Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides

Novel mutations in TLR genes cause hyporesponsiveness to Mycobacterium avium subsp. paratuberculosis infection

<p>Abstract</p> <p>Background</p> <p>Toll like receptors (TLR) play the central role in the recognition of pathogen associated molecular patterns (PAMPs). Mutations in the TLR1, TLR2 and TLR4 genes may change the ability to recognize PAMPs and cause altered responsiveness to the bacterial pathogens.</p> <p>Results</p> <p>The study presents association between TLR gene mutations and increased susceptibility to <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP) infection. Novel mutations in TLR genes (TLR1- Ser150Gly and Val220Met; TLR2 – Phe670Leu) were statistically correlated with the hindrance in recognition of MAP legends. This correlation was confirmed subsequently by measuring the expression levels of cytokines (IL-4, IL-8, IL-10, IL-12 and IFN-γ) in the mutant and wild type moDCs (mocyte derived dendritic cells) after challenge with MAP cell lysate or LPS. Further <it>in silico </it>analysis of the TLR1 and TLR4 ectodomains (ECD) revealed the polymorphic nature of the central ECD and irregularities in the central LRR (leucine rich repeat) motifs.</p> <p>Conclusion</p> <p>The most critical positions that may alter the pathogen recognition ability of TLR were: the 9^thamino acid position in LRR motif (TLR1–LRR10) and 4^thresidue downstream to LRR domain (exta-LRR region of TLR4). The study describes novel mutations in the TLRs and presents their association with the MAP infection.</p

Organometallic half-sandwich iridium anticancer complexes

The low-spin 5d6 IrIII organometallic half-sandwich complexes [(η5-Cpx)Ir(XY)Cl]0/+, Cpx = Cp*, tetramethyl(phenyl)cyclopentadienyl (Cpxph), or tetramethyl(biphenyl)cyclopentadienyl (Cpxbiph), XY = 1,10-phenanthroline (4−6), 2,2′-bipyridine (7−9), ethylenediamine (10 and 11), or picolinate (12−14), hydrolyze rapidly. Complexes with N,N-chelating ligands readily form adducts with 9-ethylguanine but not 9-ethyladenine; picolinate complexes bind to both purines. Cytotoxic potency toward A2780 human ovarian cancer cells increases with phenyl substitution on Cp*: Cpxbiph > Cpxph > Cp*; Cpxbiph complexes 6 and 9 have submicromolar activity. Guanine residues are preferential binding sites for 4−6 on plasmid DNA. Hydrophobicity (log P), cell and nucleus accumulation of Ir correlate with cytotoxicity, 6 > 5 > 4; they distribute similarly within cells. The ability to displace DNA intercalator ethidium bromide from DNA correlates with cytotoxicity and viscosity of Ir−DNA adducts. The hydrophobicity and intercalative ability of Cpxph and Cpxbiph make a major contribution to the anticancer potency of their IrIII complexes

Differences in the cellular response and signaling pathways between cisplatin and monodentate organometallic Ru(II) antitumor complexes containing a terphenyl ligand

The new monofunctional Ru(II)-arene complex [(η6-arene)Ru(II)(en)Cl]+, where en = 1,2-diaminoethane and the arene is para-terphenyl (complex 1) exhibits promising cytotoxic effects in human tumor cells including those resistant to conventional cisplatin (J. Med. Chem.2008, 51, 5310). The present study is focused on the cellular pharmacology of 1 to elucidate more deeply the mechanisms underlying its antitumor effects. We have identified several cellular mechanisms induced by 1 in human ovarian carcinoma cells, including inhibition of DNA synthesis, overexpression and activation of p53, expression of proapoptotic proteins p21WAF1 and Bax, G0/G1 arrest, and nuclear fragmentation as a result of apoptotic, and, to a much lower extent, also necrotic processes. Thus, 1 inhibits growth of the cancer cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigations have shown that 1 induces apoptosis by regulating the expression of Bcl-2 family proteins. There were significant differences in cellular responses to the treatment with 1 and with conventional cisplatin, particularly in the kinetics and the extent of these responses. In addition, the distinct p53 activation profile of 1 compared with cisplatin provides an explanation for the activity of this ruthenium drug against cisplatin-resistant cells. Hence complex 1 may provide an alternative therapy in patients with acquired cisplatin resistance, particularly with respect to its very low mutagenicity and different mode of action compared to platinum antitumor drugs in clinical use

Self-Assembly of Cationic, Hetero- or Homonuclear Ruthenium(II) Macrocyclic Rectangles and Their Photophysical, Electrochemical, and Biological Studies

A series of supramolecular rectangles, including two mixed-metal Ru/Pt complexes, have been formed by the coordination-driven self-assembly of a range of arene-Ru &quot;molecular clip&quot; acceptors (la id) with rigid dipyridyl-based ligands (2a-2d) over the course of 10 h in solution. The isolated products were characterized by multinuclear NMR ((1)H and (13)C or (31)P), HR-ESI-MS, and an X-ray diffraction study to support the ascribed two-component rectangular structures. The rectangles were further characterized by UV-vis and fluorescence studies. The redox behaviors of rectangles 3ca and 3da were also determined using cyclic voltammetry. Additionally, the antitumor activities of the suite of rectangles were determined against various human cancer cell lines, and significant activity was shown by complexes 3ca, 3da, 3cb, 3cc, and 3cd, with IC(50) values as low as 2.65 mu M.close171