Riccardo Rubbiani

Benzimidazol-2-ylidene gold(I) complexes are thioredoxin reductase inhibitors with multiple antitumor properties.

Gold(I) complexes such as auranofin have been used for decades to treat symptoms of rheumatoid arthritis and have also demonstrated a considerable potential as new anticancer drugs. The enzyme thioredoxin reductase (TrxR) is considered as the most relevant molecular target for these species. The here investigated gold(I) complexes with benzimidazole derived N-heterocyclic carbene (NHC) ligands represent a promising class of gold coordination compounds with a good stability against the thiol glutathione. TrxR was selectively inhibited by in comparison to the closely related enzyme glutathione reductase, and all complexes triggered significant antiproliferative effects in cultured tumor cells. More detailed studies on a selected complex revealed a distinct pharmacodynamic profile including the high increase of reactive oxygen species formation, apoptosis induction, strong effects on cellular metabolism (related to cell surface properties, respiration, and glycolysis), inhibition of mitochondrial respiration and activity against resistant cell lines.

Comparative in vitro evaluation of N-heterocyclic carbene gold(I) complexes of the benzimidazolylidene type.

Gold(I) complexes with a 1,3-diethylbenzimidazol-2-ylidene N-heterocyclic carbene (NHC) ligand of the type NHC-Au-L (L=-Cl, -NHC, or -PPh3) were comparatively evaluated as thioredoxin reductase (TrxR) inhibitors and antimitochondrial anticancer agents. Different effects were noted in various biochemical assays (e.g., inhibition of TrxR, cellular and mitochondrial uptake, or effects on mitochondrial membrane potential), and this was related to properties of the complexes such as bond dissociation energies and overall charge. Remarkable antiproliferative effects, a strong induction of apoptosis, and enhancement of reactive oxygen species (ROS) formation as well as other effects on tumor cell metabolism confirmed the promising potential of the complexes as novel anticancer chemotherapeutics.

Visible-Light-Induced Annihilation of Tumor Cells with Platinum–Porphyrin Conjugates†

Despite the extensive use of porphyrins in photodynamic therapy (PDT), tetraplatinated porphyrins have so far not been studied for their anticancer properties. Herein, we report the synthesis of such novel platinum-porphyrin conjugates as well as their photophysical characterization and in vitro light-induced anticancer properties. These conjugates showed only minor cytotoxicity in the dark, but IC50 values down to 19 nM upon irradiation with light at 420 nm.These values correspond to an excellent phototoxic index (PI=IC50 in the dark/IC50 in light), which reached 5000 in a cisplatin-resistant cell line. After incubation with HeLa cells, nuclear Pt concentrations were 30 times higher than with cisplatin. All of these favorable characteristics imply that tetraplatinated porphyrin complexes are worthy of exploration as novel PDT anticancer agents in vivo.

A spontaneous gold(I)-azide alkyne cycloaddition reaction yields gold-peptide bioconjugates which overcome cisplatin resistance in a p53-mutant cancer cell line

Solid-phase peptide synthesis (SPPS) is a versatile technique for the assembly of small to medium size peptides, that can help in the delivery of bound metal complexes to certain cellular compartments, for example in cancer cells. This work shows a new route to gold-peptide bioconjugates via a non-catalyzed [3 + 2] cycloaddition reaction of gold azides with alkynyl peptides. Gold(I) tetrapeptide conjugates with a mitochondria-targeting sequence were synthesized and display prolonged stability in the presence of thiol-containing biological media. Their antiproliferative potency against selected cancer cells (2–50 μM) corresponds to the lipophilicity of the conjugates. The cellular uptake of Au, determined by atomic absorption spectroscopy (AAS), shows that high initial uptake equals strong cytotoxicity. Respiration and acidification rates react immediately upon treatment with the Au-peptide conjugates, and a terminal breakdown of essential cellular functions is complete within ca. 12 h at most, as observed by online monitoring of the cancer cell metabolism in a microfluidic biosensor device (Bionas sensorchip system). The mode of action of these Au-peptide bioconjugates was elucidated by a variety of biochemical and cell biological experiments. First, a strong selective inhibition of the enzyme thioredoxin reductase (TrxR), a regulator of cellular redox processes, was found. In this context, elevated levels of reactive oxygen species (ROS) and strong effects on the respiration of isolated mouse liver mitochondria were found. These finally lead to cell death via apoptotic pathways, as indicated by flow cytometry, low mitochondrial membrane potential (MMP) and DNA fragmentation. Intriguingly, cisplatin-resistance in p53-mutant MDA-MB231 breast cancer cells could be overcome by the Au-peptide conjugates presented herein.

Photo-induced uncaging of a specific Re(I) organometallic complex in living cells

In the last decades, a large number of organometallic complexes have shown promising anti-proliferative activity towards different cancer cell lines. However, these compounds generally had low cellular uptake and low selectivity towards cancer cells over healthy cells. The use of external triggers (e.g. light, ultra-sound, temperature, etc.) to modify the cytotoxic effect of a prodrug and the coupling of a targeting vector (e.g. peptides, antibodies, etc.) to a drug were found to be very successful techniques to tackle these drawbacks. Here, we envisioned combining these two methods, namely an external trigger (i.e. light activation) and a targeting vector, in an organometallic compound. More specifically, a Re(I) tricarbonyl N,N-bis(quinolinoyl) complex (Re-NH2) was derivatised with a photo-labile protecting group (PLPG) to cage Re-NH2 by formation of Re-PLPG. For organelle/cellular specificity, Re-PLPG was then further coupled to a nuclear localization sequence (NLS) or a bombesin peptide derivative to give Re-PLPG-NLS or Re-PLPG-Bombesin, respectively. Photolysis experiments in PBS buffer (pH 7.4) demonstrated that Re-NH2 was completely photo-released from Re-PLPG-NLS and Re-PLPG-Bombesin using a very low irradiation dose (1.2 J cm−2). To the best of our knowledge, these are the first two examples of the selective photo-release of an intact organometallic compound from a bioconjugate. Of high interest, both derivatives showed toxicity comparable to that of cisplatin towards cervical cancer cells (HeLa) upon light irradiation, although the phototoxic index (PTI) varied greatly with the targeting peptide. The cell death mechanism of Re-PLPG-NLS was explored using different techniques, including fluorescence microscopy, ICP-MS, gel electrophoresis, flow cytometry and transmission electron microscopy (TEM). It could be demonstrated that HeLa cells treated with Re-PLPG-NLS in the dark and upon irradiation showed severe cell stress (nucleolar segregation, pyknosis and vacuolation). The data obtained from an Annexin V/propidium iodide (PI) assay indicated that, after an early apoptotic stage, the onset induced by Re-PLPG-NLS led to cell death, with features ascribable to late apoptosis and necrosis, which were more marked for the treatment involving irradiation.

TrxR inhibition and antiproliferative activities of structurally diverse gold N-heterocyclic carbene complexes

Gold compounds with N-heterocyclic carbene (NHC) ligands have been widely described as potent thioredoxin reductase (TrxR) inhibitors and effective anticancer agents. However, despite these promising aspects structure–activity-relationship (SAR) studies still remain limited. In this study a structurally diverse library of gold(I) and gold(III) NHC complexes was investigated for inhibitory capacity against TrxR and for antiproliferative activity in HT-29 human colon adenocarcinoma cells with the aim of identifying a valid SAR. Overall results indicated that the bioactivity, carried by the gold center, is intimately linked to the chemical properties of the residues at the NHC scaffold as well as other ligands coordinated to the gold atom. Although a direct correlation between IC50 values for cytotoxicity and enzyme inhibition could not be established, the inhibition of TrxR represents an important parameter to achieve a good cytotoxic activity.

Gold(I) complexes of water-soluble diphos-type ligands: Synthesis, anticancer activity, apoptosis and thioredoxin reductase inhibition

Gold(I) complexes of imidazole and thiazole-based diphos type ligands were prepared and their potential as chemotherapeutics investigated. Depending on the ligands employed and the reaction conditions complexes [L(AuCl)(2)] and [L(2)Au]X (X = Cl, PF(6)) are obtained. The ligands used are diphosphanes with azoyl substituents R(2)P(CH(2))(2)PR(2) {R = 1-methylimidazol-2-yl (1), 1-methylbenzimidazol-2-yl (4), thiazol-2-yl (5) and benzthiazol-2-yl (6)} as well as the novel ligands RPhP(CH(2))(2)PRPh {R = 1-methylimidazol-2-yl (3)} and R(2)P(CH(2))(3)PR(2) {R = 1-methylimidazol-2-yl (2)}. The cytotoxic activity of the complexes was assessed against three human cancer cell lines and a rat hepatoma cell line and correlated to the lipophilicity of the compounds. The tetrahedral gold complexes [(3)(2)Au]PF(6) and [(5)(2)Au]PF(6) with intermediate lipophilicity (logD(7.4) = 0.21 and 0.25) showed significant cytotoxic activity in different cell lines. Both compounds induce apoptosis and inhibit the enzymes thioredoxin reductase and glutathione reductase.

A TrxR inhibiting gold(I) NHC complex induces apoptosis through ASK1-p38-MAPK signaling in pancreatic cancer cells

BackgroundCancer cells in the advanced stage show aberrant antioxidant capacity to detoxify excessive ROS resulting in the compensation for intrinsic oxidative stress and therapeutic resistance. PDAC is one of the most lethal cancers and often associated with a high accumulation of ROS. Recent studies identified gold(I) NHC complexes as potent TrxR inhibitors suppressing cell growth in a wide spectrum of human malignant cell lines at the low micromolar concentration. However, the mechanism of action is not completely elucidated yet.MethodsTo understand the biological function of gold(I) NHC complexes in PDAC, we used a recently published gold(I) NHC complex, MC3, and evaluated its anti-proliferative effect in four PDAC cell lines, determined by MTT and SRB assays. In further detailed analysis, we analyzed cellular ROS levels using the ROS indicator DHE and mitochondrial membrane potential indicated by the dye JC-1 in Panc1. We also analyzed cell cycle arrest and apoptosis by FACS. To elucidate the role of specific cell signaling pathways in MC3-induced cell death, co-incubation with ROS scavengers, a p38-MAPK inhibitor and siRNA mediated depletion of ASK1 were performed, and results were analyzed by immunoblotting, ELISA-microarrays, qRT-PCR and immunoprecipitation.ResultsOur data demonstrate that MC3 efficiently suppressed cell growth, and induced cell cycle arrest and apoptosis in pancreatic cancer cells, in particular in the gemcitabine-resistant cancer cells Panc1 and ASPC1. Treatment with MC3 resulted in a substantial alteration of the cellular redox homeostasis leading to increased ROS levels and a decrease in the mitochondrial membrane potential. ROS scavengers suppressed ROS formation and rescued cells from damage. On the molecular level, MC3 blocked the interaction of Trx with ASK1 and subsequently activated p38-associated signaling. Furthermore, inhibition of this pathway by using ASK1 siRNA or a p38 inhibitor clearly attenuated the effect of MC3 on cell proliferation in Panc1 and ASPC1.ConclusionsOur results confirm that MC3 is a TrxR inhibitor and show MC3 induced apoptosis in gemcitabine-resistant PDACs. MC3 mediated cell death could be blocked by using anti-oxidants, ASK1 siRNA or p38 inhibitor suggesting that the Trx-ASK1-p38 signal cascade played an important role in gold(I) NHC complexes-mediated cellular damage.

Organometallic Rhenium Complexes Divert Doxorubicin to the Mitochondria.

Doxorubicin, a well-established chemotherapeutic agent, is known to accumulate in the cell nucleus. By using ICP-MS, we show that the conjugation of two small organometallic rhenium complexes to this structural motif results in a significant redirection of the conjugates from the nucleus to the mitochondria. Despite this relocation, the two bioconjugates display excellent toxicity toward HeLa cells. In addition, we carried out a preliminarily investigation of aspects of cytotoxicity and present evidence that the conjugates disrupt the mitochondrial membrane potential, are strong inhibitors of human Topoisomerase II, and induce apoptosis. Such derivatives may enhance the therapeutic index of the aggressive parent drug and overcome drug resistance by influencing nuclear and mitochondrial homeostasis.

Cytotoxic Gold(I) N-heterocyclic Carbene Complexes with Phosphane Ligands as Potent Enzyme Inhibitors

Organometallic gold complexes with N‐heterocyclic carbene (NHC) ligands have been demonstrating promising properties as novel anticancer agents. Gold(I) NHC complexes containing different phosphanes as secondary ligands were shown to trigger strong cytotoxic effects in cancer cells, and their effective uptake into the cells was quantified by atomic absorption spectroscopy. Moreover, the new compounds strongly inhibited the activity of the seleno‐enzyme thioredoxin reductase (TrxR) and of the zinc‐finger enzyme poly(ADP‐ribose) polymerase 1 (PARP‐1). In the case of TrxR inhibition, their activity depended clearly on the size of the alkyl/aryl residues of phosphorus atoms. Density functional theory (DFT) calculations showed that the AuP bond of the triphenylphosphane complex [AuI(NHC)(PPh3)]I had a lower bond dissociation energy compared to trialkylphosphane complexes [AuI(NHC)(PR3)]I, indicating a higher kinetic reactivity of this particular compound. In fact, [AuI(NHC)(PPh3)]I triggered an enhanced inhibitory activity against PARP‐1.