Pseudomonas aeruginosa and Staphylococcus aureus Display Differential Proteomic Responses to the Silver(I) Compound, SBC3.

The urgent need to combat antibiotic resistance and develop novel antimicrobial therapies has triggered studies on novel metal-based formulations. N-heterocyclic carbene (NHC) complexes coordinate transition metals to generate a broad range of anticancer and/or antimicrobial agents, with ongoing efforts being made to enhance the lipophilicity and drug stability. The lead silver(I) acetate complex, 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*) (SBC3), has previously demonstrated promising growth and biofilm-inhibiting properties. In this work, the responses of two structurally different bacteria to SBC3 using label-free quantitative proteomics were characterised. Multidrug-resistant Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) are associated with cystic fibrosis lung colonisation and chronic wound infections, respectively. SBC3 increased the abundance of alginate biosynthesis, the secretion system and drug detoxification proteins in P. aeruginosa, whilst a variety of pathways, including anaerobic respiration, twitching motility and ABC transport, were decreased in abundance. This contrasted the affected pathways in S. aureus, where increased DNA replication/repair and cell redox homeostasis and decreased protein synthesis, lipoylation and glucose metabolism were observed. Increased abundance of cell wall/membrane proteins was indicative of the structural damage induced by SBC3 in both bacteria. These findings show the potential broad applications of SBC3 in treating Gram-positive and Gram-negative bacteria.

Novel Anticancer NHC*-Gold(I) Complexes Inspired by Lepidiline A.

N-Heterocyclic carbene gold(I) complexes derived from 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene (NHC*) represent a promising class of anticancer drugs. Complexes of the type NHC*-Au-L (L = Br-, I-, C≡C-R) and [NHC*-Au-L]+ (L = NHC*, PPh3) have been synthesised. The X-ray crystal structures of all gold(I) complexes are presented; aurophilic interactions were observed in five of the complexes. The anticancer activity was assessed via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-based proliferation assays against the human colon carcinoma cell line HCT-116wt and the multidrug-resistant human breast carcinoma cell line MCF-7topo. Most complexes showed good cytotoxicity with IC50 values in the low micromolar range, while excellent sub-micromolar activity was observed for 2c, 3a and 3b. Generally, the activity of the ligands studied was as follows: carbene > phosphine > alkyne > halide, with an exception for the highly active iodido derivative 2c.

In-vitro and in-vivo investigations into the carbene-gold anticancer drug candidates NHC*-Au-SCSNMe2 and NHC*-Au-S-GLUC against advanced prostate cancer PC3.

The anticancer drug candidates 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative exhibited nanomolar in-vitro activity against prostate cancer cells advanced prostate cancer (PC3) and micromolar inhibition of mammalian thioredoxin reductase. Encouraging maximum tolerable dose experiments led to human prostate cancer subcutaneous xenograft experiments; 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative were applied twelve times at two doses in groups of n = 5 PC3 to tumor-bearing NMRI:nu/nu mice. 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative at the dose of 10 and 20 mg/kg showed good tolerability, while no significant body weight loss was seen in both groups. In particular, for the drug 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate the tumor growth inhibition suggested to be dose dependent, reflected by the respective optimal T/C values of 0.45 at the dose of 10 mg/kg and of 0.31 at the dose of 20 mg/kg. By contrast, the 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative treated groups showed no indication for dose-dependent antitumoral activity, as reflected by the optimal T/C values of 0.44 for the 10 mg/kg and for the 20 mg/kg treated mice. Immunohistochemical experiments involving Ki67 staining of tumor tissue showed that both compounds reduced PC3 cell proliferation against the difficult to treat advanced human prostate tumors derived from PC3.

Synthesis and Cytotoxicity Studies of Novel NHC*-Gold(I) Complexes Derived from Lepidiline A.

Ten novel N-heterocyclic carbene gold(I) complexes derived from lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride) are reported here with full characterisation and biological testing. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (NHC*-AuCl) (1) was modified by substituting the chloride for the following: cyanide (2), dithiocarbamates (3⁻5), p-mercaptobenzoate derivatives (12⁻14) and N-acetyl-l-cysteine derivatives (15⁻17). All complexes were synthesised in good yields of 57⁻78%. Complexes 2, 12, 13, and 14 were further characterised by X-ray crystallography. Initial evaluation of the biological activity was conducted on all ten complexes against the multidrug resistant MCF-7topo breast cancer, HCT-116wt, and p53 knockout mutant HCT-116-/- colon carcinoma cell lines. Across the three cell lines tested, mainly single-digit micromolar IC50 values were observed. Nanomolar activity was exhibited on the MCF-7topo cell line with 3 displaying an IC50 of 0.28 µM ± 0.03 µM. Complexes incorporating a Au⁻S bond resulted in higher cytotoxic activity when compared to complexes 1 and 2. Theoretical calculations, carried out at the MN15/6⁻311++G(2df,p) computational level, show that NHC* is the more favourable ligand for Au(I)-Cl when compared to PPh3.

Synthesis and Biological Evaluation of Homogeneous Thiol-Linked NHC*-Au-Albumin and -Trastuzumab Bioconjugates.

Targeted delivery of potent cytotoxic drugs to cancer cells minimizes systemic toxicity and several side effects. NHC*-Au-Cl has already been proven to be a potent anticancer agent. In this study, we explore a strategy based on chemoselective cysteine conjugation of NHC*-Au-Cl to albumin and trastuzumab (Thiomab LC-V205C) to potentiate drug-ligand ratio, pharmacokinetics, as well as drug efficacy and safety. This strategy is a step forward towards the use of gold-based anticancer agents as targeted therapies.

The non-isomorphous crystal structures of NHC-Au-Cl and NHC-Au-Br (NHC is 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene).

Gold monochloride and monobromide can be transformed into monomeric complexes by ligands such as CO, PPh3 or Me2S, and such ligand-stabilized gold monochloride compounds have been investigated as catalysts, luminescent materials and anticancer drugs, especially when coordinated to a lipophilic benzyl-substituted N-heterocyclic carbene (NHC) ligand. The triclinic structures of NHC-Au-Cl {chlorido(1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene)gold, [AuCl(C29H24N2)]} and NHC-Au-Br {bromido(1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene)gold, [AuBr(C29H24N2)]}, determined by X-ray crystallography at 100 K, have one and four molecules, respectively, in their asymmetric units. The chloride compound shows an almost linear C-Au-Cl fragment [179.76 (8)°], with an Au-C distance of 1.976 (3) Šand an Au-Cl distance of 2.3013 (6) Å, while the bromide compound shows surprisingly large geometry deviations, from 1.969 (12) to 2.016 (10) Šfor the Au-C distance and from 2.4279 (14) to 2.4796 (12) Šfor the Au-Br distance, in the four independent molecules.

N-heterocyclic carbene metal complexes as bio-organometallic antimicrobial and anticancer drugs.

Late transition metal complexes that bear N-heterocyclic carbene (NHC) ligands have seen a speedy growth in their use as both, metal-based drug candidates and potentially active homogeneous catalysts in a plethora of C-C and C-N bond forming reactions. This review article focuses on the recent developments and advances in preparation and characterization of NHC-metal complexes (metal: silver, gold, copper, palladium, nickel and ruthenium) and their biomedical applications. Their design, syntheses and characterization have been reviewed and correlated to their antimicrobial and anticancer efficacies. All these initial discoveries help validate the great potential of NHC-metal derivatives as a class of effective antimicrobial and anticancer agents.

A comparative chemical-biological evaluation of titanium(IV) complexes with a salan or cyclopentadienyl ligand.

Titanium(IV) complexes with a salan or cyclopentadienyl ligand showed different biological behaviour concerning binding to biomolecules, cellular accumulation and intracellular distribution. Binding efficacy as well as trafficking on the cellular level are crucial parameters for their biological effects.

Assessment of serum cotinine in patients with chronic heart failure: self-reported versus objective smoking behaviour.

BACKGROUND: Smoking is a major risk factor in the development of coronary artery disease and thus chronic heart failure (HF). The value of self-reported smoking behaviour has not been validated in patients with HF. We sought to assess serum cotinine levels, a marker of recent tobacco exposure, in a cohort of clinically stable patients with chronic HF. METHODS AND RESULTS: We analysed serum cotinine values in 75 patients with chronic HF [mean age ± SD 64 ± 16 years, 85 % male, left ventricular ejection fraction 30 ± 1 %, New York Heart Association class (I/II vs. III/IV) 73 %/27 %, haemoglobin (Hb) 13.4 ± 1.5 g/dL, serum creatinine 1.21 ± 0.51 mg/dL] and 30 control subjects of similar age (63 ± 11 years, 43 % male, Hb 14.1 ± 1.5 g/dL, creatinine 1.12 ± 0.92 mg/dL) using a chemiluminescence immunoassay. Patients were interviewed about their smoking habits, and routine laboratory parameters were analysed. In patients with HF, cotinine values ranged from undetectable to 829 µg/L (mean 110 ± 208 µg/L). Similar findings were evident in healthy subjects with cotinine ranging from undetectable to 860 µg/L (mean 105 ± 208 µg/L). Serum cotinine levels correlated with leukocyte count and haemoglobin concentration (both p < 0.05). Self-reported smoking behaviour did not correspond to serum cotinine level in serum in 16.9 % of the patients with chronic HF. No such finding was evident in control subjects. CONCLUSIONS: Serum cotinine measurement provides an easily applicable means to analyse smoking behaviour in patients with chronic HF. Its assessment may permit analysis of smoking deception in daily clinical routine.

Alterations of phosphoproteins in NCI-H526 small cell lung cancer cells involved in cytotoxicity of cisplatin and titanocene Y.

First-line treatment of small cell lung cancer (SCLC) with combination chemotherapy consisting of cis-diamminedichloroplatinum(II) (cisplatin) and etoposide is frequently followed by early relapses and a dismal prognosis. Survival of a fraction of tumor cells and development of chemoresistance may be influenced by an initial cellular stress response against the administered xenobiotics. Therefore, we compared the short-term effects of cisplatin and non-cross-resistant bis-[(p-methoxybenzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene Y) on phosphorylation of 46 sites of a total of 38 signaling proteins in tumor suppressor protein 53 (p53)-wild-type NCI-H526 SCLC cells. The functional significance of selected kinases for the cytotoxicity of both drugs was tested using specific inhibitors and an activator. The cisplatin-induced cellular stress response involved activation of p38α mitogen-activated protein kinase, whereas Titanocene Y-triggered signaling affected c-Jun N-terminal kinase. Phosphorylation of adenosine monophosphate (AMP)-activated protein kinase α1 (AMPKα1) was increased by both drugs, which promoted cell survival, as indicated by results obtained using AMPK inhibitor compound C and AMPK activator 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside. This is in good agreement with previous reports, where AMPKα1 was demonstrated to represent an important factor for the sensitivity to cisplatin in colon and ovarian cancers, most likely by induction of autophagy. Thus, AMPKα1 constitutes a potential target to be exploited for chemotherapeutic treatment of SCLC to circumvent resistance to metal-based compounds.

Gold(I)-NHC complexes of antitumoral diarylimidazoles: structures, cellular uptake routes and anticancer activities.

Five new heterocyclic gold carbene complexes were prepared, four chlorido-[1,3-dimethyl-4,5-diarylimidazol-2-ylidene]gold complexes 6a-d and a chlorido-[1,3-dibenzylimidazol-2-ylidene]gold complex 11, and three of them were characterised by X-ray single crystal analyses. They were tested for cytotoxicity against a panel of four human cancer cell lines and non-malignant fibroblasts, for tubulin interaction, and for the pathways of their uptake into 518A2 melanoma cells. All complexes showed cytotoxic activity in the micromolar IC(50) range with distinct selectivities for certain cell lines. In stark contrast to related metal-free 1-methyl-4,5-diarylimidazoles, the complexes 6 and 11 did not noticeably inhibit the polymerisation of tubulin to give microtubules. The cellular uptake of complexes 6 occurred mainly via the copper transporter (Ctr1) and the organic cation transporters (OCT-1/2). Complex 11 was accumulated preferentially via the organic cation transporters and by Na(+)/K(+)-dependent endocytosis. The new gold carbene complexes seem to operate by a mechanism different from that of the parent 1-methylimidazolium ligands.

Anticancer activity and mode of action of titanocene C.

Titanocenes constitute a class of metal-based anticancer agents that seem to display a mode of action distinct from that of platinum complexes and to be more tolerable with a differing spectrum of activity. In the present study, titanocene C (bis-(N,N-dimethylamino-2(N-methylpyrrolyl)-methyl-cyclopentadienyl) titanium(IV) dichloride) was shown to exhibit antiproliferative activity against human tumor cell lines with a mean IC50 value of 48.3 ± 32.5 µM. In particular, high activity was found against small cell lung cancer (SCLC) cell lines with a profile different from cisplatin. Titanocene C induced cell cycle arrest at the G1/0-S interphase. Cross-resistance to either cisplatin or oxoplatin, respectively, was low for titanocene C and absent for titanocene Y in variant HL-60 cell lines. Alterations in gene expression of NCI-H526 SCLC cells induced by titanocene C were investigated using genome-wide expression arrays. Downregulation was found for genes coding for topoisomerases I and IIα, histones of the HIST1H4 cluster, enzymes involved in glycolysis, components of the cytoskeleton and vesicular transport, among others. In contrast, expression of genes involved in apoptosis, stress response, particularly members of the metallothionein gene cluster 1, DNA damage and growth factors was upregulated following exposure to titanocene C. Approximately 50% of those genes downregulated by titanocene C and cisplatin were concordant, including the previously identified markers of cisplatin-sensitivity, tubulin and stathmin, indicating partial overlap of the pathways affected by these metal complexes. The present findings point helicases/topoisomerases and HIST1H4 core histones out as targets of titanocene C and metallothioneins as putative main effectors of drug resistance.

Effects of titanocene dichloride derivatives on prostate cancer cells, specifically DNA damage-induced apoptosis.

BACKGROUND: While locally advanced prostate cancer is initially treatable with androgen ablation, eventually cells develop a castrate-resistant phenotype. Currently, there are no effective treatments for this form of the disease with Docetaxel only providing a small survival advantage. In this study, the effects of novel derivatives of titanocene dichloride on prostate cancer cell lines has been investigated. METHODS: Cellular effects were assessed using the crystal violet assay and the clonogenic survival assay. Cell cycle and apoptosis were assessed by propidium iodide staining. DNA damage was analyzed by comet assay and Western analysis. DNA damage response inhibition was achieved by pre-incubation with an ATM/ATR inhibitor; CGK733 and DNA-PK inhibitor; DMNB. RESULTS: These analogs caused a reduction in cell number. In particular titanocene Y and C had significant effects in all cell lines. A reduction in clonogenic survival was found in response to titanocene Y in three cell lines while the PC-3 cells exhibited increased resistance.Further analysis showed no effect on cell cycle however, the analogs were found to induce apoptosis in a dose-dependent manner in all cell lines. These analogs associate with DNA, induce DNA damage and a differential damage response. Inhibition of key regulators of this DNA damage response sensitized the PC-3 cell line to titanocene-induced apoptosis and significantly reduced the clonogenic capacity of the cells. CONCLUSION: These results demonstrate the mechanism of action of these novel titanocene dichloride analogs and their potential use in castrate-independent advanced prostate cancer.

Novel benzyl-substituted N-heterocyclic carbene-silver acetate complexes: synthesis, cytotoxicity and antibacterial studies.

From the reaction of 1-methylimidazole (1a), 4,5-dichloro-1H-imidazole (1b(I)) and 1-methylbenzimidazole (1c) with p-cyanobenzyl bromide (2a), non-symmetrically substituted N-heterocyclic carbene (NHC) [(3a-c)] precursors, 5,6-dimethyl-1H-benzimidazole (1d) and 4,5-diphenyl-1H-imidazole (1e) with p-cyanobenzyl bromide (2a) and benzyl bromide (2b), symmetrically substituted N-heterocyclic carbene (NHC) [(3d-f)] precursors were synthesised. These NHC-precursors were then reacted with silver(i) acetate to yield the NHC-silver complexes (1-methyl-3-(4-cyanobenzyl)imidazole-2-ylidene)silver(i)acetate (4a), (4,5-dichloro-1-(4-cyanobenzyl)-3-methyl)imidazole-2-ylidene)silver(i)acetate (4b), (1-methyl-3-(4-cyanobenzyl)benzimidazole-2-ylidene)silver(i)acetate (4c), (1,3-bis(4-cyanobenzyl)5,6-dimethylbenzimidazole-2-ylidene) silver(i) acetate (4d), (1,3-dibenzyl-5,6-dimethylbenzimidazole-2-ylidene) silver(i) acetate (4e) and (1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene) silver(i) acetate (4f) respectively. Three NHC-precursors 3c-e and four NHC-silver complexes 4b and 4d-f were characterised by single crystal X-ray diffraction. Preliminary in vitro antibacterial activity of the NHC-precursors and NHC-silver complexes was investigated against Gram-positive bacteria Staphylococcus aureus, and Gram-negative bacteria Escherichia coli using the qualitative Kirby-Bauer disk-diffusion method. NHC-silver complexes have shown very high antibacterial activity compared to the NHC-precursors. All six NHC-silver complexes were tested for their cytotoxicity through MTT based in vitro tests on the human renal-cancer cell line Caki-1 in order to determine their IC50 values. NHC-silver complexes 4a-f were found to have IC50 values of 6.2 (±1.0), 7.7 (±1.6), 1.2 (±0.6), 10.8 (±1.9), 24.2 (±1.8) and 13.6 (±1.0) µM, respectively. These values represent improved cytotoxicity against Caki-1, most notably for 4c, which is a three times more cytotoxic than cisplatin (IC50 value = 3.3 µM) itself.

Titanocene difluorides with improved cytotoxic activity.

Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me(3)SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.

Binding and hydrolysis studies of antitumoural titanocene dichloride and Titanocene Y with phosphate diesters.

The interaction of the antitumoural metallocene dihalides, titanocene dichloride (Cp(2)TiCl(2)) and Titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) chloride), with bis(4-nitrophenyl) phosphate (BNPP), which is a widely used model for the phosphate diester linkages in DNA, has been studied. Cp(2)TiCl(2) has been shown to promote the cleavage of the phosphate diester in weakly acidic solution. At pH 4, 37 degrees C, a 10(6)-fold rate acceleration over the uncatalysed reaction was observed under pseudo-first-order conditions, when freshly prepared solutions of Cp(2)TiCl(2) were applied. The activity of aged solutions dropped significantly due to the formation of insoluble precipitates of hydrolysed Ti species. The precipitates isolated from aged solutions were shown to act as moderately active, heterogeneous catalysts for BNPP cleavage. By contrast, no hydrolysis of the phosphate diester could be observed in the presence of Titanocene Y. Implications for the mode of action of the apoptosis-inducing metallocene dihalides are discussed.

Pseudo-halide derivatives of titanocene: synthesis and cytotoxicity studies.

The well-known anticancer drug candidate bis-[(p-methoxybenzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene ) was reacted with sodium azide or potassium cyanate, thiocyanate or selenocyanate in order to give pseudo-halide analogues of Titanocene . and were characterised by single crystal X-ray diffraction, which confirmed the expected nitrogen binding of the cyanate and thiocyanate to the titanium centre. All four titanocenes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney epithelial) cell line in an MTT based assay in order to determine their IC50 values. Titanocenes were found to have IC50 values of 24 (± 8) µM, 101 (± 14) µM, 54 (± 21) µM and 27 (± 4) µM respectively. All four titanocene derivatives show significant cytotoxicity improvement when compared to unsubstituted titanocene dichloride and and showed similiar cytotoxic behaviour to Titanocene in vitro.

Bioorganometallic fulvene-derived titanocene anti-cancer drugs.

6-Substituted fulvenes are interesting and easily accessible starting materials for the synthesis of novel substituted titanocenes via reductive dimerisation, carbolithiation or hydridolithiation reactions, which are followed by a transmetallation reaction with titanium tetrachloride in the latter two cases. Depending on the substitution pattern, these titanocenes prove to be bioorganometallic anti-cancer drugs, which have significant potential against advanced or metastatic renal-cell cancer. Patients bearing these stages of kidney cancer have a poor prognosis so far and therefore real progress in the area of metal-based anti-cancer drugs may come from this simple and effective synthetic approach. This tutorial review provides an insight into the synthesis of fulvene-derived titanocenes and their activity in preclinical experiments.

Analyses of Titanocenes in the spheroid-based cellular angiogenesis assay.

Titanocene dichloride and two of its derivatives (Titanocene Y and C) were tested on human umbilical vein endothelial cells (HUVEC) sprouting in a spheroid-based cellular angiogenesis assay in order to determine IC50 values of inhibition. Titanocene dichloride and Titanocene Y inhibited HUVEC sprouting in this angiogenesis assay with IC50 values of 19 microM and 4.9 microM, while Titanocene C surprisingly showed no inhibition. This classifies Titanocene dichloride as a purely anti-angiogenic anticancer drug and Titanocene C as a purely cytotoxic anticancer drug. On the other hand, Titanocene Y combines both favourable anticancer activities and seems to be the drug candidate of choice for further optimisation.