In this study, we have used [1H, 15N] NMR spectroscopy to investigate the interactions of the trinuclear platinum anticancer drug triplatin (1) (1,0,1/t,t,t or BBR3464) with site-specific sulfated and carboxylated disaccharides. Specifically, the disaccharides GlcNS(6S)-GlcA (I) and GlcNS(6S)-IdoA(2S) (II) are useful models of longer-chain glycosaminoglycans (GAGs) such as heparan sulfate (HS). For both the reactions of 15N-1 with I and II, equilibrium conditions were achieved more slowly (65 h) compared to the reaction with the monosaccharide GlcNS(6S) (9 h). The data suggest both carboxylate and sulfate binding of disaccharide I to the Pt with the sulfato species accounting for <1% of the total species at equilibrium. The rate constant for sulfate displacement of the aqua ligand (kL2) is 4 times higher than the analogous rate constant for carboxylate displacement (kL1). There are marked differences in the equilibrium concentrations of the chlorido, aqua, and carboxy-bound species for reactions with the two disaccharides, notably a significantly higher concentration of carboxylate-bound species for II, where sulfate-bound species were barely detectable. The trend mirrors that reported for the corresponding dinuclear platinum complex 1,1/t,t, where the rate constant for sulfate displacement of the aqua ligand was 3 times higher than that for acetate. Also similar to what we observed for the reactions of 1,1/t,t with the simple anions, aquation of the sulfato group is rapid, and the rate constant k-L2 is 3 orders of magnitude higher than that for displacement of the carboxylate (k-L1). Molecular dynamics calculations suggest that extra hydrogen-bonding interactions with the more sulfated disaccharide II may prevent or diminish sulfate binding of the triplatin moiety. The overall results suggest that Pt-O donor interactions should be considered in any full description of platinum complex cellular chemistry.

Metal complexes studied to date under the framework of metalloglycomics belong to the M-NH3 general motif (polynuclear platinum compounds; Werner's complex), acting mainly as cationic hydrogen bonding species toward glycosaminoglycans (GAGs), an interaction termed metalloshielding. In this paper, we expand our studies to substitution-inert octahedral cobalt(III) and ruthenium(II) complexes bearing the non‑hydrogen-donor ligand 2,2'-bipyridine (bpy). We identified by NMR spectroscopy that [Co(bpy)3]3+ binds to the highly sulfated synthetic pentasaccharide, Fondaparinux (FPX), while no major perturbations are found in the presence of [Ru(bpy)3]2+. This result is of significance as both coordination compounds have analogous 3D structures. Although weakly binding to the model GAG, [Ru(bpy)3]2+ completely inhibits the enzymatic cleavage of FPX by the bacterial heparinase II (HepII) enzyme, which is not observed for the Co(III) analog. This observation suggests a direct inhibition of HepII by the Ru compound, through a mechanism that is unrelated to metalloshielding.

We determine that the substitution-inert polynuclear platinum complex (PPC) TriplatinNC is an antiviral agent and protects cells from enterovirus 71 and human metapneumovirus infection. This protection occurs through the formation of adducts with cell-surface glycosaminoglycans. Our detailed mechanistic investigation demonstrates that TriplatinNC blocks viral entry by shielding cells from virus attack, opening new directions for metalloshielding antiviral drug development.

1 H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1 C4 :2 S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.

We report herein a detailed NMR study of the aquation and subsequent covalent binding of the trinuclear clinical agent [{ trans-PtCl(15NH3)2}2{μ- trans-Pt(15NH3)2(15NH2(CH2)615NH2)2}]4+ (1, 1,0,1/ t, t, t or Triplatin) with three d-glucosamine residues containing varied O-sulfate and N-sulfate or N-acetyl substitutions, which represent monosaccharide fragments present within the repeating disaccharide sequences of cell surface heparan sulfate (HS). The monosaccharides GlcNS(6S), GlcNS, and GlcNAc(6S) were synthesized in good yield from a common 4,6-diol α-methyl glucopyranoside intermediate. The reactions of 15N-1 with sodium sulfate, GlcNS(6S), GlcNS, and GlcNAc(6S) were followed by 2D [1H,15N] heteronuclear single quantum coherence (HSQC) NMR spectroscopy using conditions (298 K, pH ≈5.4) similar to those previously used for other anionic systems, allowing for a direct comparison. The equilibrium constants (p K1) for the aquation of 1 in the presence of GlcNS(6S) and GlcNS were slightly higher compared to that of the aquation in a sulfate solution, while a comparable p K1 value was observed in the presence of GlcNAc(6S). A comparison of the rate constants for sulfate displacement of the aqua ligand showed preferential binding to 2- N-sulfate compared to 6- O-sulfate but a more rapid liberation. For disulfated GlcNS(6S), equilibrium conditions were achieved rapidly (9 h) and strongly favored the dichloro form, with <2% sulfato species observed. The value of kL1 was up to 15-fold lower than that for binding to sulfate, whereas the rate constant for the reverse ligation ( k-L1) was comparable. Equilibrium conditions were achieved much more slowly (∼ 100 h) for the reactions of 1 with GlcNS and GlcNAc(6S), attributed to covalent binding also to the N-donor of the sulfamate (GlcNS) group and the O-donor of the N-acetyl [GlcNAc(6S)] group. The rate constants ( kL2) were 20-40-fold lower than that for binding to the 2- N- or 6- O-sulfate, but the binding was less reversible, so that their equilibrium concentrations (5-8%) were comparable to the 2- N- or 6- O-sulfate-bound species. The results emphasize the relevance of glycans in bioinorganic chemistry and underpin a fundamental molecular description of the HS-Pt interactions that alter the profile of platinum agents from cytotoxic to metastatic in a systematic manner.

Reported herein is a detailed NMR and DFT study of the interaction of the 15N-labelled dinuclear platinum anticancer compound [{cis-PtCl(NH3)2}2{μ-H2N(CH2)6NH2}]2+ (15N-1, 1,1/c,c) with 1,2-dihexanoyl-sn-glycero-3-phosphate (DHPA), as a comparison with an earlier study of the interaction of the same water-soluble phospholipid fragment with the geometric trans isomer (1,1/t,t). The reaction of 15N-1 with the sodium salt of DHPA was studied at 298 K, pH ∼ 5.6, by [1H,15N] HSQC 2D NMR spectroscopy. The NMR data, supported by DFT models, provide evidence that the monofunctional DHPA adduct of 15N-1 exists in two conformational forms, with different orientation of the (CH2)6 linker; one has an interaction between the unbound {PtN3Cl} moiety and the coordinated DHPA molecule. Similarly, two bifunctional adduct conformers are identified, in which one has an interaction between the phosphate groups of the two bound DHPA molecules. When compared to the previously reported reactions of 1,1/t,t with DHPA, equilibrium conditions of the 1,1/c,c reaction are reached more slowly (120 h), similar to the reaction with phosphate. The rate constant for the first step of DHPA binding (kL) is slightly lower (1.6 fold) for the cis-compared to the trans-isomer, whereas the rate constant for the reverse reaction is 4-fold lower, resulting in a much greater proportion of DHPA bound species at equilibrium.

Platinum complexes with S- and N-donor small molecule ligands have received much attention with respect to understanding of Pt-protein and Pt-DNA(RNA) interactions in biology. Oxygen-donor ligands have received less attention, partly due to the fact that as a hard Lewis base, oxygen-donor interactions are expected to be less favourable for the soft Lewis acid properties of Pt(II), especially. Yet, it is now clear that for a full understanding of the cellular fate of platinum complexes, a plethora of oxygen-donor interactions are possible, considering extracellular and intracellular concentrations of simple anions in buffer. Further, the importance of the general class of glycans, the third major class of biomolecules after proteins and nucleic acids, contain many specific examples of important biomolecules such as sialic acids and sulphated glycosaminoglycans capable of metal complex interactions. In this contribution we summarise some important kinetic and thermodynamic aspects of platinum-oxygen-donor ligand interactions and their relevance to examples of biomolecular interactions contributing to the overall profile of platinum (and metal complexes in general) biology.

We report a detailed NMR and DFT study of the interaction of polynuclear platinum anticancer agents (PPCs) with negatively charged phospholipids as a mechanism for their cellular uptake. The reactions of fully 15 N-labelled [{trans-PtCl(NH3 )2 }2 (μ-trans-Pt(NH3 )2 {NH2 (CH2 )6 NH2 }2 )]4+ (15 N-1, 1,0,1/t,t,t) and the dinuclear [{trans-PtCl(NH3 )2 }2 {μ-H2 N(CH2 )6 NH2 }]2+ (15 N-2, 1,1/t,t) with the sodium salt of 1,2-dihexanoyl-sn-glycero-3-phosphate (DHPA) were studied at 298 K, pH ≈5.4, by [1 H,15 N] HSQC 2D NMR spectroscopy. Both 15 N-1 and 15 N-2 form an initial mono-adduct in which the DHPA is coordinated via the phosphate O atom. For the dinuclear 15 N-2, coordination of a second DHPA, in two different orientations, leads to two conformers of the bifunctional adduct. For 15 N-1, coordination of the second DHPA allows the central {PtN4 } coordination unit to bind electrostatically to two additional DHPA molecules via phosphate clamp interactions, in an extended network. For both 1,0,1/t,t,t (1) and 1,1/t,t (2), equilibrium conditions are obtained more slowly (>35 h) than in the presence of phosphate (12 h) and in each case the rate constant for the first step of DHPA binding (kL ) is about 8 times higher than that for phosphate, whereas the rate constants for the reverse reactions are quite similar. Reaction of 15 N-1 with the sodium salt of 1,2-dihexanoyl-sn-glycero-3-[phosphatidyl-l-serine] (DHPS) showed only minor adduct formation via coordination to the N-donor atom of the phosphoserine group.

Cleavage of heparan sulfate proteoglycans (HSPGs) by the enzyme heparanase modulates tumour-related events including angiogenesis, cell invasion, and metastasis. Metalloshielding of heparan sulfate (HS) by positively charged polynuclear platinum complexes (PPCs) effectively inhibits physiologically critical HS functions. Studies using bacterial P. heparinus heparinaseII showed that a library of Pt complexes varying in charge and nuclearity and the presence or absence of a dangling amine inhibits the cleavage activity of the enzyme on the synthetic pentasaccharide, Fondaparinux (FPX). Charge-dependent affinity of PPC for FPX was seen in competition assays with methylene blue and ethidium bromide. The dissociation constant (K-d) of TriplatinNC for FPX was directly measured by isothermal titration calorimetry (ITC). The trend in DFT calculated interaction energies with heparin fragments is consistent with the spectroscopic studies. Competitive inhibition of TAMRA-R-9 internalization in human carcinoma (HCT116) cells along with studies in HCT116, wildtype CHO and mutant CHO-pgsA745 (lacking HS/CS) cells confirm that HSPG-mediated interactions play an important role in the cellular accumulation of PPCs.

Due to the clinical success and limitations of platinum-based anticancer drugs, there has been considerable interest in the development of new therapeutic agents based upon other transition metals, in particular ruthenium. There have now been many studies demonstrating the anticancer and antimicrobial properties of kinetically-inert polypyridylruthenium(II) complexes. This review showcases the diverse range of polypyridylruthenium complexes that exhibit significant biological activity, and discusses the relationship between their chemical structure and biological processing (cellular uptake and localisation) in eukaryotic cells. Perhaps more importantly, this review also highlights several recent studies that have shown kinetically-inert polypyridylruthenium(II) complexes can exhibit anticancer activity in in vivo trials - studies that provide the "proof of concept" that this class of metal-based agent has real clinical potential. (C) 2017 Elsevier B.V. All rights reserved.

The relatively non-toxic family of cucurbit[n]uril, Q[n], have shown considerable potential in vitro as drug delivery agents, with only a few examples of pharmacokinetic (PK) studies for drug⊂Q[n]. Drug-free Q[n] PK studies are the next step in determining the pharmacological applicability in their drug delivery potential. The results for the first PK and bio-distribution of drug-free 14C-Q[7] are described for administration via intravenous (i.v.) and intraperitoneal (i.p.) dosing. A study of oral administration of drug-free 14C-Q[8] has also been undertaken to determine the time course for the gastrointestinal tract (GIT), absorption and subsequent bio-distribution. Q[10], a potential drug carrier for larger drugs, was evaluated for its effect on the PK profile of a dinuclear ruthenium complex (Rubb12), a potential antimicrobial agent. The Rubb12⊂Q[10] complex and free Rubb12 were administered by i.v. to determine differences in Rubb12 plasma concentrations and organ accumulation. Interestingly, the PK profiles and bio-distribution observed for Q[7] showed similarities to those of Rubb12⊂Q[10]. Drug-free Q[7] has a relatively fast plasma clearance and a generally low organ accumulation except for the kidneys. Drug-free Q[8] showed a low absorption from the GIT into the blood stream but the small percentage absorbed reflected the organ accumulation of Q[7]. These results provide a better understanding of the probable PK profile and bio-distribution for a drug⊂Q[n] through the influence of the drug delivery vehicle and the positive clearance of drug-free Q[n] via the kidneys supports its potential value in future drug delivery applications.

Condensation studies of chromosomal DNA in E. coli with a tetranuclear ruthenium complex are carried out and images obtained with wide-field fluorescence microscopy. Remarkably different condensate morphologies resulted, depending upon the treatment protocol. The occurrence of condensed nucleoid spirals in live bacteria provides evidence for the transertion hypothesis.

OBJECTIVESThe objectives of this study were to: (i) determine the in vitro activities of a series of di-, tri- and tetra-nuclear ruthenium complexes (Rubbn, Rubbn-tri and Rubbn-tetra) against a range of Gram-positive and -negative bacteria and compare the antimicrobial activities with the corresponding toxicities against eukaryotic cells; and (ii) compare MIC values with achievable in vivo serum concentrations for the least toxic ruthenium complex.METHODSThe in vitro activities were determined by MIC assays and time-kill curve experiments, while the toxicities of the ruthenium complexes were determined using the Alamar blue cytotoxicity assay. A preliminary pharmacokinetic study was undertaken to determine the Rubb12 serum concentration in mice as a function of time after administration.RESULTSRubb12, Rubb12-tri and Rubb12-tetra are highly active, with MIC values of 1-2 mg/L (0.5-1.5 μM) for a range of Gram-positive strains, but showed variable activities against a panel of Gram-negative bacteria. Time-kill experiments indicated that Rubb12, Rubb12-tri and Rubb12-tetra are bactericidal and kill bacteria within 3-8 h. The di-, tri- and tetra-nuclear complexes were ∼50 times more toxic to Gram-positive bacteria and 25 times more toxic to Gram-negative strains, classified as susceptible, than to liver and kidney cells. Preliminary pharmacokinetic experiments established that serum concentrations higher than MIC values can be obtained for Rubb12 with an administered dose of 32 mg/kg.CONCLUSIONSThe ruthenium complexes, particularly Rubb12, have potential as new antimicrobial agents. The structure of the dinuclear ruthenium complex can be readily further modified in order to increase the selectivity for bacteria over eukaryotic cells.

Ruthenium(II) complexes containing the tetradentate ligand bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ("bbn "; n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bbn )](2+) (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-α-[Ru(phen)(bb12 )](2+) species showed good activity against Gram-negative species. In particular, the cis-α-[Ru(phen)(bb12 )](2+) complex was two to four times more active than the cis-β-[Ru(phen)(bb12 )](2+) complex against the Gram-negative strains. The cis-α- and cis-β-[Ru(phen)(bb12 )](2+) complexes readily accumulated in the bacteria but, significantly, showed the highest level of uptake in Pseudomonas aeruginosa. Furthermore, the accumulation of the cis-α- and cis-β-[Ru(phen)(bb12 )](2+) complexes in P. aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-α-[Ru(phen)(bb12 )](2+) complex into live P. aeruginosa was confirmed by using fluorescence microscopy. The water/octanol partition coefficients (log P) were determined to gain understanding of the relative cellular uptake. The cis-α- and cis-β-[Ru(phen)(bbn )](2+) complexes exhibited relatively strong binding to DNA (Kb ≈10(6)  M(-1) ), but no significant difference between the geometric isomers was observed.

Confocal microscopy was used to study the intracellular localisation of a series of inert polypyridylruthenium(II) complexes with three eukaryotic cells lines - baby hamster kidney (BHK), human embryonic kidney (HEK-293) and liver carcinoma (Hep-G2). Co-staining experiments with the DNA-selective dye DAPI demonstrated that the di-, tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,12-dodecane bridging ligand ("bb12") showed a high degree of selectivity for the nucleus of the eukaryotic cells. Additional co-localisation experiments with the general nucleic acid stain SYTO 9 indicated that the ruthenium complexes showed a considerable preference for the RNA-rich nucleolus, rather than chromosomal DNA. No significant differences were observed in the intracellular localisation between the ΔΔ and ΛΛ enantiomers of the dinuclear complex. Cytotoxicity assays carried out over 72 hours indicated that the ruthenium complexes, particularly the tri- and tetra-nuclear species, were significantly toxic to the eukaryotic cells. However, when the activity of the least cytotoxic compound (the ΔΔ enantiomer of the dinuclear species) was determined over a 24 hour period, the results indicated that the ruthenium complex was approximately a 100-fold less toxic to liver and kidney cells than to Gram positive bacteria. Circular dichroism (CD) spectroscopy was used to examine the effect of the ΔΔ and ΛΛ enantiomers of the dinuclear complex on the solution conformations of RNA and DNA. The CD experiments indicated that the RNA maintained the A-type conformation, and the DNA the B-type structure, upon binding by the ruthenium complexes.

The dinuclear ruthenium complex with X = H is four-times more cytotoxic than cisplatin against breast cancer cell lines; however, when X = NO2 the ruthenium complex is less active than cisplatin.

A series of inert tri- and tetra-nuclear polypyridylruthenium(II) complexes that are linked by the bis[4(4'-methyl-2,2'-bipyridyl)]-1,n-alkane ligand ("bb(n)" for n = 10, 12 and 16) have been synthesised and their potential as antimicrobial agents examined. Due to the modular nature of the synthesis of the oligonuclear complexes, it was possible to make both linear and non-linear tetranuclear ruthenium species. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria--Gram positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). In order to gain an understanding of the relative antimicrobial activities, the cellular uptake and water-octanol partition coefficients (log P) were determined for a selection of the ruthenium complexes. Although the trinuclear complexes were the most lipophilic based upon log P values and showed the greatest cellular uptake, the linear tetranuclear complexes were generally more active, with MIC values <1 μM against the Gram positive bacteria. Similarly, although the non-linear tetranuclear complexes were slightly more lipophilic and were taken up to a greater extent by the bacteria, they were consistently less active than their linear counterparts. Of particular note, the cellular accumulation of the oligonuclear ruthenium complexes was greater in the Gram negative strains compared to that in the Gram positive S. aureus and MRSA. The results demonstrate that the lower antimicrobial activity of polypyridylruthenium(II) complexes towards Gram negative bacteria, particularly P. aeruginosa, is not strongly correlated to the cellular accumulation but rather to a lower intrinsic ability to kill the Gram negative cells.