Target-guided selection of DNA-encoded chemical libraries for macrocycle ligand discovery

Abstract

Macrocycles are emerging as a promising class of drugs. Occupying the chemical space beyond the Rule of Five, macrocycles present potential opportunities to modulate 'difficult-to-drug' targets. However, macrocyclic drug discovery is challenging due to the synthetic complexities. Integrating DNA-encoded chemical library (DECL) technique shows promise in addressing these issues, enabling rapid synthesis and high-throughput screening of extensive libraries of compounds. Nevertheless, reported macrocycle DECLs still face synthetic cyclisation challenges, where incomplete cyclisation yields linear impurities that can affect selections. Additionally, each macrocycle ligand can exist in multiple conformations but one of which may bind to the target preferentially. However, the energy required to change to the favourite spatial arrangement can be too high because of the restricted conformation of the pre-cyclised structure. This thesis introduces a novel design of DECL tailored for macrocycle ligand discovery to help to overcome these obstacles. The proposed DECL is a linear precursor library, differing from the usual approach of pre-cyclised macrocycle DECLs. The library incorporates the principle of target-guided synthesis, allowing the cyclisation of library members under the protein template during the selection process. The thesis details the design and synthesis of a 1 million-member linear precursor DECL for macrocycle ligand discovery. The library was selected against ten challenging targets. The most outstanding selection results were obtained from the Bcl-xL and Src proteins. Selected 'hits' were synthesised and the binding affinities were determined using surface plasmon resonance. Notably, the cyclised 'hits' exhibited 10 to 30 fold increase in affinity compared to their linear precursors. The most promising binder identified for the Src target exhibited a KD value of 164 nM, while its linear form demonstrated a substantially weaker affinity with a KD value of 5.08 µM. This approach tackles the challenges and expand the scope of DECL macrocycle. The identification of a nanomolar binder from this macrocycle library underscores the promising application of DECL in macrocycle drug discovery.