Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.

TRPV6 is a calcium-selective ion channel implicated in epithelial Ca2+ uptake. TRPV6 inhibitors are needed for the treatment of a broad range of diseases associated with disturbed calcium homeostasis, including cancers. Here we combine cryo-EM, calcium imaging, and mutagenesis to explore molecular bases of human TRPV6 inhibition by the antifungal drug econazole and the universal ion channel blocker ruthenium red (RR). Econazole binds to an allosteric site at the channel's periphery, where it replaces a lipid. In contrast, RR inhibits TRPV6 by binding in the middle of the ion channel's selectivity filter and plugging its pore like a bottle cork. Despite different binding site locations, both inhibitors induce similar conformational changes in the channel resulting in closure of the gate formed by S6 helices bundle crossing. The uncovered molecular mechanisms of TRPV6 inhibition can guide the design of a new generation of clinically useful inhibitors.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with cardinal features of cognitive dysfunction in an individual. Recently, the blockade of mitochondrial calcium uniporter (MCU) exhibits neuroprotective activity in experimental animals. However, the therapeutic potential of MCU has not yet been established in the management of AD. Therefore, the present study explored the therapeutic potential of either Ruthenium red (RR), a MCU blocker, or Spermine, a MCU opener, on the extent of mitochondrial calcium accumulation, function, integrity and bioenergetics in hippocampus, pre-frontal cortex and amygdale of ICV-STZ challenged rats. Experimental AD was induced in male rats by intracerebroventricular injection of streptozotocin (ICV-STZ) on day-1 (D-1) of the experimental protocol at a sub-diabetogenic dose (3 mg/kg) twice at an interval of 48 h into both rat lateral ventricles. RR attenuated ICV-STZ-induced memory-related behavioral abnormalities in Morris water maze and Y-maze tests. RR also attenuated ICV-STZ-induced decrease in the level of acetylcholine and activity of choline acetyltransferase and, increase in the activity of acetylcholinestarase in memory-sensitive rat brain regions. Further, RR attenuated mitochondrial toxicity in terms of reducing mitochondrial calcium accumulation and improving the mitochondrial function, integrity and bioenergetics in memory-sensitive brain regions of ICV-STZ challenged rats. Furthermore, RR attenuated the percentage of apoptotic cells in ICV-STZ challenged rat brain regions. However, Spermine did not alter ICV-STZ-induced behavioral, biochemical and molecular observations in any of the brain regions. These observations indicate the fact that the MCU blockage could be a potential therapeutic option in the management of sporadic type of AD.

Calcium homeostasis modulator 1 (CALHM1) is a voltage-dependent channel involved in neuromodulation and gustatory signaling. Despite recent progress in the structural biology of CALHM1, insights into functional regulation, pore architecture, and channel blockade remain limited. Here we present the cryo-EM structure of human CALHM1, revealing an octameric assembly pattern similar to the non-mammalian CALHM1s and the lipid-binding pocket conserved across species. We demonstrate by MD simulations that this pocket preferentially binds a phospholipid over cholesterol to stabilize its structure and regulate the channel activities. Finally, we show that residues in the amino-terminal helix form the channel pore that ruthenium red binds and blocks.

Pharmacological separation of the background potassium currents of closely related K2P channels is a challenging problem. We previously demonstrated that ruthenium red (RR) inhibits TASK-3 (K2 P 9.1), but not TASK-1 (K2 P 3.1) channels. RR has been extensively used to distinguish between TASK currents in native cells. In the present study, we systematically investigate the RR sensitivity of a more comprehensive set of K2 P channels.

1. The effect of ruthenium red on calcium and sodium currents was studied in guinea-pig isolated ventricular heart cells with the whole cell patch-clamp technique. 2. Ruthenium red very efficiently blocked the L-type calcium current in a dose-dependent manner. A significant block was observed for concentrations as low as 0.3 microM. Analysis of the dose-response curve with the logistic equation indicated an EC50 of 0.8 microM, a maximum inhibition of 85% reached at 5 microM, and a coefficient of 2.37. 3. There was no shift in the voltage-dependence of the Ca current activation, nor in that of its steady-state inactivation determined with a 1 s prepulse. However, removal of Ca current inactivation at positive voltage was considerably reduced in the presence of concentrations of ruthenium red above 1 microM. A slowing of the time-course of inactivation of the Ca current was also observed. 4. At 10 microM, a concentration generally used to block the sarcoplasmic Ca release channels or the mitochondrial Ca uptake, ruthenium red blocked 26.7+/-4.3% (n=8) of the sodium current, and slowed its inactivation time-course. No effect was observed on the voltage-dependence of the current activation or inactivation. The peak sodium current was also decreased at a 10 times lower concentration by 7.6+/-2.7% (n=3). 5. Thus, at concentrations used to assess intracellular Ca movements, ruthenium red induced in heart cells a significant block of both Ca and Na channels.

Sponges (phylum Porifera) are highly specialized filter-feeding metazoans, pumping and filtering water with a network of canals and chambers, the aquiferous system. Most sponges have a leuconoid aquiferous system, characterized by choanocytes organized in small spherical chambers connected with ambient water by a complex net of canals. Such organization requires substantial pressure difference to drive water through an elaborate system of canals, so the choanocytes in leuconoid sponges have several structural features to generate pressure difference. In contrast, it is generally accepted that asconoid and syconoid sponges with long choanocyte tubes or large choanocyte chambers have no similar structures in their choanocytes. The present study is devoted to the detailed ultrastructural analysis of the choanocytes and their filter apparatus in the asconoid calcareous sponge Leucosolenia variabilis. The general structure of L. variabilis choanocytes is similar to that described for other sponge species. However, the fixation with 0.1% ruthenium red allowed us to reveal for the first time a complex of glycocalyx structures (vanes on the flagella, a fine glycocalyx sealing microvilli in the collar, and a glycocalyx strainer, embedding the apical parts of neighboring collars) in the choanocytes of L. variabilis, which are traditionally associated with the pumping and filtration process in leuconoid demosponges. All revealed glycocalyx structures have dimensions and locations similar to those found in the choanocyte chambers of some demosponges. The data suggest that L. variabilis utilizes the principles of water pumping and filtration similar to those in demosponges and revealed glycocalyx structures are potentially crucial for these processes. It seems that sponges from distant phylogenetic lineages and with different body plans rely on common principles of choanoderm organization for effective pumping and filtration of water. However, while some adaptation for effective pumping and filtration of water have possibly arisen before the diversification of Porifera, others have appeared independently in different lineages.

Ruthenium red (RR) inhibits calcium (Ca2+) entry from the cytoplasm to the mitochondria, and is involved in maintenance of Ca2+ homeostasis in mammalian cells. Ca2+ homeostasis is very important for further embryonic development of fertilized oocytes. However, the effect of RR on mitochondria-Ca2+ (mito-Ca2+) levels during in vitro fertilization (IVF) on subsequent blastocyst developmental capacity in porcine is unclear. The present study explored the regulation of mito-Ca2+ levels using RR and/or histamine in fertilized oocytes and their influence on blastocyst developmental capacity in pigs. Red fluorescence intensity by the mito-Ca2+ detection dye Rhod-2 was significantly increased (P < 0.05) in zygotes 6 h after IVF compared to mature oocytes. Based on these results, we investigated the changes in mito-Ca2+ by RR (10 and 20 μM) in presumptive zygotes using Rhod-2 staining and mito-Ca2+ uptake 1 (MICU1) protein levels as an indicator of mito-Ca2+ uptake using western blot analysis. As expected, RR-treated zygotes displayed decreased protein levels of MICU1 and Rhod-2 red fluorescence intensity compared to non-treated zygotes 6 h after IVF. Blastocyst development rate of 20 μM RR-treated zygotes was significantly increased 6 h after IVF (P < 0.05) due to improved mitochondrial functions. Conversely, the blastocyst development rate was significantly decreased in histamine (mito-Ca2+ inhibitor, 100 nM) treated zygotes (P < 0.05). The collective results demonstrate that RR improves blastocyst development in porcine embryos by regulating mito-Ca2+ and MICU1 expression following IVF.

Osteoporosis is a disorder of bone metabolism that is extremely common in elderly patients as well as in postmenopausal women. The main manifestation is that the bone resorption capacity is greater than the bone formation capacity, which eventually leads to a decrease in bone mass, increasing the risk of fracture. There is growing evidence that inhibiting osteoclast formation and resorption ability can be effective in treating and preventing the occurrence of osteoporosis. Our study is the first time to explore the role of the mitochondrial calcium uniporter (MCU) and its inhibitor ruthenium red (RR) in bone metabolism, clarifying the specific mechanism by which it inhibits osteoclast formation in vitro and plays a therapeutic role in osteoporosis in vivo. We verified the suppressive effects of RR on the receptor activator of nuclear factor-κB ligand (RANKL-)-induced differentiation and bone resorption function of osteoclasts in vitro. The reactive oxygen species (ROS) production stimulated by RANKL and the expression level of P38 MAPK/NFATc1 were also found to be inhibited by RR. Moreover, the promotion of RR on osteogenesis differentiation was investigated by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and the detection of osteogenesis-specific gene expression levels by quantitative polymerase chain reaction (qPCR) and western blotting. Moreover, in ovariectomy (OVX-)-induced osteoporosis models, RR can downregulate the expression and function of the MCU, relieving bone loss and promoting osteogenesis to present a therapeutic effect on osteoporosis. This new finding will provide an important direction for the study of RR and MCU in the study of bone metabolism therapy targets.

Synthesis, characterization, electrochemistry, and photophysics of homo- and heteroleptic ruthenium(II) complexes [Ru(cpmp)2 ]2+ (22+ ) and [Ru(cpmp)(ddpd)]2+ (32+ ) bearing the tridentate ligands 6,2''-carboxypyridyl-2,2'-methylamine-pyridyl-pyridine (cpmp) and N,N'-dimethyl-N,N'-dipyridin-2-ylpyridine-2,6-diamine (ddpd) are reported. The complexes possess one (32+ ) or two (22+ ) electron-deficient dipyridyl ketone fragments as electron-accepting sites enabling intraligand charge transfer (ILCT), ligand-to-ligand charge transfer (LL'CT) and low-energy metal-to-ligand charge transfer (MLCT) absorptions. The latter peak around 544 nm (green light). Complex 22+ shows 3 MLCT phosphorescence in the red to near-infrared spectral region at room temperature in deaerated acetonitrile solution with an emission quantum yield of 1.3 % and a 3 MLCT lifetime of 477 ns, whereas 32+ is much less luminescent. This different behavior is ascribed to the energy gap law and the shape of the parasitic excited 3 MC state potential energy surface. This study highlights the importance of the excited-state energies and geometries for the actual excited-state dynamics. Aromatic and aliphatic amines reductively quench the excited state of 22+ paving the way to photocatalytic applications using low-energy green light as exemplified with the green-light-sensitized thiol-ene click reaction.

The trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the "Keystone inhibitor site" under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR "super-responders" having nanomolar sensitivity. Together, the data define a "finger in the dam" inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.

The number of cancer cases continues to increase worldwide, and unfortunately the main systemic treatments available have numerous of side effects. Ruthenium complexes have shown to be promising chemotherapeutic agents, since they present low toxicity and are more selective for tumor tissues. We report the synthesis, characterization and biological properties of two new ruthenium (II) complexes containing Lapachol and Lawsone as ligands: (1) [Ru(Law)(dppb)(phen)]PF6 and (2) [Ru(Lap)(dppb)(phen)]PF6, where Law = Lawsone, Lap = Lapachol, dppb = 1,4-bis(diphenylphosphine)butane and phen = 1,10-phenanthroline. The ability of the complexes (1) and (2) to interact with CT-DNA (Calf Thymus) was investigated, and the results indicate that the complexes have shown a weak interaction with this macromolecule. Complexes (1) and (2) showed a moderate interaction with BSA, via a spontaneous process with the involvement of van der Waals and hydrogen bond interactions. Both complexes were tested against human lung cancer cell lines, chronic human myeloid leukemia, murine melanoma and human cervical and non-tumoral murine fibroblast adenocarcinoma, human lung fibroblasts and monkey kidney epithelia. The potential for cytotoxicity was tested out using the MTT assay and the neutral red test, to calculate inhibitory concentrations (IC50) and selectivity indices (IS). Both complexes showed a higher selectivity index of 1.17 and 10.91, respectively, for the HeLa tumor line. Studies of toxicological evaluation, using the micronucleus test and the comet assay against non-tumor cells, as well as an assessment of the potential for acute toxicity and neurotoxicity in zebrafish (Danio rerio). In the in vitro micronucleus test, complex (1) showed the least genotoxic potential, and in the in vitro comet assay both compounds had revealed a genotoxic potential at 0.5 and 1.0 mg L-1, with no difference between 24 h and 48 h exposure times. In the acute toxicity tests on zebrafish embryos, complex (1) showed sublethal effects such as decreased blood circulation and heartbeat rate, which were less pronounced than with complex (2). In contrast to complex 2, which caused lethality even before 48h, complex (1) did not cause the death of the embryos at concentrations up to (2.0 mg L-1). Complex (2) also lead to a delay in the embryo. Cell based in vitro methods thus proved able to provide specific toxicological data, allowing a significant reduction in ∖animal experimentation. Given that in vitro tests cannot completely replace animal tests, the use of less advanced developmental stages such as zebrafish embryos, which - at least in the European Union - are not regarded protected, could be shown to be an excellent alternative for testing with, e.g., mammals.

Ruthenium nitrosyl complexes of the general formulas (cation)(+)[cis-RuCl4(NO)(Hazole)](-), where (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (Hind) (1c), (cation)(+) = (H2pz)(+), Hazole = 1H-pyrazole (Hpz) (2c), (cation)(+) = (H2bzim)(+), Hazole = 1H-benzimidazole (Hbzim) (3c), (cation)(+) = (H2im)(+), Hazole = 1H-imidazole (Him) (4c) and (cation)(+)[trans-RuCl4(NO)(Hazole)](-), where (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (1t), (cation)(+) = (H2pz)(+), Hazole = 1H-pyrazole (2t), as well as osmium analogues of the general formulas (cation)(+)[cis-OsCl4(NO)(Hazole)](-), where (cation)(+) = (n-Bu4N)(+), Hazole =1H-indazole (5c), 1H-pyrazole (6c), 1H-benzimidazole (7c), 1H-imidazole (8c), (cation)(+) = Na(+); Hazole =1H-indazole (9c), 1H-benzimidazole (10c), (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (11c), (cation)(+) = H2pz(+), Hazole = 1H-pyrazole (12c), (cation)(+) = (H2im)(+), Hazole = 1H-imidazole (13c), and (cation)(+)[trans-OsCl4(NO)(Hazole)](-), where (cation)(+) = n-Bu4N(+), Hazole = 1H-indazole (5t), 1H-pyrazole (6t), (cation)(+) = Na(+), Hazole = 1H-indazole (9t), (cation)(+) = (H2ind)(+), Hazole = 1H-indazole (11t), (cation)(+) = (H2pz)(+), Hazole = 1H-pyrazole (12t), have been synthesized. The compounds have been comprehensively characterized by elemental analysis, ESI mass spectrometry, spectroscopic techniques (IR, UV-vis, 1D and 2D NMR) and X-ray crystallography (1c·CHCl3, 1t·CHCl3, 2t, 3c, 6c, 6t, 8c). The antiproliferative activity of water-soluble compounds (1c, 1t, 3c, 4c and 9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c) in the human cancer cell lines A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma) has been assayed. The effects of metal (Ru vs Os), cis/trans isomerism, and azole heterocycle identity on cytotoxic potency and cell line selectivity have been elucidated. Ruthenium complexes (1c, 1t, 3c, and 4c) yielded IC50 values in the low micromolar concentration range. In contrast to most pairs of analogous ruthenium and osmium complexes known, they turned out to be considerably more cytotoxic than chemically related osmium complexes (9c, 9t, 10c, 11c, 11t, 12c, 12t, 13c). The IC50 values of Os/Ru homologs differ by factors (Os/Ru) of up to ~110 and ~410 in CH1 and SW480 cells, respectively. ESI-MS studies revealed that ascorbic acid may activate the ruthenium complexes leading to hydrolysis of one M-Cl bond, whereas the osmium analogues tend to be inert. The interaction with myoglobin suggests nonselective adduct formation; i.e., proteins may act as carriers for these compounds.

A collection of luminescent metal complexes have been widely used as oxygen probes in the biomedical field. However, single intensity-based detection approach usually suffered from errors caused by the signal heterogeneity or fluctuation of the optoelectronic system. In this work, respective ruthenium (II) and terbium (III) complexes were chosen to coordinate a bipyridine-branched copolymer, so that to produce oxygen-sensitive metallopolymer (Ru-Poly) and oxygen-insensitive metallopolymer (Tb-Poly). Based on the hydrophobic Ru-Poly and Tb-Poly, a ratiometric luminescent oxygen nanoprobe was facilely prepared by a nanoprecipitation method. The nanoprobes have a typical size of ~100 nm in aqueous solution, exhibiting a green-red dual-wavelength emission under the excitation of 300 nm and 460 nm, respectively. The red emission is strongly quenched by dissolved oxygen while the green one is rather stable, and the ratiometric luminescence was well fitted by a linear Stern-Volmer equation. Using the ratiometric biocompatible nanoprobes, the distribution of intracellular oxygen within three-dimensional multi-cellular tumor spheroids was successfully imaged.

This work reports the in vitro activity against Plasmodium falciparum blood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.

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 αIIbβ3), 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.

Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of ∼2 μM tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii) chloride (RPC2) retards or arrests microtubule motion as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins. Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase, suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy, these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an associative binding constant of 4.8 × 106 M-1 for RPC2 to preformed microtubules and support a 1 : 1 RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is co-localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule stabilizing agent and sharing many similarities with cells treated with paclitaxel.

Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking.

Cyclometallated ruthenium complexes typically exhibit red-shifted absorption bands and lower photolability compared to their polypyridyl analogues. They also have lower symmetry, which sometimes makes their synthesis challenging. In this work, the coordination of four N,S bidentate ligands, 3-(methylthio)propylamine (mtpa), 2-(methylthio)ethylamine (mtea), 2-(methylthio)ethyl-2-pyridine (mtep), and 2-(methylthio)methylpyridine (mtmp), to the cyclometallated precursor [Ru(bpy)(phpy)(CH3 CN)2 ]+ (bpy=2,2'-bipyridine, Hphpy=2-phenylpyridine) has been investigated, furnishing the corresponding heteroleptic complexes [Ru(bpy)(phpy)(N,S)]PF6 ([2]PF6 -[5]PF6 , respectively). The stereoselectivity of the synthesis strongly depended on the size of the ring formed by the Ru-coordinated N,S ligand, with [2]PF6 and [4]PF6 being formed stereoselectively, but [3]PF6 and [5]PF6 being obtained as mixtures of inseparable isomers. The exact stereochemistry of the air-stable complex [4]PF6 was established by a combination of DFT, 2D NMR, and single-crystal X-ray crystallographic studies. Finally, [4]PF6 was found to be photosubstitutionally active under irradiation with green light in acetonitrile, which makes it the first cyclometallated ruthenium complex capable of undergoing selective photosubstitution of a bidentate ligand.

In vivo data are rare but essential for establishing the clinical potential of ruthenium-based photoactivated chemotherapy (PACT) compounds, a new family of phototherapeutic drugs that are activated via ligand photosubstitution. Here a novel trisheteroleptic ruthenium complex [Ru(dpp)(bpy)(mtmp)](PF6)2 ([2](PF6)2, dpp = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2'-bipyridine, mtmp = 2-methylthiomethylpyridine) was synthesized and its light-activated anticancer properties were validated in cancer cell monolayers, 3D tumor spheroids, and in embryonic zebrafish cancer models. Upon green light irradiation, the non-toxic mtmp ligand is selectively cleaved off, thereby releasing a phototoxic ruthenium-based photoproduct capable notably of binding to nuclear DNA and triggering DNA damage and apoptosis within 24-48 h. In vitro, fifteen minutes of green light irradiation (21 mW cm-2, 19 J cm-2, 520 nm) were sufficient to generate high phototherapeutic indexes (PI) for this compound in a range of cancer cell lines including lung (A549), prostate (PC3Pro4), conjunctival melanoma (CRMM1, CRMM2, CM2005.1) and uveal melanoma (OMM1, OMM2.5, Mel270) cancer cell lines. The therapeutic potential of [2](PF6)2 was further evaluated in zebrafish embryo ectopic (PC3Pro4) or orthotopic (CRMM1, CRMM2) tumour models. The ectopic model consisted of red fluorescent PC3Pro4-mCherry cells injected intravenously (IV) into zebrafish, that formed perivascular metastatic lesions at the posterior ventral end of caudal hematopoietic tissue (CHT). By contrast, in the orthotopic model, CRMM1- and CRMM2-mCherry cells were injected behind the eye where they developed primary lesions. The maximally-tolerated dose (MTD) of [2](PF6)2 was first determined for three different modes of compound administration: (i) incubating the fish in prodrug-containing water (WA); (ii) injecting the prodrug intravenously (IV) into the fish; or (iii) injecting the prodrug retro-orbitally (RO) into the fish. To test the anticancer efficiency of [2](PF6)2, the embryos were treated 24 h after engraftment at the MTD. Optimally, four consecutive PACT treatments were performed on engrafted embryos using 60 min drug-to-light intervals and 90 min green light irradiation (21 mW cm-2, 114 J cm-2, 520 nm). Most importantly, this PACT protocol was not toxic to the zebrafish. In the ectopic prostate tumour models, where [2](PF6)2 showed the highest photoindex in vitro (PI > 31), the PACT treatment did not significantly diminish the growth of primary lesions, while in both conjunctival melanoma orthotopic tumour models, where [2](PF6)2 showed more modest photoindexes (PI ∼ 9), retro-orbitally administered PACT treatment significantly inhibited growth of the engrafted tumors. Overall, this study represents the first demonstration in zebrafish cancer models of the clinical potential of ruthenium-based PACT, here against conjunctival melanoma.