| Author: | Zhang, Xiaozhe |
| Title: | Structure-based design of novel mitochondria-targeting stapled peptides and mechanistic study of their therapeutic application in cancer |
| Advisors: | Zhao, Yanxiang (ABCT) |
| Degree: | Ph.D. |
| Year: | 2022 |
| Subject: | Mitochondria Mitochondrial pathology Cancer -- Molecular aspects Cancer -- Treatment Hong Kong Polytechnic University -- Dissertations |
| Department: | Department of Applied Biology and Chemical Technology |
| Pages: | xvi, 169 pages : color illustrations |
| Language: | English |
| Abstract: | Mitochondria are cellular organelles dedicated to the oxidative phosphorylation (OXPHOS) process for ATP production. Additionally, mitochondria also play crucial roles in essential cellular processes such as apoptosis, necrosis and calcium signaling. Cancer cells are particularly dependent on mitochondria to support their excessive proliferation. Such dependence has sparked intensive effort to develop mitochondria-targeting entities as novel cancer therapeutics. It is technically challenging to target mitochondria for cancer therapy. Most commercially available mitochondrial OXPHOS inhibitors, such as the commercially available FCCP, rotenone and oligomycin, are non-selective against cancer cells and show overt toxicity. The double-membraned structure of mitochondria also poses as a physical barrier that prevents the free diffusion of therapeutic entities to IMS, IMM or matrix. While positively charged lipophilic compounds such as rhodamine and triphenylphosphonium (TPP) have been shown to accumulate in active mitochondria driven by membrane potential, they also accumulate in other subcellular locations that are electrostatically compatible. In this project, we propose to develop mitochondria-targeting peptide mimetics by mimicking the N-terminal type of Mitochondrial Targeting Sequence (MTS). This type of MTS is a short segment of 15-30 residues commonly found in the N-terminal region of cytosolic proteins destined for import into mitochondria. This MTS forms a short positively charged alpha helix and binds to the receptor protein TOM20 on mitochondria outer membrane. This MTS-TOM20 interaction will then guide the target protein into mitochondria through the TOM40 channel. We reason peptide scaffolds that mimic this MTS would serve as specific mitochondria-targeting entities. Here we present our structure-based rational design of mitochondria-targeting stapled peptides (Mito-SPs) followed by functional studies to characterize their anti-proliferative potency in cell- and animal-based models of triple negative breast cancer (TNBC). The scaffold of our Mito-SPs consists of two segments, including the N-terminal positively charged TAT sequence to facilitate cell penetration and the C-terminal hydrocarbon stapled peptide to mimic the alpha helical MTS. Additionally, the TAT sequence is believed to cause deleterious effect on essential mitochondria metabolic activities due to its poly-arginine sequence. Our results show that the designed Mito-SPs interacted with TOM20 with micromolar affinity and specifically co-localized with mitochondria in multiple TNBC cell lines tested. Mito-SPs also disrupted mitochondria membrane potential and impaired OXPHOS activity in dosage-dependent manner. Furthermore, Mito-SPs caused calcium-induced membrane permeability transition in mitochondria, possibly due to excessive release of calcium from ER, and induced to necrotic cell death. Highly proliferative and metastatic cancer cells are known to maintain low levels of calcium in mitochondria as an anti-apoptosis mechanism. This feature may render them particularly sensitive to the deleterious effect of Mito-SPs. Indeed, Mito-SPs showed potent anti-proliferative effect in TNBC cell lines and inhibited tumor growth in mouse xenograft models for TNBC without obvious toxicity. In summary, our work has demonstrated the feasibility of designing mitochondria-targeting stapled peptides by mimicking MTS. Our designed peptides have shown potent anti-proliferative efficacy in TNBC cancer cells by attacking their mitochondria vulnerability. Our study suggests a new approach to target mitochondria for cancer drug discovery. |
| Rights: | All rights reserved |
| Access: | open access |
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