The ruthenium-based anticancer agent BOLD-100/ KP1339 has shown promising results in several in vitro and in vivo tumour models as well as in early clinical trials. However, its mode of action remains to be fully elucidated. Recent evidence identified stress induction in the endoplasmic reticulum (ER) and concomitant down-modulation of HSPA5 (GRP78) as key drug effects. By exploiting the naturally formed adduct between BOLD-100 and human serum albumin as an immobilization strategy, we were able to perform target-profiling experiments that revealed the ribosomal proteins RPL10, RPL24, and the transcription factor GTF2I as potential interactors of this ruthenium(III) anticancer agent. Integrating these findings with proteomic profiling and transcriptomic experiments supported ribosomal disturbance and concomitant induction of ER stress. The formation of polyribosomes and ER swelling of treated cancer cells revealed by TEM validated this finding. Thus, the direct interaction of BOLD-100 with ribosomal proteins seems to accompany ER stress-induction and modulation of GRP78 in cancer cells. The ruthenium complex sodium trans-[tetrachlorido-bis(1Hindazole)ruthenate(III)] (BOLD-100/KP1339, Figure 1) is among the most widely investigated non-platinum metalbased anticancer drugs. [1] It has shown promising anticancer effects in an autochthonous tumour model [2] and its safety has been proven in clinical trials [3] with clinical phase 1b trials under way (NCT04421820). In contrast to the clinically widely administered platinum-based anticancer agents that target DNA, this ruthenium(III) anticancer drug candidate acts on other biomolecules. [4] Recently, the down-modulation of the glucose-regulated protein of 78 kDa (GRP78, HSPA5) combined with endoplasmic reticulum (ER) stress, as well as the induction of reactive oxygen species (ROS) emerged as accepted drug effects in several 2D and 3D tumour models. [5] However, the mechanism of ER-stress induction and possible targets of BOLD-100 remained rather elusive despite intense research efforts. Metalloproteomics is an emerging tool to elucidate potential targets and mechanisms of action for metallodrugs, and also in conjunction with other omics techniques. [1b, 6] Recently, solutions were found to identify and validate protein targets of largely substitution-inert metal-based anticancer agents based on gold [7] and platinum, [8] but also of labile organoruthenium prodrugs. [9] Moreover, metallopro

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.