Microtubule-Targeting NAP Peptide-Ru(II)-polypyridyl Conjugate As a Bimodal Therapeutic Agent for Triple Negative Breast Carcinoma
Atin Chatterjee, Sandip Sarkar, Sangheeta Bhattacharjee, Arpan Bhattacharyya, Surajit Barman, Uttam Pal, Raviranjan Pandey, Anitha Ethirajan, Batakrishna Jana, Benu Brata Das, Amitava Das
Abstract
Triple-negative breast cancer (TNBC) poses significant treatment challenges due to its high metastasis, heterogeneity, and poor biomarker expression. The N-terminus of an octapeptide NAPVSIPQ ( NAP ) was covalently coupled to a carboxylic acid derivative of Ru(2,2′-bipy) 3 2+ ( Rubpy ) to synthesize an N-stapled short peptide-Rubpy conjugate ( Ru-NAP ). This photosensitizer (PS) was utilized to treat TNBC through microtubule (MT) targeted chemotherapy and photodynamic therapy (PDT). Ru-NAP formed more elaborate molecular aggregates with fibrillar morphology as compared to NAP . A much higher binding affinity of Ru-NAP over NAP toward β-tubulin ( K Ru-NAP: (6.8 ± 0.55) × 10 6 M –1; K NAP: (8.2 ± 1.1) × 10 4 M –1 ) was observed due to stronger electrostatic interactions between the MT with an average linear charge density of ∼85 e/nm and the cationic Rubpy part of Ru-NAP . This was also supported by docking, simulation, and appropriate imaging studies. Ru-NAP promoted serum stability, specific binding of NAP to the E-site of the β III -tubulin followed by the disruption of the MT network, and effective singlet oxygen generation in TNBC cells (MDA-MB-231), causing cell cycle arrest in the G2/M phase and triggering apoptosis. Remarkably, MDA-MB-231 cells were more sensitive to Ru-NAP compared to noncancerous human embryonic kidney (HEK293 cells) when exposed to light ( Light IC 50 Ru-NAP [HEK293]: 17.2 ± 2.5 μM, compared to Light IC 50 Ru-NAP [MDA-MB-231]: 32.5 ± 7.8 nM, Dark IC 50 Ru-NAP [HEK293]: > 80 μM, compared to Dark IC 50 Ru-NAP [MDA-MB-231]: 2.9 ± 0.5 μM). Ru-NAP also effectively inhibited tumor growth in MDA-MB-231 xenograft models in nude mice. Our findings provide strong evidence that Ru-NAP has a potential therapeutic role in TNBC treatment.