Microtubule-Targeting NAP Peptide-Ru(II)-polypyridyl Conjugate As a Bimodal Therapeutic Agent for Triple Negative Breast Carcinoma

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) × 10⁶ M^-1; K_NAP: (8.2 ± 1.1) × 10⁴ 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 (^LightIC₅₀^RU-NAP [HEK293]: 17.2 ± 2.5 μM, compared to ^LightIC₅₀ ^RU-NAP [MDA-MB-231]: 32.5 ± 7.8 nM, ^DarkIC₅₀^RU-NAP [HEK293]: > 80 μM, compared to ^DarkIC₅₀^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.