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 beta-tubulin (KRu-NAP: (6.8 +/- 0.55) x 10(6) M-1; K-NAP: (8.2 +/- 1.1) x 10(4) M-1) was observed due to stronger electrostatic interactions between the MT with an average linear charge density of similar to 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 beta(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 ((IC50Ru-NAP)-I-Light[HEK293]: 17.2 +/- 2.5 mu M, compared to (IC50Ru-NAP)-I-Light[MDA-MB-231]: 32.5 +/- 7.8 nM, (IC50Ru-NAP)-I-Dark[HEK293]: > 80 mu M, compared to (IC50Ru-NAP)-I-Dark[MDA-MB-231]: 2.9 +/- 0.5 mu M). 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.