Abstract by Angelika Christensen

Obesity is a growing global health crisis driven by genetic, environmental, and lifestyle factors that alter the balance of energy and increase the risk of diabetes, cardiovascular disease, and other diseases. The leptin–melanocortin pathway, and in particular the MC4R is central to hypothalamic control of appetite and energy expenditure. MC4R variants are among the most common monogenic causes of severe early obesity, making selective modulation of MC4R a therapeutic strategy. To accelerate the discovery of peptide modulators, we developed a miniaturized 96-platform cyclization cleavage that yields bicyclic peptides with high crude purity material for direct biological screening. From an initial bicyclic library screened against MC3R/MC4R, four hit sequences were selected for resynthesis and quantitative functional tests. cAMP assays showed that all four were highly potent MC4R agonists with selectivity over MC3R, and single residue changes around the conserved pharmacophore produced changes in potency, intrinsic efficacy, and receptor selectivity. Inspired by the rationale for structure activity, the substitution of D-Phe with the larger D-2-Nal in the pharmacophore converted several ligands into full antagonists, demonstrating a steric mechanism that preserves binding while stabilizing inactive receptor states. We have determined a 3D structure using NMR for compound 2.2, which reveals a partially populated helical motif.

In the second project, we similarly aimed to modulate MC4R activity, however, we decided to investigate alternative scaffolds, specifically kalata B1 (kB1) and Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). We grafted both scaffolds with the pharmacophore in loop VI and interestingly MCoTI-II showed MC4R agonism with EC50=2.45 nM and negligible MC3R activity.

Finally, to demonstrate a broader applicability and enable the screening of expanded libraries, the miniaturized bicyclic synthesis platform was applied to synthesize inhibitors against HDACs, where the library of 270 incorporated two non-canonical amino acids. Screening across representative HDAC isoforms identified hits with micromolar IC50 for HDAC1, revealed differential isoform sensitivities (HDAC4 largely unresponsive, HDAC6 and HDAC11 showing distinct activity patterns), and confirmed that the miniaturized workflow on resin can rapidly generate a library.