Aquaporin-3 is involved in NLRP3-inflammasome activation contributing to the setting of inflammatory response.

Inflammasomes are large immune multiprotein complexes that tightly regulate the production of the pro-inflammatory cytokines, being dependent on cell regulatory volume mechanisms. Aquaporins (AQPs) are protein channels that facilitate the transport of water and glycerol (aquaglyceroporins) through membranes, essential for cell volume regulation. Although these membrane proteins are highly expressed in monocytes and macrophages, their role in the inflammatory process is still unclear. Here, we investigated the role of aquaglyceroporin AQP3 in NLRP3-inflammasome activation by complementary approaches based either on shRNA silencing or on AQP3 selective inhibition. The latter has been achieved using a reported potent gold-based inhibitor, Auphen. AQP3 inhibition or silencing partially blocked LPS-priming and decreased production of IL-6, proIL-1ß, and TNF-α, suggesting the possible involvement of AQP3 in macrophage priming by Toll-like receptor 4 engagement. Moreover, AQP3-dependent cell reswelling increased IL-1ß release through caspase-1 activation. NLRP3-inflammasome activation induced by reswelling, nigericin, and ATP was also blocked when AQP3 was inhibited or silenced. Altogether, these data point towards AQPs as potential players in the setting of the inflammatory response.

Synthesis, characterization, DFT study, DNA/BSA-binding affinity, and cytotoxicity of some dinuclear and trinuclear gold(III) complexes.

In this study, we have synthesized a series of dinuclear and trinuclear gold(III) complexes of the general formula [Au2(N-N)Cl6] (1-3) for dinuclear and [Au3(N-N)2Cl8]+ (4-6) for trinuclear compounds, respectively, in which N-N is a bidentate ligand (1,4-diaminobutane; 1,6-diaminohexane or 1,8-diaminooctane). These complexes were characterized by elemental analysis, molar conductivity, and spectroscopic techniques (IR, UV-Vis, 1H NMR, ESI-MS). We performed DFT calculations to get insight into the geometry of the studies complexes. DNA-binding studies were performed by UV-Vis spectrophotometry and fluorescence spectroscopy. The results of competitive reactions between gold(III) complexes and ethidium bromide (EB) towards DNA have shown that selected complexes can displace EB from DNA-EB adduct. In addition, these experiments confirm that polynuclear gold(III) complexes interact with DNA covalently or via intercalation. Furthermore, high values of binding constants of gold(III) complexes towards bovine serum albumin (BSA) protein indicate good binding affinity. In addition, redox stability of complexes in the presence of DNA/BSA was confirmed by cyclic voltammetry. Results of the interactions between gold(III) complexes with DNA/BSA were discussed in reference to molecular docking data obtain by Molegro virtual docker. The cytotoxic activity of synthesized gold(III) complexes was evaluated on human breast cancer cell line (MDA-MB-231), human colorectal cancer cell line (HCT-116), and normal human lung fibroblast cell line (MRC-5). All complexes dose-dependently reduced cancer and normal cells viabilities, with significant cytotoxic effects (IC50 < 25 µM) for trinuclear gold(III) complexes (4, 5) on HCT-116 cells.

Protein-mediated disproportionation of Au(i): insights from the structures of adducts of Au(iii) compounds bearing N,N-pyridylbenzimidazole derivatives with lysozyme.

Au(iii) compounds bearing N,N-pyridylbenzimidazole derivatives with the ethyl (1) or propyl sulfonate (2) appendage react with the model protein hen egg white lysozyme (HEWL), forming adducts with different gold-containing fragments. The conformation of the enzyme, the exact gold binding sites and the oxidation state of Au in the adducts are unknown. Here we report a structural study on the reaction of 1 and 2 with HEWL in solution and solid state. In agreement with previously reported electrospray ionization mass spectra, the compounds degrade in their interaction with the protein. In the structure derived from HEWL crystals exposed to 1 for less than one day, three Au binding sites were identified: Au(i) ions are bound to the side chain of His15 and to the side chains of His15 and Asn93. The third gold centre is buried in the hydrophobic pocket of the protein via the binding to the side chain of Met105 and the trapping between the side chains of Trp28, Trp108 and Trp111. In a second crystal fished three hours later from the same drop, only one Au ion, probably in the +1 oxidation state, is observed; it binds the protein close to the side chains of Asn93 and His15. After three days of soaking, the colour of HEWL crystals obtained in the presence of 1 turned violet. In these crystals, anomalous signals attributable to Au are found on the protein surface; gold atoms are not directly coordinated to residue side chains. Longer exposure of HEWL crystals to 1 produces gold-free crystals. In the adduct of HEWL exposed to 2 for one day, three Au(i) ions are detected close to the side chains of both Asn93 and His15, the side chain of His15 and that of Met105. Longer exposure of HEWL crystals to 2 affords gold-free crystals. These structural data and those of the other protein/gold adducts available at the Protein Data Bank suggest that the reduction of Au(iii) into Au(i) is the basis of the mechanism of action of the biologically active gold(iii) compounds. Besides, Au(i) ions can undergo disproportionation into Au(iii) and Au(0) that can diffuse away from the protein crystals.

Acridine-decorated cyclometallated gold(iii) complexes: synthesis and anti-tumour investigations.

(C^N) and (C^N^C) cyclometalated Au(iii) represent a highly promising class of potential anticancer agents. We report here the synthesis of seven new cyclometalated Au(iii) complexes with five of them bearing an acridine moiety attached via (N^O) or (N^N) chelates, acyclic amino carbenes (AAC) and N-heterocyclic carbenes (NHC). The antiproliferative properties of the different complexes were evaluated in vitro on a panel of cancer cells including leukaemia, lung and breast cancer cells. We observed a trend between the cytotoxicity and the intracellular gold uptake of some representative compounds of the series. Some of the acridine-decorated complexes were demonstrated to interact with ds-DNA using FRET-melting techniques.

Synthesis and in vitro Evaluation of ADAM10 and ADAM17 Highly Selective Bioimaging Probes.

A disintegrin and metalloproteinase (ADAMs) are membrane-bound metalloproteases responsible for the ectodomain shedding of various transmembrane proteins and play important roles in multiple relevant biological processes. Their altered expression is involved in several pathological conditions, and in particular ADAM10 or ADAM17 overexpression is found in various forms of cancer. To better understand how they are regulated in the cellular context, it is useful to visualize the specific ADAMs pathway by means of molecular imaging techniques. For this purpose, we synthesized bioactive fluorescent probes suitable for cell imaging and that are able to specifically target ADAM10 or ADAM17. Two previously developed ADAM17- and ADAM10-selective inhibitors were chosen for conjugation, respectively, to a Cy5.5 dye and to Cy5.5 and FITC dyes. Herein we also report the synthesis of a gold-labeled compound as an additional bioimaging probe for ADAM10. The newly synthesized ligands were found to be active in vitro on human recombinant ADAM10 and/or ADAM17, showing IC50 values in the nanomolar range and a good selectivity over matrix metalloproteinases (MMPs). Finally, these newly developed probes were successfully used for ADAMs staining on different lymphoma cell lines and lymph node mesenchymal stromal cells.

Structural evidences for a secondary gold binding site in the hydrophobic box of lysozyme.

A new crystal structure is reported here for the adduct formed in the reaction between NH4 [Au(Sac)2], AuSac2, a cytotoxic homoleptic gold(I) complex with the saccharinate ligand, and the model protein hen egg white lysozyme. To produce this adduct, AuSac2 breaks down and releases both saccharinate ligands. The resulting Au(I) ions bind the protein to ND1 and NE2 atoms of His15 but also to SD atom of the zero-solvent accessible Met105 side chain, which is located in the protein hydrophobic box. The unexpected existence of this secondary gold(I) binding site is confirmed by spectroscopic and spectrometric measurements in solution.

Atypical fluoroquinolone gold(III) chelates as potential anticancer agents: relevance of DNA and protein interactions for their mechanism of action.

Quinolones are known for their antimicrobial and antitumor activities. Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. In this work three gold(III) complexes of the fluoroquinolones antimicrobial agents norfloxacin (NOR), levofloxacin (LEVO) and sparfloxacin (SPAR) were prepared and characterized with physicochemical and spectroscopic techniques. In these complexes, NOR, LEVO and SPAR act as bidentate neutral ligands bound to gold(III) through the nitrogen atoms of the piperazine ring, which is an unusual mode of coordination for this class of compounds. Two chloride ions occupy the remaining coordination sites. The cytotoxic activity of the fluoroquinolones and their gold(III) complexes was tested against the A20 (murine lymphoma), B16-F10 (murine melanoma) and K562 (human myeloid leukemia) tumor cell lines as well as the L919 (murine lung fibroblasts) and MCR-5 (human lung fibroblasts) normal cells lines. All complexes were more active than their corresponding free ligands. Complex [AuCl(2)(LEVO)]Cl was selected for DNA fragmentation and cell cycle analysis. Spectroscopic titration with calf-thymus DNA (CT DNA) showed that the complexes can bind weakly to CT DNA, probably by an external contact (electrostatic or groove binding). The complexes exhibit good binding propensity to bovine serum albumin (BSA) having relatively high binding constant values.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Synthesis, characterization, and in vitro studies of bis[1,3-diethyl-4,5-diarylimidazol-2-ylidene]gold(I/III) complexes.

Cationic bis[1,3-diethyl-4,5-diarylimidazol-2-ylidene]gold(I) complexes with 4-OCH(3) or 4-F substituents in the aromatic rings and Br(-) (3a,b) or BF(4)(-) (7a,b) counterions were synthesized, characterized, and investigated for tumor growth inhibitory properties in vitro. Analogous to auranofin, the N-heterocyclic carbenes (NHCs) were also combined with a phosphine ligand (triphenylphosphine, 4a,b) and 2',3',4',6'-tetra-O-acetyl-ß-D-glucopyranosyl-1-thiolate (5a,b). The growth inhibitory effect against MCF-7, MDA-MB 231, and HT-29 cells, which is more than 10-fold higher than that of cisplatin or 5-FU, was independent of the oxidation state (Au(III), 6a,b) and the anionic counterion. Bis[1,3-diethyl-4,5-bis(4-fluorophenyl)imidazol-2-ylidene]gold(I) bromide 3b as the most cytotoxic compound reduced the growth of MCF-7 cells with IC(50) = 0.10 µM (cisplatin, 1.6 µM; 5-FU, 4.7 µM). The thioredoxin reductase (TrxR), the estrogen receptor (ER), and the cyclooxygenase (COX) enzymes, which have to be considered as possible targets based on the drug design, can be excluded from being involved in the mode of action.

Molecular mechanisms and proposed targets for selected anticancer gold compounds.

Nowadays, gold compounds constitute a family of very promising experimental agents for cancer treatment. Indeed, several gold(I) and gold(III) compounds were shown to manifest outstanding antiproliferative properties in vitro against selected human tumor cell lines and some of them performed remarkably well even in tumor models in vivo. Notably, the peculiar chemical properties of the gold centre impart innovative pharmacological profiles to gold-based metallodrugs most likely in relation to novel molecular mechanisms. The precise mechanisms through which cytotoxic gold compounds produce their biological effects are still largely unknown. Within this frame, the major aim of this review is to define the possible modes of action and the most probable biomolecular targets for a few representative gold compounds on which extensive biochemical and cellular data have been gathered. In particular, we will focus on auranofin and analogues, on gold(III) porphyrins and gold(III) dithiocarbamates. For these three families markedly distinct molecular mechanisms were recently invoked: a direct mitochondrial mechanism involving thioredoxin reductase inhibition in the case of the gold(I) complexes, the influence on some apoptotic proteins--i.e. MAPKs and Bcl-2--for gold(III) porphyrins, and the proteasome inhibition for gold(III) dithiocarbamates. In a few cases the distinct mechanisms may overlap. The general perspectives for the development of new gold compounds as effective anticancer agents with innovative modes of action are critically discussed.

Enhancing the contrast of ApoB to locate the surface components in the 3D density map of human LDL.

A 26 Å resolution map of the structure of human low-density lipoprotein (LDL) was obtained from electron cryomicroscopy and single-particle image reconstruction. The structure showed a discoidal-shaped LDL particle with high-density regions mainly distributed at the edge of the particle and low-density regions at the flat surface that covers the core region. To determine the chemical components that correspond to these density regions and to delineate the distribution of protein and phospholipid located at the particle surface at the resolution of the map, we used Mono-Sulfo-NHS-Undecagold labeling to increase preferentially the contrast of the apolipoprotein B component on the LDL particle. In the three-dimensional map from the image reconstruction of the undecagold-labeled LDL particles, the high-density region from the undecagold label was distributed mainly at the edge of the particle, and lower density regions were found at the flat surfaces that cover the neutral lipid core. This suggests that apolipoprotein B mainly encircles LDL at the edge of the particle and the phospholipid monolayers are located at the flat surfaces, which are parallel to the cholesterol ester layers in the core and may interact with the core lipid layers through the acyl chains.

Latest insights into the anticancer activity of gold(III)-dithiocarbamato complexes.

In this review paper we aim at giving a detailed overview on our research work devoted to the design of gold-based anticancer agents. In particular, during the last decade, we have been developing some gold(III)-dithiocarbamato derivates showing outstanding in vitro and in vivo antitumor properties and reduced, or even no, systemic and renal toxicity, compared to the reference clinically-established anticancer drug cisplatin. Starting from the rationale behind our investigations, we here summarize the results achieved so far, focusing on the latest in-depth mechanistic studies that have recently provided insights into their mechanism of action, thus opening up new prospects for further pharmacological testing and, hopefully, to enter clinical trials.

Gold(I) phosphine mediated selective inhibition of lymphoid tyrosine phosphatase.

Selective protein tyrosine phosphatase (PTP) inhibition is often difficult to achieve owing to the high degree of similarity of the catalytic domains of this family of enzymes. Selective inhibitors of the lymphoid specific tyrosine phosphatase, LYP, are of great interest due to the involvement of LYP in several autoimmune disorders. This manuscript describes a study into the mechanistic details of selective LYP inhibition by a Au(I)-phosphine complex. The complex, [Au((CH(2)CH(2)CN)(2)PPh)Cl], selectively inhibits LYP activity both in vitro and in cells, but does not inhibit other T-cell derived PTPs including the highly homologous PTP-PEST. The mode of inhibition was probed by investigating inhibition of LYP, the LYP mutant C129/231S, and PTP-PEST. Inhibition of LYP and PTP-PEST was competitive, while the LYP double mutant appeared mixed. Wild-type LYP was inhibited more potently than LYP C129/231S, indicating an important role for at least one of these residues in Au(I) binding. Coordination of Au(I) by both the active site cysteine residue as well as either Cys129 or 231 is suggested as a potential mechanism for LYP selective inhibition.