Rhenium fac-Tricarbonyl Bisimine Chalcogenide Complexes: Synthesis, Photophysical Studies, and Confocal and Time-Resolved Cell Microscopy.

We describe the preparation, characterization, and imaging studies of rhenium carbonyl complexes with a pyta (4-(2-pyridyl)-1,2,3-triazole) or tapy (1-(2-pyridyl)-1,2,3-triazole)-based heteroaromatic N∧N ligand and thiolate or selenoate X ligand. The stability and photophysical properties of the selenolate complexes are compared with parent chloride complexes and previously described analogues with benzenethiolate ligands. Two complexes were imaged in A549 cells upon excitation at 405 nm. Colocalization studies suggest a lysosomal accumulation, while one parent chloride complex was described to localize at the Golgi apparatus. Preliminary fluorescence lifetime measurements and imaging demonstrate potential for application in time-resolved microscopy techniques due to the long and variable lifetimes observed in cellular environments, including an increase in lifetime between the solution and solid state many times larger than previously reported.

A New Manganese Superoxide Dismutase Mimetic Improves Oxaliplatin-Induced Neuropathy and Global Tolerance in Mice.

Reactive oxygen species (ROS) are produced by every aerobic cell during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Superoxide Dismutases (SOD) are antioxidant proteins that convert superoxide anions (O2•-) to hydrogen peroxide (H2O2) and dioxygen. Using the differential in the level of oxidative stress between normal and cancer cells, SOD mimetics can show an antitumoral effect and prevent oxaliplatin-induced peripheral neuropathy. New Pt(IV) conjugate prodrugs (OxPt-x-Mn1C1A (x = 1, 1-OH, 2)), combining oxaliplatin and a Mn SOD mimic (MnSODm Mn1C1A) with a covalent link, were designed. Their stability in buffer and in the presence of sodium ascorbate was studied. In vitro, their antitumoral activity was assessed by the viability and ROS production of tumor cell lines (CT16, HCT 116, KC) and fibroblasts (primary culture and NIH 3T3). In vivo, a murine model of colorectal cancer was created with subcutaneous injection of CT26 cells in Balb/c mice. Tumor size and volume were measured weekly in four groups: vehicle, oxaliplatin, and oxaliplatin associated with MnSODm Mn1C1A and the bis-conjugate OxPt-2-Mn1C1A. Oxaliplatin-induced peripheral neuropathy (OIPN) was assessed using a Von Frey test reflecting chronic hypoalgesia. Tolerance to treatment was assessed with a clinical score including four items: weight loss, weariness, alopecia, and diarrhea. In vitro, Mn1C1A associated with oxaliplatin and Pt(IV) conjugates treatment induced significantly higher production of H2O2 in all cell lines and showed a significant improvement of the antitumoral efficacy compared to oxaliplatin alone. In vivo, the association of Mn1C1A to oxaliplatin did not decrease its antitumoral activity, while OxPt-2-Mn1C1A had lower antitumoral activity than oxaliplatin alone. Mn1C1A associated with oxaliplatin significantly decreased OIPN and also improved global clinical tolerance of oxaliplatin. A neuroprotective effect was observed, associated with a significantly improved tolerance to oxaliplatin without impairing its antitumoral activity.

SOD mimics: From the tool box of the chemists to cellular studies.

Superoxide dismutases (SODs) are metalloproteins that protect cells against oxidative stress by controlling the concentration of superoxide (O2-) through catalysis of its dismutation. The activity of superoxide dismutases can be mimicked by low-molecular-weight complexes having potential therapeutic applications. This review presents recent strategies for designing efficient SOD mimics, from molecular metal complexes to nanomaterials. Studies of these systems in cells reveal that some SOD mimics, designed to react directly with superoxide, may also indirectly enhance the cellular antioxidant arsenal. Finally, a good understanding of the bioactivity requires information on the cell-penetration, speciation, and subcellular location of the SOD mimics: we will describe recent studies and new techniques that open opportunities for characterizing SOD mimics in biological environments.

Cellular Detection of a Mitochondria Targeted Brominated Vinyl Triphenylamine Optical Probe (TP-Br) by X-Ray Fluorescence Microscopy.

Triphenylamine (TP) derivatives such as two-branch cationic vinylbenzimidazolium triphenylamine TP-2Bzim are promising turn-on fluorescent probes suitable for two-photon imaging, labelling mitochondria in live cells. Here, we designed two TP-2Bzim derivatives as bimodal probes suitable for X-ray fluorescence imaging. The conjugation of the TP core with a rhenium tricarbonyl moiety in the TP-RePyta probe altered the localisation in live cells from mitochondria to lysosomes. The introduction of bromine on the TP core generated the TP-Br probe retaining good photophysical properties and mitochondria labelling in live cells. The influence of calcium channels in the uptake of TP-Br was studied. Synchrotron Radiation X-ray Fluorescence (SXRF) imaging of bromine enabled the detection of TP-Br and suggested a negligible presence of the probe in an unbound state in the incubated cells, a crucial point in the development of these probes. This study paves the way towards the development of TP probes as specific organelle stainers suitable for SXRF imaging.

Understanding the g-tensors of perchlorotriphenylmethyl and Finland-type trityl radicals.

The 285 GHz EPR spectra of perchlorotriphenylmethyl and tetrathiatriarylmethyl radicals in frozen solution have been accurately measured. The relationship between their molecular structures and their g-tensors has been investigated with the aid of DFT calculations, revealing that the degree of spin density delocalization away from the central methylene carbon is an important determining factor of the g-anisotropy. In particular, the small amount of spin densities on the Cl or S heteroatoms at the 2 and 6 positions with respect to the central carbon have the strongest influence. Furthermore, the amount of spin densities on these heteroatoms and thus the anisotropy can be modulated by the protonation (esterification) state of the carboxylate groups at the 4 position. These results provide unique insights into the g-anisotropy of persistent trityl radicals and how it can be tuned.

Anti-inflammatory activity of superoxide dismutase mimics functionalized with cell-penetrating peptides.

A superoxide dismutase mimic (Mn1) was functionalized with three positively charged-peptides: RRRRRRRRR (Mn1-R9), RRWWWRRWRR (Mn1-RW9) or Fx-r-Fx-K (Mn1-MPP). Characterization of the physico-chemical properties of the complexes show that they share similar binding affinity for Mn2+, apparent reduction potential and intrinsic superoxide dismutase activity. However, their accumulation in cells is different (Mn1-R9 < Mn1-MPP < Mn1-RW9 < Mn1), as well as their subcellular distribution. In addition, the three functionalized-complexes display a better anti-inflammatory activity than Mn1 when assayed at 10 µM. This improvement is due to a combination of an anti-inflammatory effect of the peptidyl moiety itself, and of the SOD mimic for Mn1-RW9 and Mn1-MPP. In contrast, the enhanced anti-inflammatory activity of Mn1-R9 is solely due to the SOD mimic.

Oxaliplatin-induced neuropathy: the preventive effect of a new super-oxide dismutase modulator.

By using the differential in level of oxidative status between normal and cancer cells, SuperOxide Dismutase (SOD) mimetics can have anti-tumor efficacy and prevent oxaliplatin-induced peripheral neuropathy. Our objective was to evaluate the neuroprotective efficacy of MAG, a new SOD mimic. In vitro, the effects of MAG alone or with oxaliplatin were studied on colon cancer cells (HT29 and CT26) and on normal fibroblast cells (NIH3T3). The cell viability (by crystal violet) as well as the production of reactive forms of oxygen and glutathione (by spectrofluorimetric assay) was measured. In vivo, efficacy on tumor growth was assessed in mice grafted with CT26 colon cancer cells. The effects on induced neurotoxicity were measured by specific behavioral Von Frey nociception, cold-plate tests, specific functional neuromuscular assay and electron microscopy. In vitro, MAG induced a production of hydrogen peroxide in all cells. At 24 h-incubation, MAG exhibits a cytotoxic activity in all cell lines. A cytotoxic additive effect of MAG and oxaliplatin was observed through oxidative burst. In vivo, oxaliplatin-treated mice associated with MAG did not counteract oxaliplatin's antitumoral efficacy. After 4 weeks of treatment with oxaliplatin combined with MAG, behavioral and functional tests showed a decrease in peripheral neuropathy induced by oxaliplatin in vivo. Electron microscopy analyses on sciatic nerves revealed an oxaliplatin-induced demyelination which is prevented by the association of MAG to this chemotherapy. In conclusion, MAG prevents the appearance of sensitive axonal neuropathy and neuromuscular disorders induced by oxaliplatin without affecting its antitumor activity.

From Naproxen Repurposing to Naproxen Analogues and Their Antiviral Activity against Influenza A Virus.

The nucleoprotein (NP) of influenza A virus (IAV) required for IAV replication is a promising target for new antivirals. We previously identified by in silico screening naproxen being a dual inhibitor of NP and cyclooxygenase COX2, thus combining antiviral and anti-inflammatory effects. However, the recently shown strong COX2 antiviral potential makes COX2 inhibition undesirable. Here we designed and synthesized two new series of naproxen analogues called derivatives 2, 3, and 4 targeting highly conserved residues of the RNA binding groove, stabilizing NP monomer without inhibiting COX2. Derivative 2 presented improved antiviral effects in infected cells compared to that of naproxen and afforded a total protection of mice against a lethal viral challenge. Derivative 4 also protected infected cells challenged with circulating 2009-pandemic and oseltamivir-resistant H1N1 virus. This improved antiviral effect likely results from derivatives 2 and 4 inhibiting NP-RNA and NP-polymerase acidic subunit PA N-terminal interactions.

Labeling of Hyaluronic Acids with a Rhenium-tricarbonyl Tag and Percutaneous Penetration Studied by Multimodal Imaging.

Hyaluronic acids were labeled with a rhenium-tricarbonyl used as single core multimodal probe for imaging and their penetration into human skin biopsies was studied using IR microscopy and fluorescence imaging (labeled SCoMPI). The penetration was shown to be dependent on the molecular weight of the molecule and limited to the upper layer of the skin.

Direct Keap1-Nrf2 disruption as a potential therapeutic target for Alzheimer's disease.

Nrf2, a transcriptional activator of cell protection genes, is an attractive therapeutic target for the prevention of neurodegenerative diseases, including Alzheimer's disease (AD). Current Nrf2 activators, however, may exert toxicity and pathway over-activation can induce detrimental effects. An understanding of the mechanisms mediating Nrf2 inhibition in neurodegenerative conditions may therefore direct the design of drugs targeted for the prevention of these diseases with minimal side-effects. Our study provides the first in vivo evidence that specific inhibition of Keap1, a negative regulator of Nrf2, can prevent neuronal toxicity in response to the AD-initiating Aß42 peptide, in correlation with Nrf2 activation. Comparatively, lithium, an inhibitor of the Nrf2 suppressor GSK-3, prevented Aß42 toxicity by mechanisms independent of Nrf2. A new direct inhibitor of the Keap1-Nrf2 binding domain also prevented synaptotoxicity mediated by naturally-derived Aß oligomers in mouse cortical neurons. Overall, our findings highlight Keap1 specifically as an efficient target for the re-activation of Nrf2 in AD, and support the further investigation of direct Keap1 inhibitors for the prevention of neurodegeneration in vivo.

Rhenium Complexes Based on 2-Pyridyl-1,2,3-triazole Ligands: A New Class of CO2 Reduction Catalysts.

A series of [Re(N^N)(CO)3(X)] (N^N = diimine and X = halide) complexes based on 4-(2-pyridyl)-1,2,3-triazole (pyta) and 1-(2-pyridyl)-1,2,3-triazole (tapy) diimine ligands have been prepared and electrochemically characterized. The first ligand-based reduction process is shown to be highly sensitive to the nature of the isomer as well as to the substituents on the pyridyl ring, with the peak potential changing by up to 700 mV. The abilities of this class of complexes to catalyze the electroreduction and photoreduction of CO2 were assessed for the first time. It is found that only Re pyta complexes that have a first reduction wave with a peak potential at ca. -1.7 V vs SCE are active, producing CO as the major product, together with small amounts of H2 and formic acid. The catalytic wave that is observed in the CVs is enhanced by the addition of water or trifluoroethanol as a proton source. Long-term controlled potential electrolysis experiments gave total Faradaic yield close to 100%. In particular, functionalization of the triazolyl ring with a 2,4,6-tri-tert-butylphenyl group provided the catalyst with a remarkable stability.

Indolin-2-one compounds targeting thioredoxin reductase as potential anticancer drug leads.

Several compounds bearing the indolinone chemical scaffold are known to possess anticancer properties. For example, the tyrosine kinase inhibitor sunitinib is an arylideneindolin-2-one compound. The chemical versatility associated with structural modifications of indolinone compounds underlies the potential to discover additional derivatives possessing anticancer properties. Previously synthesized 3-(2-oxoethylidene)indolin-2-one compounds, also known as supercinnamaldehyde (SCA) compounds in reference to the parent compound 1 [1-methyl-3(2-oxopropylidene)indolin-2-one], bear a nitrogen-linked α,ß-unsaturated carbonyl (Michael acceptor) moiety. Here we found that analogs bearing N-substituents, in particular compound 4 and 5 carrying an N-butyl and N-benzyl substituent, respectively, were strongly cytotoxic towards human HCT 116 colorectal and MCF-7 breast carcinoma cells. These compounds also displayed strong thioredoxin reductase (TrxR) inhibitory activity that was likely attributed to the electrophilicity of the Michael acceptor moiety. Their selectivity towards cellular TrxR inhibition over related antioxidant enzymes glutathione reductase (GR), thioredoxin (Trx) and glutathione peroxidase (GPx) was mediated through targeting of the selenocysteine (Sec) residue in the highly accessible C-terminal active site of TrxR. TrxR inhibition mediated by indolin-2-one compounds led to cellular Trx oxidation, increased oxidative stress and activation of apoptosis signal-regulating kinase 1 (ASK1). These events also led to activation of p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways, and cell death with apoptotic features of PARP cleavage and caspase 3 activation. In conclusion, these results suggest that indolin-2-one-based compounds specifically targeting TrxR may serve as novel drug leads for anticancer therapy.

A Bis-Manganese(II)-DOTA Complex for Pulsed Dipolar Spectroscopy.

High-spin gadolinium(III) and manganese(II) complexes have emerged as alternatives to standard nitroxide radical spin labels for measuring nanometric distances by using pulsed electron-electron double resonance (PELDOR or DEER) at high fields/frequencies. For certain complexes, particularly those with relatively small zero-field splitting (ZFS) and short distances between the two metal centers, the pseudosecular term of the dipolar coupling Hamiltonian is non-negligible. However, in general, the contribution from this term during conventional data analysis is masked by the flexibility of the molecule of interest and/or the long tethers connecting them to the spin labels. The efficient synthesis of a model system consisting of two [Mn(dota)](2-) (MnDOTA; DOTA(4-) =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) directly connected to the ends of a central rodlike oligo(phenylene-ethynylene) (OPE) spacer is reported. The rigidity of the OPE is confirmed by Q-band PELDOR measurements on a bis-nitroxide analogue. The Mn(II) -Mn(II) distance distribution profile determined by W-band PELDOR is in reasonable agreement with one simulated by using a simple rotamer analysis. The small degree of flexibility arising from the linking MnDOTA arm appears to outweigh the contribution from the pseudosecular term at this interspin distance. This study illustrates the potential of MnDOTA-based spin labels for measuring fairly short nanometer distances, and also presents an interesting candidate for in-depth studies of pulsed dipolar spectroscopy methods on Mn(II) -Mn(II) systems.

Bimodal X-ray and Infrared Imaging of an Organometallic Derivative of Praziquantel in Schistosoma mansoni.

An organometallic derivative of praziquantel was studied directly in worms by using inductively coupled plasma-mass spectrometry (ICP-MS) for quantification and synchrotron-based imaging. X-ray fluorescence (XRF) and IR absorption spectromicroscopy were used for the first time in combination to directly locate this organometallic drug candidate in schistosomes. The detection of both CO (IR) and Cr (XRF) signatures proved that the Cr(CO)3 core remained intact in the worms. Images showed a preferential accumulation at the worm's tegument, consistent with a possible targeting of the calcium channel but not excluding other biological targets inside the worm.

The Use of Mn(II) Bound to His-tags as Genetically Encodable Spin-Label for Nanometric Distance Determination in Proteins.

A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 µM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.

Design, Synthesis, and Evaluation of Triazole Derivatives That Induce Nrf2 Dependent Gene Products and Inhibit the Keap1-Nrf2 Protein-Protein Interaction.

The transcription factor Nrf2 regulates the expression of a large network of cytoprotective and metabolic enzymes and proteins. Compounds that directly and reversibly inhibit the interaction between Nrf2 and its main negative regulator Keap1 are potential pharmacological agents for a range of disease types including neurodegenerative conditions and cancer. We describe the development of a series of 1,4-diphenyl-1,2,3-triazole compounds that inhibit the Nrf2-Keap1 protein-protein interaction (PPI) in vitro and in live cells and up-regulate the expression of Nrf2-dependent gene products.

Nanometric distance measurements between Mn(ii)DOTA centers.

Pulse electron-electron double resonance (PELDOR) is a versatile technique for probing the structures and functions of complex biological systems. Despite the recent interest in high-spin metal-ions for high field/frequency applications, PELDOR measurements of Mn(ii) remain relatively underexplored. Here we present Mn(ii)-Mn(ii) PELDOR distance measurements at 94 GHz on polyproline II (PPII) helices doubly spin-labeled with Mn(ii)DOTA, which are distinguished by their small zero-field interaction. The measured Mn-Mn distances and distribution profiles were in good agreement with the expected values from molecular models. Additional features in the frequency-domain spectra became apparent at certain combinations of detect and pump frequencies. Spin-Hamiltonian calculations showed that they likely arose from contributions from the pseudo-secular component of the dipolar interaction that were found to be non-negligible for Mn(ii)DOTA. However, the influence of the pseudo-secular component on the distance distribution profiles apparently was limited. The results show the potential of Mn(ii)DOTA spin labels for high-field PELDOR distance measurements in proteins and other biological systems.

Luminescence modulations of rhenium tricarbonyl complexes induced by structural variations.

Octahedral d(6) low-spin Re(I) tricarbonyl complexes are of considerable interest as noninvasive imaging probes and have been deeply studied owing to their biological stability, low toxicity, large Stokes shifts, and long luminescence lifetimes. We reported recently the bimodal IR and luminescence imaging of a Re(I) tricarbonyl complex with a Pyta ligand (4-(2-pyridyl)-1,2,3-triazole) in cells and labeled such metal-carbonyl complexes SCoMPIs for single-core multimodal probes for imaging. Re(I) tricarbonyl complexes have unique photophysical properties allowing for their unequivocal detection in cells but also present some weaknesses such as a very low luminescence quantum yield in aqueous medium. Further optimizations would thus be desirable. We therefore developed new Re(I) tricarbonyl complexes prepared from different ancillary ligands. Complexes with benzothiadiazole-triazole ligands show interesting luminescent quantum yields in acetonitrile and may constitute valuable luminescent metal complexes in organic media. A series of complexes with bidentate 1-(2-quinolinyl)-1,2,3-triazole (Taquin) and 1-(2-pyridyl)-1,2,3-triazole (Tapy) ligands bearing various 4-substituted alkyl side chains has been designed and synthesized with efficient procedures. Their photophysical properties have been characterized in acetonitrile and in a H2O/DMSO (98/2) mixture and compared with those of the parent Quinta- and Pyta-based complexes. Tapy complexes bearing long alkyl chains show impressive enhancement of their luminescent properties relative to the parent Pyta complex. Theoretical calculations have been performed to further characterize this new class of rhenium tricarbonyl complexes. Preliminary cellular imaging studies in MDA-MB231 breast cancer cells reveal a strong increase in the luminescence signal in cells incubated with the Tapy complex substituted with a C12 alkyl chain. This study points out the interesting potential of the Tapy ligand in coordination chemistry, which has been so far underexploited.

Peptide inhibitors of the Keap1-Nrf2 protein-protein interaction.

Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the cap'n'collar basic-region leucine-zipper transcription factor Nrf2 is a potential strategy to enhance expression of antioxidant and free radical detoxification gene products regulated by Nrf2. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer-chemopreventive agents. We describe the structure-activity relationships for a series of peptides based upon regions of the Nrf2 Neh2 domain, of varying length and sequence, that interact with the Keap1 Kelch domain and disrupt the interaction with Nrf2. We have also investigated sequestosome-1 (p62) and prothymosin-α sequences that have been reported to interact with Keap1. To achieve this we have developed a high-throughput fluorescence polarization (FP) assay to screen inhibitors. In addition to screening synthetic peptides, we have used a phage display library approach to identify putative peptide ligands with non-native sequence motifs. Candidate peptides from the phage display library screening protocol were evaluated in the FP assay to quantify their binding activity. Hybrid peptides based upon the Nrf2 "ETGE" motif and the sequestosome-1 "Keap1-interaction region" have superior binding activity compared to either native peptide alone.