Microtubule-Targeting\nNAP Peptide-Ru(II)-polypyridyl\nConjugate As a Bimodal Therapeutic Agent for Triple Negative Breast\nCarcinoma

Triple-negative breast cancer (TNBC) poses significant\ntreatment\nchallenges due to its high metastasis, heterogeneity, and poor biomarker\nexpression. The N-terminus of an octapeptide NAPVSIPQ (<b>NAP</b>) was covalently coupled to a carboxylic acid derivative of Ru(2,2′-bipy)<sub>3</sub><sup>2+</sup> (<b>Rubpy</b>) to synthesize an N-stapled\nshort peptide-Rubpy conjugate (<b>Ru-NAP</b>). This photosensitizer\n(PS) was utilized to treat TNBC through microtubule (MT) targeted\nchemotherapy and photodynamic therapy (PDT). <b>Ru-NAP</b> formed\nmore elaborate molecular aggregates with fibrillar morphology as compared\nto <b>NAP</b>. A much higher binding affinity of <b>Ru-NAP</b> over <b>NAP</b> toward β-tubulin (<i>K</i><sub>Ru‑NAP</sub>: (6.8 ± 0.55) × 10<sup>6</sup> M<sup>–1</sup>; <i>K</i><sub>NAP</sub>: (8.2 ±\n1.1) × 10<sup>4</sup> M<sup>–1</sup>) was observed due\nto stronger electrostatic interactions between the MT with an average\nlinear charge density of ∼85 e/nm and the cationic <b>Rubpy</b> part of <b>Ru-NAP</b>. This was also supported by docking,\nsimulation, and appropriate imaging studies. <b>Ru-NAP</b> promoted\nserum stability, specific binding of <b>NAP</b> to the E-site\nof the β<sup>III</sup>-tubulin followed by the disruption of\nthe MT network, and effective singlet oxygen generation in TNBC cells\n(MDA-MB-231), causing cell cycle arrest in the G2/M phase and triggering\napoptosis. Remarkably, MDA-MB-231 cells were more sensitive to <b>Ru-NAP</b> compared to noncancerous human embryonic kidney (HEK293\ncells) when exposed to light (<sup>Light</sup>IC<sub>50</sub><sup>Ru‑NAP</sup>[HEK293]: 17.2 ± 2.5 μM, compared to <sup>Light</sup>IC<sub>50</sub><sup>Ru‑NAP</sup>[MDA-MB-231]: 32.5\n± 7.8 nM, <sup>Dark</sup>IC<sub>50</sub><sup>Ru‑NAP</sup>[HEK293]: > 80 μM, compared to <sup>Dark</sup>IC<sub>50</sub><sup>Ru‑NAP</sup>[MDA-MB-231]: 2.9 ± 0.5 μM). <b>Ru-NAP</b> also effectively inhibited tumor growth in MDA-MB-231\nxenograft models in nude mice. Our findings provide strong evidence\nthat <b>Ru-NAP</b> has a potential therapeutic role in TNBC\ntreatment.