Development of fragment-bearing cyclic peptides as first-in-class therapeutics against ALT-positive osteosarcomas

Abstract

Osteosarcoma is the most common type of primary bone malignancy that arises during the adolescent growth spurt and disproportionately affects young people. With no targeted therapy available, patients diagnosed with osteosarcoma are predominantly treated with high-dose chemotherapy combined with disabling amputation or limb salvage surgery, leaving significant burden on survivors. A novel strategy for treating osteosarcoma is to target a DNA repair mechanism known as Alternative Lengthening of Telomeres (ALT), which is required by over 60% of osteosarcomas for continued growth. Since there is no evidence of ALT activation in healthy somatic cells, inhibition of ALT offers a significant window for therapeutic intervention against osteosarcoma. ALT activity can be targeted by disrupting two well-defined protein-protein interactions (PPIs), FANCM-BTR and TRF2. Both PPIs exhibit well-defined binding interfaces, each providing a suitable site for competitive targeting that is structurally defined and ready for novel inhibitor discovery. The FANCM-BTR target is mediated by hydrophobic interactions between a 12-mer peptide from the FANCMMM2 domain and the RMI1/2 heterodimer, while TRF2 recruits other telomeric protein components via its TRFH domain that prefers a conserved binding motif. This thesis reports first-in-class de novo cyclic peptide inhibitors against FANCM-BTR and TRF2TRFH by implementation of fragment-based drug discovery and mRNA display. These cyclic peptides serve as novel chemical tools for further investigation on the biochemical mechanism of FANCM-RMI1/2 and TRF2TRFH in regulating ALT activity and potential targeted therapy for ALTpositive osteosarcomas. The proof-of-concept fragment-incorporated RaPID screen also provides a novel platform for hit-to-lead generation and structure-activity relationship studies of de novo cyclic peptides containing fragment-bearing amino acids as intracellular PPI inhibitors with high affinity and target specificity.