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 (<b>NAP</b>) was covalently coupled to a carboxylic acid derivative of Ru(2,2'-bipy)₃²⁺ (<b>Rubpy</b>) to synthesize an N-stapled short peptide-Rubpy conjugate (<b>Ru-NAP</b>). This photosensitizer (PS) was utilized to treat TNBC through microtubule (MT) targeted chemotherapy and photodynamic therapy (PDT). <b>Ru-NAP</b> formed more elaborate molecular aggregates with fibrillar morphology as compared to <b>NAP</b>. A much higher binding affinity of <b>Ru-NAP</b> over <b>NAP</b> toward β-tubulin (<i>K</i>_Ru-NAP: (6.8 ± 0.55) × 10⁶ M^-1; <i>K</i>_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 <b>Rubpy</b> part of <b>Ru-NAP</b>. This was also supported by docking, simulation, and appropriate imaging studies. <b>Ru-NAP</b> promoted serum stability, specific binding of <b>NAP</b> 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 <b>Ru-NAP</b> 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). <b>Ru-NAP</b> also effectively inhibited tumor growth in MDA-MB-231 xenograft models in nude mice. Our findings provide strong evidence that <b>Ru-NAP</b> has a potential therapeutic role in TNBC treatment.