Design and development of proteasome inhibitors with improved efficacy in breast cancer cells: 3D tissue models and detailed molecular mechanistic studies

Abstract

The proteasome is an enzyme complex that regulates the degradation of intracellular proteins, maintaining cell cycle regulation, protein quality, and cellular homeostasis. Proteasome inhibition has gained popularity as a cancer treatment strategy due to its ability to alter protein turnover in rapidly proliferating tumor cells. Bortezomib, the first FDA-approved proteasome inhibitor, established the potential of this technique, but it has considerable side effects and is not effective against solid tumors. Thus, there is a need for the development of better inhibitors that have both high selectivity and fewer side effects. Accordingly, this thesis focuses on the design, synthesis, and evaluation of novel boronopeptide-based proteasome inhibitors (PI). A series of boronopeptides were synthesized, and the hydrophobicity and hydrophilicity of these peptides were finetuned by judiciously varying the amino acid residues at the P2 position. The P3 position is secured with thiazole moiety, which has a very high therapeutic index. All the peptides were evaluated for inhibitory activity against the human 20S proteasome using model substrates for catalytic active sites β1, β2, and β5. The results showed that the newly synthesized peptides selectively and effectively inhibit the major catalytic active site β5 over β1 and β2. We have also studied the potency of these PIs in a series of cancer cell lines such as HeLa, HepG2, A549, & HEK293T and found that these boronopeptides selectively kill the MCF7 breast cancer cells over other cells. Furthermore, surpassing the commercial drug bortezomib, these PIs showed >100 times selectivity towards the MCF7 breast cancer variant over the normal breast variant cells HMF & MCF10A. Interestingly, these PIs showed higher cytotoxicity in 3D tumor models than bortezomib, highlighting their potential for enhanced tumor penetration and efficacy in physiologically relevant models. Encouraged by these results, we have carried out a series of detailed biochemical analyses such as cell cycle analysis, DNA, protein and reactive oxygen species (ROS) quantification, proteomics, mitochondrial potential analysis, and computational studies, including DFT, covalent-protein docking and ADMET analysis. These studies revealed that proteasomal inhibition led to the accumulation of essential regulatory proteins such as polyubiquitinated substrates, BAX, XBP1, p21, p53, cleaved caspase-9, cleaved caspase-3, thus inducing mitochondrial dysfunction, ROS production, ER stress, cell cycle arrest, and, subsequent apoptosis. These findings showed that the newly synthesized PIs are promising candidates for advancing targeted cancer therapy, offering insights into their application in physiologically relevant models and clinical translation. These intriguing results is discussed in detail in this thesis.