Time-dependent shotgun proteomics revealed distinct effects of an organoruthenium prodrug and its activation product on colon carcinoma cells

Abstract

Time-dependent shotgun proteomics revealed distinct effects of an organoruthenium prodrug and its activation product on colon carcinoma cellsThe unravelling of target interactions in cancer cells is a crucial process for identifying promising metal-based anticancer agents. Therefore, target profiles were analysed in a time-dependent manner using proteomics techniques by accounting for the hydrolysis kinetics of plecstatin, an organometallic ruthenium(arene) derivative. While the intact prodrug provoked a cytoprotective integrated stress response, which was further verified by gene expression studies, the activated species was responsible for the target engagement with plectin, the previously validated protein target. As featured in:ISSN 1756-591X Activation kinetics of metallo-prodrugs control the types of possible interactions with biomolecules. The intact metallo-prodrug is able to engage with potential targets by purely non-covalent bonding, while the activated metallodrug can form additional coordination bonds. It is hypothesized that the additional coordinative bonding might be favourable with respect to the target selectivity of activated metallodrugs. Thus, a time-dependent shotgun proteomics study was conducted in HCT116 colon carcinoma cells with plecstatins, which are organoruthenium anticancer drug candidates. First, the target selectivity was evaluated in a time-dependent fashion, which accounted for their hydrolysis kinetics. The binding selectivity increased from 50-to 160-fold and the average specificity from 0.72 to 0.86, respectively, from the 2 h to the 4 h target profiling experiment. Target profiling after 19 h did not reveal significant enrichments, possibly due to deactivation of the probe via arene cleavage. Up to 450 interactors were identified in the target profiling experiments. A plecstatin analogue that substituted a hydrogen bond acceptor with a hydrogen bond donor abrogated the target selectivity for plectin in HCT116 whole cell lysates, underlining the necessity of this hydrogen bond acceptor for a strong interaction between plecstatin and plectin. Second, time-dependent response profiling experiments provided evidence that plecstatin-2 induced an integrated stress response (ISR) in HCT116 cell culture. The phosphorylation of eIF2a, a key mediator of the ISR, after 3 h treatment indicated that this perturbation was initiated by the intact plecstatin-2 prodrug, while the effects of plectin-targeting are mediated by activated plecstatin-2. Significance to metallomicsCell-level investigations of metallodrugs by shotgun proteomics are still scarce despite the evident advantages of these methods. We performed time-dependent target profiling experiments with organoruthenium-based plecstatins in cell lysates of HCT116 colon carcinoma cells. The changes in target selectivity over time correlated with the activation status of the metallo-prodrug. The target engagement of the plecstatins was tested by substituting a hydrogen bond acceptor with a hydrogen bond donor, which abrogated the plect...

Interaction of Oxidative Stress and Misfolded Proteins in the Mechanism of Neurodegeneration

“…Importantly, α-synuclein (oligomer)-induced ROS production in salt solution or in cells can be blocked by chelators of copper, iron, or zinc [ 22 , 26 ]. This ROS production is less likely to be produced only by transition metals in the medium, because the same medium was used for monomers and fibrils which did not produce ROS and is possibly connected to some structural changes in the α-synuclein molecule induced by these metals ions, although α-synuclein binds these metals in very small amounts [ 27 , 28 , 29 ].…”

Plecstatin-1 induces an immunogenic cell death signature in colorectal tumour spheroids

“…Previous studies showed the target selectivity of plecstatin-1 towards plectin. 10,21 Therefore, we aimed at better understanding whether and how the treatment with plecstatin-1 would impact biological processes in cancer cells. Our work employed multicellular spheroids to further characterize the response of cancer cells upon treatment with plecstatin-1.…”

“…Generation of ROS was previously observed for plecstatin-1 in cell culture monolayers. 10,21 Together with a mitochondrial stress signature related to the cytoprotective integrated stress response (ISR), these processes culminated in the phosphorylation of eIF2a. 21 Importantly, eIF2a is a master regulator of several stress response pathways, also including endoplasmic reticulum (ER) stress upon increased ROS, production as part of the unfolded protein response (UPR).…”

“…10,21 Together with a mitochondrial stress signature related to the cytoprotective integrated stress response (ISR), these processes culminated in the phosphorylation of eIF2a. 21 Importantly, eIF2a is a master regulator of several stress response pathways, also including endoplasmic reticulum (ER) stress upon increased ROS, production as part of the unfolded protein response (UPR). 64,65 ISR is an integral part of cellular responses to diverse stresses including those affecting the mitochondria.…”

“…10 Moreover, treating HCT116 colon carcinoma cells in monolayers with plecstatin-1 led to the phosphorylation of eukaryotic initiation factor 2-alpha (eIF2a) and down-stream activation of the cytoprotective integrated stress response (ISR), characterized by mitochondrial stress. 21 EIF2a is also involved in the mode of action of NKP-1339 6,8 and a cyclometalated gold(III) N-heterocyclic carbene anticancer drug. 16 Interestingly, eIF2a is a master regulator of cellular stress management.…”