Design and Evaluation of Light-Responsive Vinyl Sulfone Proteasome Inhibitors for Targeted Cytotoxicity

Abstract

Design and Evaluation of Light-Responsive Vinyl Sulfone Proteasome Inhibitors for Targeted Cytotoxicity The Proteasome is a multi-catalytic enzyme complex found in the nucleus and cytoplasm of all eukaryotic cells. It is the primary intracellular proteolytic system involved in intracellular proteolysis.1 The Proteasome degrades damaged, oxidized, or misfolded proteins and plays a vital role in regulating proteins that control the cell cycle, transcription factors, and cell growth. Therefore, the continued health of the malignant cells, as opposed to normal cells, may depend on the degradation of damaged proteins.2 So, proteasome inhibition is a targeted therapy for cancer to promote cell cycle arrest or apoptosis. Thus, the precise regulation of proteasome activity has become central to current research, particularly its implications in cancer treatment. Bortezomib is used for treating multiple myeloma and is found to be ineffective against solid tumors.3 A spatiotemporal control over the proteasome is one of the solutions to resolve these issues using external stimuli, such as light. Thus, we designed and synthesized azobenzene-containing tripeptide vinyl sulfones as the azobenzene moiety can impart E↔Z isomerism upon exposure to UV light. Further, the hydrophobicity of these peptides was fine-tuned by systematically varying the size of hydrophobic amino acids at the P1, P2, and P3 positions. The light-induced Z isomers of these photopeptides showed excellent cellular potency in HeLa, A549 especially in MCF-7 cell lines. Photopeptide with valine at the proximal position, phenylalanine at P2, and leucine at the P1 positions exhibited nearly 20- and 6.6-fold cellular potency in MCF-7 and A549 cells, respectively.4 Taking cue from these results, we next investigated the substituents' effect on photoswitchability and cytotoxicity of a new set of photopeptides. We systematically changed the substitution position on the azobenzene moiety of the newly synthesised peptides. This modular approach helped us to understand the role of electronic and steric perturbations on the photo-conversion rate and their cytotoxicity. Furthermore, through cell cycle analysis, we established that the newly synthesised compounds cause cell cycle arrest in the G2 phase and activate apoptosis in breast cancer cells. These results are significant, as they demonstrate that light-controlled proteasome inhibition can be effectively translated to cellular systems, offering a promising approach for spatiotemporally regulated cancer therapy. All these intriguing results are presented in this thesis.5 References: 1. Ravid, T.; Hochstrasser, M. Diversity of Degradation Signals in the Ubiquitin-Proteasome System. Nat. Rev. Mol. Cell Biol. 2008, 9 (9), 679–690. 2. Marques, A.-J.; Palanimurugan, R.; Matias, A.-C.; Ramos, P.-C.; Dohmen, R.-J. Catalytic Mechanism and Assembly of the Proteasome. Chem. Rev. 2009, 109 (4), 1509–1536. 3. Russo, A.; Bronte, G.; Fulfaro, F.; Cicero, G.; Adamo, V.; Gebbia, N.; Rizzo, S. Bortezomib: A New Pro-Apoptotic Agent in Cancer Treatment. Curr. Cancer Drug Targets 2010, 10 (1), 55–67. 4. Pradhan, S.; Sarker, S.; Thilagar, P. Azobenzene-Tagged Photopeptides Exhibiting Excellent Selectivity and Light-Induced Cytotoxicity in MCF-7 Cells over HeLa and A549. J. Med. Chem. 2024, 67 (21), 18794–18806. 5. Pradhan, S.; Sarker, S.; Thilagar, P. Substituted Azobenzene Exhibits Enhanced Photoconversion, Spatiotemporal Control of the Proteasome, and Light-Induced Cytotoxicity. 2025, Manuscript in preparation.