De novo developed small cyclic peptides that are orally available

Cyclic peptides are ring-shaped molecules that emerged as a promising class of therapeutics. While it is often difficult to find small molecule binders for challenging disease targets, cyclic peptides can bind to featureless surfaces or inhibit protein-protein interactions. However, due to their typically large size and polar surface area, most peptides have a limited membrane permeability which restricts them to inhibiting extracellular targets and prevents an oral administration. The few peptides that are orally bioavailable generally have a molecular weight being below 1 kDa, five or less hydrogen-bond donors, and a polar surface area smaller than 200 Ã 2, which indicates the requirements for cyclic peptides to be orally available. The de novo development of cyclic peptides that bind to new disease targets and that fulfill those requirements is challenging due to the lack of sufficiently large libraries that could be screened. A limiting factor in accessing such libraries is the low synthetic throughput associated with challenging cyclization reactions and time-consuming chromatographic purifications.

The overall goal of my thesis was to establish a procedure that enables de novo development of biologically active cyclic peptides that can be administrated orally. Towards this aim, I build onto the nanomole-scale cyclic peptide synthesis approach established in our group and implemented a dithiol cyclization with bis-electrophilic crosslinkers. Using a two-step combinatorial synthesis, I cyclized 384 peptide sequences with 2 crosslinkers and diversified a peripheral amino group via N-acylation with 11 carboxylic acids to generate a library of 8,448 cyclic peptides. This approach implements acoustic droplet ejection technology (ADE) for combinatorial dispending of nanoliter droplet of the reactants. Screening of the 8,448-member cyclic peptide library against human ï ¡-thrombin identified a double digit nanomolar inhibitor with a molecular weight of 700 Da. I then applied the approach to improve the membrane permeability and metabolic stability in iterative synthesis and screening cycles. This resulted in the de novo development of a cyclic peptide that inhibits human ï ¡-thrombin with nanomolar affinity (Ki = 65 nM) and has an oral bioavailability of 18% in rats.

The automated nanoscale-synthesis on the acoustic dispenser can rapidly generate large and diverse libraries that may even develop ligands for challenging targets such as protein-protein interactions. The established method is general and may be applied for generating orally bioavailable peptide ligands for many other targets.