Peptide and Nonpeptide Approaches to Target Protein-protein Interactions for the Treatment of Cancer.

Abstract

Protein-protein interactions (PPIs) are macromolecular contacts critical in physiological processes and they play a role in both normal cellular processes and disease pathogenesis. Targeting PPIs represents a new but poorly explored therapeutic strategy. In this thesis, I have investigated peptide and nonpeptide approaches to two separate PPIs. Repressor activator protein 1 (RAP1) is a binding partner of telomeric repeat-binding factor 2 (TRF2) for the regulation of telomere function. A previous study has suggested that RAP1 recruits IκB kinases (IKK) to NF-κB complex phosphorylate p65 S536 and activates NF-κB pathway. Knocking down RAP1 sensitizes breast cancer cells apoptosis via NF-κB suppression. We employed a structure-based design strategy to develop a series of triazole-stapled α-helical peptides based upon the TRF2 sequence to block both RAP1/TRF2 and RAP1/IKK interactions. The most potent peptide synthesized binds to RAP1 with a Ki value of 7 nM, and is 400-fold more potent than the initial TRF2 peptide. Cellular studies show that an optimized peptide dose-dependently down-regulates p65 phosphorylation and inhibits cell growth in the HeLa and MDA-MB-157 cancer cell lines. This study generates the first-in-class inhibitor targeting RAP1, which can be used as a pharmacological tool to study NF-κB signaling in certain type of cancer. Bromodomain and extra terminal (BET) proteins, including BRD2, BRD3 and BRD4, are epigenetic readers and play a key role in regulation of gene transcription by binding to acetylated lysine residues on histone tails. Small-molecule inhibitors of BET have therapeutic potential for the treatment of human cancers and other diseases. We have carried out structure-based design, synthesis and evaluation of γ-carboline-containing compounds as a new class of small molecule BET inhibitors. Our most potent inhibitor (compound 18, RX-37) binds to BET bromodomain proteins (BRD2, BRD3 and BRD4) with Ki values of 3.2-24.7 nM and demonstrates high selectivity over other bromodomain proteins. RX-37 potently and selectively inhibits cell growth in human acute leukemia cell lines harboring rearranged mixed lineage leukemia 1 gene. Further modification of RX-37 yielded RX-201 which shows high oral bioavailability and in vivo efficacy in mice. This study has introduced a new chemical scaffold with potential therapeutic value in clinic.