Cyclic Ruthenium-Peptide Conjugates as Integrin-Targeting Phototherapeutic Prodrugs for the Treatment of Brain Tumors.

To investigate the potential of tumor-targeting photoactivated chemotherapy, a chiral ruthenium-based anticancer warhead, Λ/Δ-[Ru(Ph2phen)2(OH2)2]2+, was conjugated to the RGD-containing Ac-MRGDH-NH2 peptide by direct coordination of the M and H residues to the metal. This design afforded two diastereoisomers of a cyclic metallopeptide, Λ-[1]Cl2 and Δ-[1]Cl2. In the dark, the ruthenium-chelating peptide had a triple action. First, it prevented other biomolecules from coordinating with the metal center. Second, its hydrophilicity made [1]Cl2 amphiphilic so that it self-assembled in culture medium into nanoparticles. Third, it acted as a tumor-targeting motif by strongly binding to the integrin (Kd = 0.061 µM for the binding of Λ-[1]Cl2 to αIIbß3), which resulted in the receptor-mediated uptake of the conjugate in vitro. Phototoxicity studies in two-dimensional (2D) monolayers of A549, U87MG, and PC-3 human cancer cell lines and U87MG three-dimensional (3D) tumor spheroids showed that the two isomers of [1]Cl2 were strongly phototoxic, with photoindexes up to 17. Mechanistic studies indicated that such phototoxicity was due to a combination of photodynamic therapy (PDT) and photoactivated chemotherapy (PACT) effects, resulting from both reactive oxygen species generation and peptide photosubstitution. Finally, in vivo studies in a subcutaneous U87MG glioblastoma mice model showed that [1]Cl2 efficiently accumulated in the tumor 12 h after injection, where green light irradiation generated a stronger tumoricidal effect than a nontargeted analogue ruthenium complex [2]Cl2. Considering the absence of systemic toxicity for the treated mice, these results demonstrate the high potential of light-sensitive integrin-targeted ruthenium-based anticancer compounds for the treatment of brain cancer in vivo.

Model-based translation of DNA damage signaling dynamics across cell types.

Interindividual variability in DNA damage response (DDR) dynamics may evoke differences in susceptibility to cancer. However, pathway dynamics are often studied in cell lines as alternative to primary cells, disregarding variability. To compare DDR dynamics in the cell line HepG2 with primary human hepatocytes (PHHs), we developed a HepG2-based computational model that describes the dynamics of DDR regulator p53 and targets MDM2, p21 and BTG2. We used this model to generate simulations of virtual PHHs and compared the results to those for PHH donor samples. Correlations between baseline p53 and p21 or BTG2 mRNA expression in the absence and presence of DNA damage for HepG2-derived virtual samples matched the moderately positive correlations observed for 50 PHH donor samples, but not the negative correlations between p53 and its inhibitor MDM2. Model parameter manipulation that affected p53 or MDM2 dynamics was not sufficient to accurately explain the negative correlation between these genes. Thus, extrapolation from HepG2 to PHH can be done for some DDR elements, yet our analysis also reveals a knowledge gap within p53 pathway regulation, which makes such extrapolation inaccurate for the regulator MDM2. This illustrates the relevance of studying pathway dynamics in addition to gene expression comparisons to allow reliable translation of cellular responses from cell lines to primary cells. Overall, with our approach we show that dynamical modeling can be used to improve our understanding of the sources of interindividual variability of pathway dynamics.

Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes.

Toxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery. Graphical abstract.

Cell viability imaging in tumor spheroids via DNA binding of a ruthenium(II) light-switch complex.

The famous ''light-switch'' ruthenium complex [Ru(bpy)2(dppz)](PF6)2 (1) has been long known for its DNA binding properties in vitro. However, the biological utility of this compound has been hampered by its poor cellular uptake in living cells. Here we report a bioimaging application of 1 as cell viability probe in both 2D cells monolayer and 3D multi-cellular tumor spheroids of various human cancer cell lines (U87, HepG2, A549). When compared to propidium iodide, a routinely used cell viability probe, 1 was found to enhance the staining of dead cells in particular in tumor spheroids. 1 has high photostability, longer Stokes shift, and displays lower cytotoxicity compared to propidium iodide, which is a known carcinogenic. Finally, 1 was also found to displace the classical DNA binding dye Hoechst in dead cells, which makes it a promising dye for time-dependent imaging of dead cells in cell cultures, including multi-cellular tumor spheroids.

A network-based transcriptomic landscape of HepG2 cells uncovering causal gene-cytotoxicity interactions underlying drug-induced liver injury.

Drug-induced liver injury (DILI) remains the main reason for drug development attritions largely due to poor mechanistic understanding. Toxicogenomic to interrogate the mechanism of DILI has been broadly performed. Gene coregulation network-based transcriptome analysis is a bioinformatics approach that potentially contributes to improve mechanistic interpretation of toxicogenomic data. Here we performed an extensive concentration time course response-toxicogenomic study in the HepG2 cell line exposed to 20 DILI compounds, 7 reference compounds for stress response pathways, and 10 agonists for cytokines and growth factor receptors. We performed whole transcriptome targeted RNA sequencing to more than 500 conditions and applied weighted gene coregulated network analysis to the transcriptomics data followed by the identification of gene coregulated networks (modules) that were strongly modulated upon the exposure of DILI compounds. Preservation analysis on the module responses of HepG2 and PHH demonstrated highly preserved adaptive stress response gene coregulated networks. We correlated gene coregulated networks with cell death onset and causal relationships of 67 critical target genes of these modules with the onset of cell death was evaluated using RNA interference screening. We identified GTPBP2, HSPA1B, IRF1, SIRT1, and TSC22D3 as essential modulators of DILI compound-induced cell death. These genes were also induced by DILI compounds in PHH. Altogether, we demonstrate the application of large transcriptome datasets combined with network-based analysis and biological validation to uncover the candidate determinants of DILI.

Oxygen-Dependent Interactions between the Ruthenium Cage and the Photoreleased Inhibitor in NAMPT-Targeted Photoactivated Chemotherapy.

Photoactivated chemotherapy agents form a new branch of physically targeted anticancer agents with potentially lower systemic side effects for patients. On the other hand, limited information exists on the intracellular interactions between the photoreleased metal cage and the photoreleased anticancer inhibitor. In this work, we report a new biological study of the known photoactivated compound Ru-STF31 in the glioblastoma cancer cell line, U87MG. Ru-STF31 targets nicotinamide phosphoribosyltransferase (NAMPT), an enzyme overexpressed in U87MG. Ru-STF31 is activated by red light irradiation and releases two photoproducts: the ruthenium cage and the cytotoxic inhibitor STF31. This study shows that Ru-STF31 can significantly decrease intracellular NAD+ levels in both normoxic (21% O2) and hypoxic (1% O2) U87MG cells. Strikingly, NAD+ depletion by light activation of Ru-STF31 in hypoxic U87MG cells could not be rescued by the addition of extracellular NAD+. Our data suggest an oxygen-dependent active role of the ruthenium photocage released by light activation.

Protectiveness of NAM-based hazard assessment - which testing scope is required?

Hazard assessment (HA) requires toxicity tests to allow deriving protective points of departure (PoDs) for risk assessment irrespective of a compound's mode of action (MoA). The scope of in vitro test batteries (ivTB) thereby necessitated for systemic toxicity is still unclear. We explored the protectiveness regarding systemic toxicity of an ivTB with a scope, which was guided by previous findings from rodent studies, where examining six main targets, including liver and kidney, was sufficient to predict the guideline scope-based PoD with high probability. The ivTB comprises human in vitro models representing liver, kidney, lung and the neuronal system covering transcriptome, mitochondrial dysfunction and neuronal outgrowth. Additionally, 32 CALUX®- and 10 HepG2 BAC-GFP reporters cover a broad range of disturbance mechanisms. Eight compounds were chosen for causing adverse effects such as immunotoxicity or anemia in vivo, i.e., effects not directly covered by assays in the ivTB. PoDs derived from the ivTB and from oral repeated dose studies in rodents were extrapolated to maximum unbound plasma concentrations for comparison. The ivTB-based PoDs were one to five orders of magnitude lower than in vivo PoDs for six of eight compounds, implying that they were protective. The extent of in vitro response varied across test compounds. Especially for hematotoxic substances, the ivTB showed either no response or only cytotoxicity. Assays better capturing this type of hazard would be needed to complement the ivTB. This study highlights the potentially broad applicability of ivTBs for deriving protective PoDs of compounds with unknown MoA.

Animal tests are used to determine which amount of a chemical is toxic ('threshold of toxicity') and which organs are affected. In principle, the threshold can also be derived solely from tests with cultured cells. However, only a limited number of cell types can practically be tested, so one challenge is to determine how many and which types shall be tested. In animal studies, only few organs including liver and kidney are regularly among those most sensitively affected. We explored whether a cell-based test battery representing these sensitive organs and covering important mechanisms of toxicity can be used to derive protective human thresholds. To challenge this approach, eight chemicals were tested that primarily cause effects in organs not directly represented in our test battery. Results provided protective thresholds for most of the investigated compounds and gave indications how to further improve the approach towards a full-fledged replacement for animal tests.

Application of high-throughput transcriptomics for mechanism-based biological read-across of short-chain carboxylic acid analogues of valproic acid.

Chemical read-across is commonly evaluated without specific knowledge of the biological mechanisms leading to observed adverse outcomes in vivo. Integrating data that indicate shared modes of action in humans will strengthen read-across cases. Here we studied transcriptomic responses of primary human hepatocytes (PHH) to a large panel of carboxylic acids to include detailed mode-of-action data as a proof-of-concept for read-across in risk assessment. In rodents, some carboxylic acids, including valproic acid (VPA), are known to cause hepatic steatosis, whereas others do not. We investigated transcriptomics responses of PHHs exposed for 24 h to 18 structurally different VPA analogues in a concentration range to determine biological similarity in relation to in vivo steatotic potential. Using a targeted high-throughput screening assay, we assessed the differential expression of ~3,000 genes covering relevant biological pathways. Differentially expressed gene analysis revealed differences in potency of carboxylic acids, and expression patterns were highly similar for structurally similar compounds. Strong clustering occurred for steatosis-positive versus steatosis-negative carboxylic acids. To quantitatively define biological read-across, we combined pathway analysis and weighted gene co-expression network analysis. Active carboxylic acids displayed high similarity in gene network modulation. Importantly, free fatty acid synthesis modulation and stress pathway responses are affected by active car­boxylic acids, providing coherent mechanistic underpinning for our findings. Our work shows that transcriptomic analysis of cultured human hepatocytes can reinforce the prediction of liver injury outcome based on quantitative and mechanistic biological data and support its application in read-across.

Dynamic modeling of Nrf2 pathway activation in liver cells after toxicant exposure.

Cells are exposed to oxidative stress and reactive metabolites every day. The Nrf2 signaling pathway responds to oxidative stress by upregulation of antioxidants like glutathione (GSH) to compensate the stress insult and re-establish homeostasis. Although mechanisms describing the interaction between the key pathway constituents Nrf2, Keap1 and p62 are widely reviewed and discussed in literature, quantitative dynamic models bringing together these mechanisms with time-resolved data are limited. Here, we present an ordinary differential equation (ODE) based dynamic model to describe the dynamic response of Nrf2, Keap1, Srxn1 and GSH to oxidative stress caused by the soft-electrophile diethyl maleate (DEM). The time-resolved data obtained by single-cell confocal microscopy of green fluorescent protein (GFP) reporters and qPCR of the Nrf2 pathway components complemented with siRNA knock down experiments, is accurately described by the calibrated mathematical model. We show that the quantitative model can describe the activation of the Nrf2 pathway by compounds with a different mechanism of activation, including drugs which are known for their ability to cause drug induced liver-injury (DILI) i.e., diclofenac (DCF) and omeprazole (OMZ). Finally, we show that our model can reveal differences in the processes leading to altered activation dynamics amongst DILI inducing drugs.

Development of a Retinal-Based Probe for the Profiling of Retinaldehyde Dehydrogenases in Cancer Cells.

Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts.