A novel rhodium(iii) complex Rh
(H
L
)Cl
(1) (H
L
= 2,6-bis(5-tert-butyl-1H-pyrazol-3-yl)pyridine) containing a pincer type, tridentate nitrogen-donor chelate system was synthesized. Single crystal ...X-ray structure analysis revealed that 1 crystallizes in the orthorhombic space group Pbcn with a = 20.7982(6), b = 10.8952(4), c = 10.9832(4) Å, V = 2488.80(15) Å
, and eight molecules in the unit cell. The rhodium center in the complex Rh
(H
L
)Cl
(1) is coordinated in a slightly distorted octahedral geometry by the tridentate N,N,N-donor and three chloro ligands, adopting a mer arrangement with an essentially planar ligand skeleton. Due to the tridentate coordination of the N,N,N-donor, the central nitrogen atom N1 is located closer to the Rh
center. The reactivity of the synthesized complex toward small biomolecules (l-methionine (l-Met), guanosine-5'-monophosphate (5'-GMP), l-histidine (l-His) and glutathione (GSH)) and to a series of duplex DNAs and RNA was investigated. The order of reactivity of the studied small biomolecules is: 5'-GMP > GSH > l-Met > l-His. Duplex RNA reacts faster with the Rh
(H
L
)Cl
complex than duplex DNA, while shorter duplex DNA (15mer GG) reacts faster compared with 22mer GG duplex DNA. In addition, a higher reactivity is achieved with a DNA duplex with a centrally located GG-sequence than with a 22GTG duplex DNA, in which the GG-sequence is separated by a T base. Furthermore, the interaction of this metal complex 1 with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) was examined by absorption (UV-Vis) and emission spectral studies (EthBr displacement studies). Overall, the studied complex exhibited good DNA and BSA interaction ability.
The kinetics for the binding of cisplatin to duplex RNAs, two fully complementary model systems and mature miR-146a, exhibits a linear dependence on cisplatin concentration and results in duplex ...dissociation at 38 °C.
The ability of the anticancer active drug cisplatin to exert biological activity through interference with nucleic acid function is well documented. Since kinetics play a key role in determining ...product distributions in these systems, methods for accurate documentation of reactivity serve the purpose to identify preferential metal binding sites. In the present study, the aim has been to further explore a recently communicated approach (C. Polonyi and S. K. C. Elmroth, J. Chem. Soc., Dalton Trans., 2013, 42, 14959-14962) utilizing UV/vis spectroscopy and metal induced duplex RNA melting for monitoring of kinetics. More specifically, the sensitivity of the UV/vis-methodology has been evaluated by investigation of how overall length and changes of base-pairing in the close vicinity of a centrally located GG-site affect the rate of cisplatin binding, using the intracellularly active mono-aquated form of cisplatin (cis-Pt(NH3)2Cl(OH2)(+), ()) as the platination reagent. For this purpose, the reactivity of five different 13- to 17 base-pair duplex RNAs was monitored at 38 °C. A common trend of a ca. 10-fold reduction in reactivity was found to accompany an increase of bulk sodium concentration from CNa+ = 122 mM to 1.0 M. Typical half-lives are exemplified by the interaction of with the fully complementary 15-mer RNA-1 with t1/2 = ca. 0.5 and 4.8 hours, at CNa+ = 122 mM and 1.0 M respectively, and C = 45 μM. Lowering of melting temperature (Tm) was found to promote reactivity regardless of whether the change involved a decrease or increase of the RNA length. For example, at CNa+ = 1.0 M, truncation of the fully complementary and GG-containing 15-mer RNA-1 (Tm = 68.9 °C) to the 13-mer RNA-1-1-S (Tm = 63.9 °C) resulted in an increase of k2,app from ca. 0.9 M(-1) s(-1) to 2.0 M(-1) s(-1). Further, the 17-mer RNA-1-4 (Tm = 42.0 °C) with a central U4 bulge exhibited the highest reactivity of the sequences studied with k2,app = 4.0 M(-1) s(-1). The study shows that the reactivity of GG-sequences in RNA exhibit a strong variation depending on exact sequence context, and with imperfectly matched and/or stacked regions as particularly reactive sites.
Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be ...identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3′ UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.
The cytotoxicity and photocytotoxicity of trans-RuCl2(DMSO)4 and cis-RuCl2(DMSO)4 complexes was tested in two melanoma cell lines, human (SK-MEL 188) and mouse (S91). The trans isomer was found to be ...more effective for cell growth inhibition than its cis analogue both in the presence and in the absence of illumination. However, the antiproliferative activity of both isomers was significantly enhanced after irradiation with UVA light in comparison with their activity observed in the dark. The influence of light on the reaction of both ruthenium(II) isomers with the single-stranded hexanucleotide d(T2GGT2), chosen as a model system for DNA, was also studied using chromatography and mass spectrometry techniques. The photochemical reaction of the ruthenium(II) complexes with the oligonucleotide d(T2GGT2) resulted in the formation of Ru(G−N7)2 adducts, which was not observed in the same time scale in thermal reactions. The initial short irradiation of the inert cis isomer was found to facilitate the covalent adduct formation with d(T2GGT2) in the secondary thermal reactions and with a rate comparable to that found for the trans isomer, which is ca. 5−10 times more reactive in the dark.
Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a ...platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.
Nucleic acids are well recognized targets for platinum-based anticancer drugs, with RNA and DNA being kinetically comparable. In the case of RNA, previous studies have shown that the reaction between ...small duplex RNAs (dsRNAs) and monoaquated cisplatin (cis-Pt(NH3)2Cl(OH2)(+), ) can be followed by the metal induced hyperchromicity occurring directly after addition of to e.g. microRNA mimics. In the present study, we have used this approach to compare thermal stability and reactivity between intracellularly- and extracellularly relevant salt concentration (CNa(+) and CK(+)ca. 0.1 M), and also as a function of increased hydrophobicity (10% v/v EtOH). In addition, reactivity was studied as a function of temperature in the interval ca. 5-20 °C below the respective dsRNA melting temperatures (Tms). Four different 13- to 20-mer dsRNAs with two different central sequence motifs were used as targets containing either a central r(GG)·r(CC)- or r(GG)·r(UAU)-sequence. The reactions exhibited half-lives in the minute- to hour range at 38 °C in the presence of excess in the μM range. Further, a linear dependence was found between C and the observed pseudo-first-order rate constants. The resulting apparent second-order rate constants were significantly larger for the lower melting r(GG)·r(UAU)-containing sequences compared with that of the fully complementary ones; the higher and lower reactivities represented by RNA-1-3 and RNA-1-1 with k2,appca. 30 and 8 M(-1) s(-1) respectively at CNa(+) = 122 mM. For all RNAs a common small, but significant, trend was observed with increased reactivity in the presence of K(+) compared with Na(+), and decreased reactivity in the presence of EtOH. Finally, the temperature dependence of k2,app was evaluated using the Eyring equation. The retrieved activation parameters reveal positive values for both ΔH(≠) and ΔS(≠) for all dsRNAs, in the range ca. 23-34 kcal mol(-1) and 22-57 cal K(-1) mol(-1) respectively. These values indicate solvational effects to be important for the rate determining step of the reaction, and thus in support of a structural change of the dsRNA to take place in parallel with the adduct formation step.
Thermodynamic parameters are presented here illustrating the effects caused by the two anticancer active metal complexes cisplatin and oxaliplatin after introduction into four closely related RNA and ...DNA duplexes. The duplexes used are blunt end, fully complementary 15‐mer duplexes with a centrally located either GG‐ or GNG (here: N = T or U) binding site. For all duplexes, a common trend of reduced melting temperature was observed after platination. Analysis of the thermodynamic parameters for the duplex dissociation reactions showed good correlation between variations in melting temperatures (Tm) and ground state enthalpies (ΔH) in both DNA‐ and RNA duplexes. The melting temperatures of the native duplexes were found to be determined by their chemical nature, i.e. with observed Tm ‐values of ca. 50 °C for DNA and ca. 61 °C for RNA (CT = 2 μM and CNa+ = 129 mM, pH 6.3). Of the two types of nucleic acids, RNA is the one that exhibits the most pronounced sensitivity towards introduction of the platinum complexes, and with oxaliplatin as the more influential metalation reagent. Of note is that the thermal destabilization caused by oxaliplatin interacting with a centrally located GUG‐sequence results in a duplex stability below native DNA.
The DNA interference pathways exhibited by cisplatin and related anticancer active metal complexes have been extensively studied. Much less is known to what extent RNA interaction pathways may ...operate in parallel, and perhaps contribute to both antineoplastic activity and toxicity. The present study was designed with the aim of comparing the reactivity of two model systems comprising RNA and DNA hairpins, r(CGCGUUGUUCGCG) and d(CGCGTTGTTCGCG), towards a series of platinum(II) complexes. Three platinum complexes were used as metallation reagents; cis-PtCl(NH3)2(OH2)+ (1), cis-PtCl(NH3)(c-C6H11NH2)(OH2)+ (2), and trans-PtCl(NH3)(quinoline)(OH2)+ (3). The reaction kinetics were studied at pH 6.0, 25 degrees C, and 1.0 mM < or = I < or = 500 mM. For both types of nucleic acid targets, compound 3 was found to react about 1 order of magnitude more rapidly than compounds 1 and 2. Further, all platinum compounds exhibited a more pronounced salt dependence for the interaction with r(CGCGUUGUUCGCG). Chemical and enzymatic cleavage studies revealed similar interaction patterns with r(CGCGUUGUUCGCG) after long exposure times to 1 and 2. A substantial decrease of cleavage intensity was found at residues G4 and G7, indicative of bifunctional adduct formation. Circular dichroism studies showed that platinum adduct formation leads to a structural change of the ribonucleic acid. Thermal denaturation studies revealed platination to cause a decrease of the RNA melting temperatures by 5-10 degrees C. Our observations therefore suggest that RNA is a kinetically competitive target to DNA. Furthermore, platination causes destabilization of RNA structural elements, which may lead to deleterious intracellular effects on biologically relevant RNA targets.
Modification and optimization of the anticancer drug cisplatin is of interest with respect to selective cell targeting and DNA binding efficiency. Attractive approaches contain both, modification of ...the platinum coordination sphere and design of hybrid molecules of the cisplatin binding moiety including peptide motifs. Peptides with cell penetrating, directing or recognizing properties can be implemented. In this study, positively charged peptide sequences were investigated with the potential of inducing DNA structural distortions caused by charge neutralization of the dsDNA helix. Association of charged peptides is likely to increase the flexibility of the DNA thereby facilitating platinum binding. The synthesis and DNA interaction of five new cisplatin–peptide hybrids with enhanced solubility and potential antitumor activity is presented. Propylenediamine or bisimidazole units were used as bisdentate platinum ligands and were coupled to a peptide sequence in the final elongation step of the solid‐phase peptide synthesis (SPPS). Agarose and polyacrylamide gel electrophoresis, fluorescence intercalation, and thermal UV melting studies, all support the presence of covalently formed platinum DNA adducts in a reaction mediated by the positively charged peptide.
Cisplatin analogs based on cationic peptide/metal binding hybrids and differing in the number of peptide charges and the platinum coordination sites were prepared. Their cooperativity was investigated with respect to covalent DNA modification considering the influence of DNA conformational change by charge‐charge interaction and the significance of platinumligation.
PDF
