Selectivity for TP53 signalling drives the mode of action of a highly potent <i>N</i> , <i>O</i> , <i>O</i> -tridentate naphthoquinone-based organo-ruthenium anticancer drug candidate

The metallodrug candidate [(3-ethyl-4-oxo-(pyrazolyl)-dihydronaphthalene)(cymene)ruthenium(ii)] (1a) was recently shown to exhibit exceptional antiproliferative activity towards the chemo-resistant SW480 cancer cell line with nanomolar potency. This study was conducted to elucidate the determining parameters of the mode of action of this <i>N</i>,<i>O</i>,<i>O</i>-tridentate organoruthenium compound <i>in vitro</i> and <i>in vivo</i>. Four metal(arenes) based on 3-ethyl naphthoquinone (3-Et-NQ, a) and 3-morpholine naphthoquinone (3-Morph-NQ, b) with ruthenium (1) and osmium (2) were synthesized and characterized. The 3-Morph-NQ ligand increased the solubility of the complexes, but showed a 30-fold reduction in antiproliferative activity compared to the 3-Et-NQ ligand and its complexes served as biologically inactive analogues. The solution reactivity of the four compounds was ligand- and metal-dependent, but they all showed selectivity for amino acids over nucleotides at biologically relevant concentrations. Drug effects were elucidated by proteome profiling at subcellular resolution and showed a pronounced ligand-dependent impact. The 3-Et-NQ containing ruthenium- and osmium(arenes) down-regulated TP53 as a central hub in the perturbation network, connected to down-regulated proliferative MAPK3 signalling. Complex 1a strongly down-regulated TP53 and potently inhibited cell cycle progression at the G2/M phase. Furthermore, 1a was found to disrupt the TP53-DDX3X-p21 signalling axis by direct interaction with DDX3X and loss of p21 expression. The 3-Et-NQ complexes, particularly 1a, showed tumour inhibitory effects <i>in vivo</i> in a CT26 colon carcinoma mouse model, while the 3-Morph-NQ complexes were inactive. Tissue proteome analyses of livers of 1a-treated mice displayed similar stress responses as observed <i>in vitro</i>. Finally, tumour tissue of 1a-treated mice revealed down-regulated EGFR, consistent with the impact on the TP53 signalling axis <i>in vitro</i>.