chloroalkane-tagged biomolecules.[1] We have established a method to synthesize thousands of diverse chloroalkane-tagged peptidic macrocycles to determine their cytosolic permeability using CAPA. This has given us new insights into the structure-permeability relationships of an unprecedented number of macrocycles and provides a clearer picture of what features govern permeability of macrocyclic compounds in cellular systems.