Probing Disaccharide Binding to Triplatin as Models for Tumor Cell Heparan Sulfate (GAG) Interactions.

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.

On the Biology of Werner's Complex.

Werner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof-of-principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non-covalent hydrogen-bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound.

Conformational Modulation of Iduronic Acid-Containing Sulfated Glycosaminoglycans by a Polynuclear Platinum Compound and Implications for Development of Antimetastatic Platinum Drugs.

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.

Glycans as Ligands in Bioinorganic Chemistry. Probing the Interaction of a Trinuclear Platinum Anticancer Complex with Defined Monosaccharide Fragments of Heparan Sulfate.

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.

Biological relevance of interaction of platinum drugs with O-donor ligands.

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.

Structural Factors Affecting Binding of Platinum Anticancer Agents with Phospholipids: Influence of Charge and Phosphate Clamp Formation.

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.

Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate.

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 heparinase II 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 (Kd ) 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-R9 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.

Chimeric platinum-polyamines and DNA binding. Kinetics of DNA interstrand cross-link formation by dinuclear platinum complexes with polyamine linkers.

The first observation of a polyamine-DNA interaction using 2D [(1)H, (15)N] HSQC NMR spectroscopy allows study of the role of the linker in polynuclear platinum-DNA interactions and a novel "anchoring" of the polyamine by Pt-DNA bond formation allows examination of the details of conformational B → Z transitions induced by the polyamine. The kinetics and mechanism of the stepwise formation of 5'-5' 1,4-GG interstrand cross-links (IXLs) by fully (15)N-labeled [{trans-PtCl((15)NH(3))(2)}(2){µ-((15)NH(2)(CH(2))(6)(15)NH(2)(CH(2))(6)(15)NH(2))}](3+) (1,1/t,t-6,6, 1) and [{trans-PtCl((15)NH(3))(2)}(2){µ-((15)NH(2)(CH(2))(6)(15)NH(2)(CH(2))(2)(15)NH(2)(CH(2))(6)(15)NH(2))}](4+) (1,1/t,t-6,2,6, 1') with the self-complementary oligonucleotide 5'-{d(ATATGTACATAT)(2)} (duplex I) are compared to the analogous reaction with 1,0,1/t,t,t (BBR3464) under identical conditions (pH 5.4, 298 K). Initial electrostatic interactions with the DNA are delocalized and followed by aquation to form the monoaqua monochloro species. The rate constant for monofunctional adduct formation, k(MF), for 1 (0.87 M(-1) s(-1)) is 3.5 fold higher than for 1,0,1/t,t,t (0.25 M(-1) s(-1); the value could not be calculated for 1' due to peak overlap). The evidence suggests that several conformers of the bifunctional adduct form, whereas for 1,0,1/t,t,t only two discrete conformers were observed. The combined effect of the conformers observed for 1 and 1' may play a crucial role in the increased potency of these novel complexes compared to 1,0,1/t,t,t. Treated as a single final product, the rate of formation of the 5'-5' 1,4-GG IXL, k(CH), for 1 (k(CH) = 4.37 × 10(-5) s(-1)) is similar to that of 1,0,1/t,t,t, whereas the value for 1' is marginally higher (k(CH) = 5.4 × 10(-5) s(-1)).

Metalloglycomics of tris(2,2'-bipyridyl) cobalt and ruthenium compounds.

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 non­hydrogen-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.

Platinum complexes act as shielding agents against virus infection.

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.

The trans influence in the modulation of platinum anticancer agent biology: the effect of nitrite leaving group on aquation, reactions with S-nucleophiles and DNA binding of dinuclear and trinuclear compounds.

To examine the effect of leaving group and trans influence on the general reactivity of polynuclear platinum antitumor agents we investigated substitution of the chloride leaving groups with nitrite ion, which forms strong bonds to Pt. It was of interest to explore whether nitrite could be used to modulate biological properties of these agents, in particular the deactivating reactions that occur on reaction with S-nucleophiles, involving loss of the linking diamine under the trans influence of sulfur. Reported herein is a study of the synthesis, aquation, DNA binding and reactions with glutathione (GSH), methionine (Met) and acetylmethione (AcMet) of nitrito derivatives of di- and trinuclear platinum antitumor compounds: [{trans-PtNO(2)(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](NO(3))(2) (1-NO(2)) and [{trans-PtNO(2)(NH(3))(2)}(2)(mu-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](NO(3))(4) (1'-NO(2)). {(1)H,(15)N}-HSQC NMR studies revealed that 1-NO(2) is inert to aquation reactions, even after prolonged incubation at physiological pH. Monitoring of the interaction of 1-NO(2) with the duplex 5'-d(ATATGTACATAT)(2) (I) showed only unreacted complex, consistent with activation by aquation being a requirement for covalent DNA binding. The reaction of 1-NO(2) with GSH was studied by (1)H, (195)Pt, (15)N and {(1)H,(15)N}-HSQC NMR spectroscopy. For the parent dichlorido compounds (1 and 1') substitution of chloride by GS(-) leads to drug degradation involving liberation of the diamine linker. While the same final products trans-[Pt(SG)(2)(NH(3))(2)] (5) and trans-[{Pt(SG)(NH(3))(2)}(2)-mu-SG] (6) are formed, different mechanisms are involved, consistent with the trans influence NO(2)(-) > Cl(-); the half-life is slightly longer for 1-NO(2) (1.8 h) compared with 1 (1.3 h). Identification of the intermediate trans-[Pt(NH(3))(2)(NO(2))(SG)] (4) shows that the nitrito group remains coordinated while the linker amine is substituted by coordination of GS(-), and then trans labilization of the nitrito group occurs leading to 5 and 6. Reaction of the trinuclear 1'-NO(2) with GSH follows essentially the same reaction pathway. Reaction of 1-NO(2) with Met and AcMet is much slower and only 20 % liberated amine was observed after reaction with Met for 24 h at 37 degrees C. The final product from reaction with AcMet is trans-[Pt(NH(3))(2)(NO(2))(AcMet)], as in this case coordination of the S-nucleophile does not lead to trans labilization of the nitrito group.

Determination of the kinetic profile of a dinuclear platinum anticancer complex in the presence of sulfate: introducing a new tool for the expedited analysis of 2D [(1)H,( 15)N] HSQC NMR spectra.

Two-dimensional (2D) [(1)H, (15)N] heteronuclear single-quantum coherence (HSQC) NMR experiments of the kinetics of aquation and sulfation of the dinuclear platinum anticancer complex [{trans-PtCl(NH(3))(2)}(2)(µ-NH(2)(CH(2))(6)NH(2))](2+) (1,1/t,t, 1) in 15 mM sulfate solution are reported using conditions (298 K, pH 5.4) identical to those previously used for other anionic systems (phosphate and acetate), allowing for a direct comparison. Sulfate is the fourth most abundant anion in human plasma. The rate constant for the aquation step (k(H)) is higher than that previously found in the presence of phosphate, but the anation rate constants are similar. The rate constant for sulfate displacement of the aqua ligand (k(L)) is approximately three times higher than that of phosphate, and a further major difference between these two anions is the very high k(-L) for loss of sulfate, suggesting that when formed in plasma the sulfato species will be substitution labile. We also introduce a novel (free) plug-in, '2D NMR analysis', developed for the expedited integration and analysis of 2D [(1)H, (15)N] HSQC NMR spectra. We have found that this plug-in significantly reduces the amount of time taken in the analysis of experiments with no loss to the quality of the data.

Anticancer activity of a Gold(I) phosphine thioredoxin reductase inhibitor in multiple myeloma.

Multiple myeloma (MM), the second most common haematological malignancy, is a clonal plasma B-cell neoplasm that forms within the bone marrow. Despite recent advancements in treatment, MM remains an incurable disease. Auranofin, a linear gold(I) phosphine compound, has previously been shown to exert a significant anti-myeloma activity by inhibiting thioredoxin reductase (TrxR) activity. A bis-chelated tetrahedral gold(I) phosphine complex [Au(d2pype)2]Cl (where d2pype is 1,2-bis(di-2-pyridylphosphino)ethane) was previously designed to improve the gold(I) compound selectivity towards selenol- and thiol-containing proteins, such as TrxR. In this study, we show that [Au(d2pype)2]Cl significantly inhibited TrxR activity in both bortezomib-sensitive and resistant myeloma cells, which led to a significant reduction in cell proliferation and induction of apoptosis, both of which were dependent on ROS. In clonogenic assays, treatment with [Au(d2pype)2]Cl completely abrogated the tumourigenic capacity of MM cells, whereas auranofin was less effective. We also show that [Au(d2pype)2]Cl exerted a significant anti-myeloma activity in vivo in human RPMI8226 xenograft model in immunocompromised NOD/SCID mice. The MYC oncogene, known to drive myeloma progression, was downregulated in both in vitro and in vivo models when treated with [Au(d2pype)2]Cl. This study highlights the "proof of concept" that improved gold(I)-based compounds could potentially be used to not only treat MM but as an alternative tool to understand the role of the Trx system in the pathogenesis of this blood disease.

Factors affecting DNA-DNA interstrand cross-links in the antiparallel 3'-3' sense: a comparison with the 5'-5' directional isomer.

Reported herein is a study of the unusual 3'-3' 1,4-GG interstrand cross-link (IXL) formation in duplex DNA by a series of polynuclear platinum anticancer complexes. To examine the effect of possible preassociation through charge and hydrogen-bonding effects the closely related compounds [{trans-PtCl(NH(3))(2)}(2)(mu-trans-Pt(NH(3))(2){NH(2)(CH(2))(6)NH(2)}(2))](4+) (BBR3464, 1), [{trans-PtCl(NH(3))(2)}(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (BBR3005, 2), [{trans-PtCl(NH(3))(2)}(2)(mu-H(2)N(CH(2))(3)NH(2)(CH(2))(4))](3+) (BBR3571, 3) and [{trans-PtCl(NH(3))(2)}(2){mu-H(2)N(CH(2))(3)-N(COCF(3))(CH(2))(4)}](2+) (BBR3571-COCF(3), 4) were studied. Two different molecular biology approaches were used to investigate the effect of DNA template upon IXL formation in synthetic 20-base-pair duplexes. In the "hybridisation directed" method the monofunctionally adducted top strands were hybridised with their complementary 5'-end labelled strands; after 24 h the efficiency of interstrand cross-linking in the 5'-5' direction was slightly higher than in the 3'-3' direction. The second method involved "postsynthetic modification" of the intact duplex; significantly less cross-linking was observed, but again a slight preference for the 5'-5' duplex was present. 2D [(1)H, (15)N] HSQC NMR spectroscopy studies of the reaction of [(15)N]-1 with the sequence 5'-d{TATACATGTATA}(2) allowed direct comparison of the stepwise formation of the 3'-3' IXL with the previously studied 5'-5' IXL on the analogous sequence 5'-d(ATATGTACATAT)(2). Whereas the preassociation and aquation steps were similar, differences were evident at the monofunctional binding step. The reaction did not yield a single distinct 3'-3' 1,4-GG IXL, but numerous cross-linked adducts formed. Similar results were found for the reaction with the dinuclear [(15)N]-2. Molecular dynamics simulations for the 3'-3' IXLs formed by both 1 and 2 showed a highly distorted structure with evident fraying of the end base pairs and considerable widening of the minor groove.

1,2-Bis(di-2-pyridylphosphino-yl)ethane.

The crystal structure of the title compound, C(22)H(20)N(4)O(2)P(2), consists of two independent half-mol-ecules, both of which lie on crystallographic inversion centres. There are no significant differences between the two mol-ecules.

Influence of geometric isomerism on the binding of platinum anticancer agents with phospholipids.

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.

Bioenergetic differences selectively sensitize tumorigenic liver progenitor cells to a new gold(I) compound.

A hallmark of cancer cells is their ability to evade apoptosis and mitochondria play a critical role in this process. Delineating mitochondrial differences between normal and cancer cells has proven challenging due to the lack of matched cell lines. Here, we compare two matched liver progenitor cell (LPC) lines, one non-tumorigenic [p53-immortalized liver (PIL) 4] and the other tumorigenic (PIL2). Analysis of these cell lines and a p53 wild-type non-tumorigenic cell line [bipotential murine oval liver (BMOL)] revealed an increase in expression of genes encoding the antiapoptotic proteins cellular inhibitor of apoptosis protein (cIAP) 1 and yes associate protein in the PIL2 cells, which resulted in an increase in the protein encoded by these genes. PIL2 cells have higher mitochondrial membrane potential (Deltapsi(m)) compared with PIL4 and BMOL and had greater levels of reactive oxygen species, despite the fact that the mitochondrial antioxidant enzyme, manganese superoxide disumutase, was elevated at transcript and protein levels. Taken together, these results may account for the observed resistance of PIL2 cells to apoptotic stimuli compared with PIL4. We tested a new gold compound to show that hyperpolarized Deltapsi(m) led to its increased accumulation in mitochondria of PIL2 cells. This compound selectively induces apoptosis in PIL2 cells but not in PIL4 or BMOL. The gold compound depolarized the Deltapsi(m), depleted the adenosine triphosphate pool and activated caspase-3 and caspase-9, suggesting that apoptosis was mediated via mitochondria. This investigation shows that the non-tumorigenic and tumorigenic LPCs are useful models to delineate the role of mitochondrial dysfunction in tumorigenesis and for the future development of mitochondria-targeted chemotherapeutics that selectively target tumor cells.

Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols.

A family of lipophilic, cationic Au(I) complexes of N-heterocyclic carbenes (NHCs) have been designed as new mitochondria-targeted antitumor agents that combine both selective mitochondrial accumulation and selective thioredoxin reductase inhibition properties within a single molecule. Two-step ligand exchange reactions with cysteine (Cys) and selenocysteine (Sec) occur with release of the NHC ligands. At physiological pH the rate constants for the reactions with Sec are 20- to 80-fold higher than those with Cys. The complexes are selectively toxic to two highly tumorigenic breast cancer cell lines and not to normal breast cells, and the degree of selectivity and potency are optimized by modification of the substituent on the simple imidazolium salt precursor. The lead compound is shown to accumulate in mitochondria of cancer cells, to cause cell death through a mitochondrial apoptotic pathway and to inhibit the activity of thioredoxin reductase (TrxR) but not the closely related and Se-free enzyme glutathione reductase.

[1H, 15N] heteronuclear single quantum coherence NMR study of the mechanism of aquation of platinum(IV) ammine complexes.

The aquation and hydrolysis of a series of platinum(IV) complexes of the general form cis, trans, cis-[PtCl 2(X) 2( (15)NH 3) 2] (X = Cl (-), O 2CCH 3 (-), OH (-)) have been followed by [ (1)H, (15)N] Heteronuclear Single Quantum Coherence NMR spectroscopy. Negligible aquation (<5%) is observed for the complexes where X = O 2CCH 3 (-) or OH (-) over 3-4 weeks. Aquation of cis-[PtCl 4( (15)NH 3) 2] ( 1) is observed, and the rate of aquation increases with increasing pH and upon the addition of 0.01 mol equiv of the platinum(II) complex cis-[PtCl 2( (15)NH 3) 2] (cisplatin). The first aquated species formed from cis-[PtCl 4(NH 3) 2] has one of the axial chloro groups (relative to the equatorial NH 3 ligands) replaced by an aqua/hydroxo ligand. The second observed substitution occurs in an equatorial position. Peaks that are consistent with five of the eight possible aquation species were observed in the NMR spectra.

In vitro antitumour and hepatotoxicity profiles of Au(I) and Ag(I) bidentate pyridyl phosphine complexes and relationships to cellular uptake.

In this study we characterised the in vitro antitumour and hepatotoxicity profiles of a series of Au(I) and Ag(I) bidentate phenyl and pyridyl complexes in a panel of cisplatin-resistant human ovarian cancer cell-lines, and in isolated rat hepatocytes. The gold and silver compounds overcame cisplatin-resistance in the CH1-cisR, 41M-cisR and SKOV-3 cell-lines, and showed cytotoxic potencies strongly correlated with their lipophilicity. Complexes with phenyl or 2-pyridyl ligands had high antitumour and hepatotoxic potency and low selectivity between different cell-lines. Their cytotoxicity profiles were similar to classic mitochondrial poisons and an example of this type of compound was shown to accumulate preferentially in the mitochondria of cancer cells in a manner that depended upon the mitochondrial membrane potential. In contrast, complexes with 3- or 4-pyridyl ligands had low antitumour and hepatotoxic potency and cytotoxicity profiles similar to 2-deoxy-D-glucose. In addition, they showed high selectivity between different cell-lines that was not attributable to variation in uptake in different cell-types. The in vitro hepatotoxic potency of the series of gold and silver compounds varied by over 61-fold and was closely related to their lipophilicity and hepatocyte uptake. In conclusion, Au(I) and Ag(I) bidendate pyridyl phosphine complexes demonstrate activity against cisplatin-resistant human cancer cells and in vitro cytotoxicity that strongly depends upon their lipophilicity.