Macrocyclic peptide drug discovery has become a powerful, multidisciplinary and worldwide endeavor to advance a therapeutic modality superclass. Innovative platforms have enabled the advancement of novel molecules of varying shapes and sizes. Peptide drug hunters are engaged in this effort to address both the challenges and opportunities to exploit this macrocyclic peptide superclass to address target space, cell permeability, and drug delivery. This presentation will overview macrocyclic peptide drugs.

Our laboratory has recently developed a new method for generating ligands based on sub-kilodalton cyclic peptides (Kale, S., et al., Science Advances, 2019). In brief, we combinatorially assembled building blocks in microwell plates to obtain macrocycles at good yield and high purity, and screened the crude reactions. Omission of a purification step allowed us to generate and screen large libraries and to find binders to important targets.

The structural and sequence information of cellular processes involving protein interactions and modifications is a valuable starting-point for the design of novel peptide therapeutics. This work exemplifies how peptide enzyme substrates can be turned into therapeutically relevant molecules.  We demonstrate how we exploit structural and sequence information on a protein mechanism and substrate specificity to convert linear peptide substrates into cyclic peptide inhibitors.

Cyclic peptides are an important tool for development of peptide therapeutics and peptide cyclizations can be achieved several ways. Here we describe several fully automated synthesis optimization methods, some using induction heating, for therapeutically relevant peptides including APY-d4, and RFP14. Other examples in this presentation include Melanotan II, NYAD1 and Agardhipeptin A cyclic peptide sequences.

The design of potent macrocyclic peptides able to efficiently modulate intracellular protein-protein interactions remains a major challenge. However, a technology platform named stapled peptides has emerged during the past 10 years that has showed promises in addressing and solving inherent limitations of peptides particularly their poor cell permeability. The unique drug-like properties of ALRN-6924, a stabilized cell-permeating α-helical peptide that is currently being investigated as a chemoprotective drug for patients with TP53-mutant cancers, will be discussed.

The primary cause of death among COVID-19 patients is the development of acute respiratory distress syndrome (ARDS), an inflammatory disease of the lungs. We have developed a peptidyl inhibitor against the calcineurin-NFAT interaction, CNI103, which specifically blocks the activation of NFATs and the production of inflammatory cytokines (e.g., TNF-alpha and IL-6). CNI103 has demonstrated excellent efficacy for prevention of acute lung injury/ARDS in a mouse model. In this talk, I will discuss how the peptidyl inhibitor was developed and its in vitro and in vivo characterization.

Macrocyclization is a promising strategy to stabilize bioactive conformations and to improve several properties for lead optimization. A general computational workflow for the recognition and evaluation of sites for macrocyclization, the identification of compatible chemical linkers, and their conformational and enthalpic scoring has been developed. The broader applicability of this approach will be demonstrated with application to different case studies, e.g., GLP1/glucagon and small molecules (TAFIa) inhibitors.

Hepcidin mimetic peptides that are orally stable and systemically active will mark a paradigm change in management of blood disorders that exhibit aberrant iron homeostasis. Protagonist Therapeutics’ Rusfertide is an injectable hepcidin mimetic, currently in Phase 2 clinical studies for polycythemia vera and hereditary hemochromatosis. I will describe 2nd Gen hepcidin mimetic peptides that are orally stable, systemically absorbed, and show improvement in disease parameters in pre-clinical mouse models.

Mining of macrocyclic natural products provided sets of cores for in silico screening to find leads for difficult-to-drug targets. Docking of the cores led to the discovery of a structurally unique, uncharged macrocyclic inhibitor of the Keap1-Nrf2 protein-protein interaction; a particularly challenging target due to its highly polar binding site. Design of a macrocycle collection inspired by the structure of one of the cores provided submicromolar inhibitors of Leishmania infections.

We have designed new methods for isolating unique and previously understudied conformations of macrocycles. The observation of unusual conformations was made possible by controlling their interconversion using dominant rotors, which represent tunable atropisomeric constituents with high rotational barriers. Evidence suggests that amino acid residues can be forced into rare turn motifs not observed in linear counterparts and homodetic rings. These findings should unlock new avenues for studying the conformation-activity relationships.

Drug Discovery is a discipline of compromises to achieve efficacy and safety with drug molecules. The balancing of properties required to enable passage across biological membranes is considered in the context of debates over the mechanism of crossing membranes, be this via the bilayer or carrier proteins. In particular, the evolutionary consequences of the latter are explored, suggesting opportunities for better biomimetic designs in molecules large and small.