Platinum binding preferences dominate the binding of novel polyamide amidine anthraquinone platinum(II) complexes to DNA.

Complexes incorporating a threading anthraquinone intercalator with pyrrole lexitropsin and platinum(II) moieties attached were developed with the goal of generating novel DNA binding modes, including the targeting of AT-rich regions in order to have high cytotoxicities. The binding of the complexes to DNA has been investigated and profiles surprisingly similar to that for cisplatin were observed; the profiles were different to those for a complex lacking the pyrrole lexitropsin component. The lack of selective binding to AT-rich regions suggests the platinum binding was dominating the sequence selectivity, and is consistent with the pyrrole lexitropsin slowing intercalation. The DNA unwinding profiles following platinum binding were evaluated by gel electrophoresis and suggested that intercalation and platinum binding were both occurring.

Warburg Effect Targeting Co(III) Cytotoxin Chaperone Complexes.

A glucose-based vector for targeting cancer cells conjugated to a tris(methylpyridyl)amine (tpa) ligand to generate targeted chaperone and caging complexes for active anticancer agents is described. The ligand, tpa(CONHPEGglucose)1, inhibits hexokinase, suggesting that it will be phosphorylated in the cell. A Co(III) complex incorporating this ligand and coumarin-343 hydroximate (C343ha), [Co(C343ha){tpa(CONHPEGglucose)1}]Cl, is shown to exhibit glucose-dependent cellular accumulation in DLD-1 colon cancer cells. Cellular accumulation of [Co(C343ha){tpa(CONHPEGglucose)1}]+ is slower than for the glucose null and glucosamine analogues, and the glucose complex also exhibits a lower ability to inhibit antiproliferative activity. Distributions of cobalt (X-ray fluorescence) and C343ha (visible light fluorescence) in DLD-1 cancer cell spheroids are consistent with uptake of [Co(C343ha){tpa(CONHPEGglucose)1}]+ by rapidly dividing cells, followed by release and efflux of C343ha and trapping of the Co{tpa(CONHPEGglucose)1} moiety. The Co{tpa(CONHPEGglucose)1} moiety is shown to have potential for the caged and targeted delivery of highly toxic anticancer agents.

Novel polyamide amidine anthraquinone platinum(II) complexes: cytotoxicity, cellular accumulation, and fluorescence distributions in 2D and 3D cell culture models.

1- and 1,5-Aminoalkylamine substituted anthraquinones (AAQs, 1C3 and 1,5C3) were peptide coupled to 1-, 2-, and 3-pyrrole lexitropsins to generate compounds that incorporated both DNA minor groove and intercalating moieties. The corresponding platinum(II) amidine complexes were synthesized through a synthetically facile amine-to-platinum mediated nitrile 'Click' reaction. The precursors as well as the corresponding platinum(II) complexes were biologically evaluated in 2D monolayer cells and 3D tumour cell models. Despite having cellular accumulation levels that were up to five-fold lower than that of cisplatin, the platinum complexes had cytotoxicities that were only three-fold lower. Accumulation was lowest for the complexes with two or three pyrrole groups, but the latter was the most active of the complexes exceeding the activity of cisplatin in the MDA-MB-231 cell line. All compounds showed moderate to good penetration into spheroids of DLD-1 cells with the distributions being consistent with active uptake of the pyrrole containing complexes in regions of the spheroids starved of nutrients.

Water-Soluble α-Amino Acid Complexes of Molybdenum as Potential Antidotes for Cyanide Poisoning: Synthesis and Catalytic Studies of Threonine, Methionine, Serine, and Leucine Complexes.

Water-soluble complexes are desirable for the aqueous detoxification of cyanide. Molybdenum complexes with α-amino acid and disulfide ligands with the formula K[(L)Mo2O2(µ-S)2(S2)] (L = leu (1), met (2), thr (3), and ser (4)) were synthesized in a reaction of [(DMF)3MoO(µ-S)2(S2)] with deprotonated α-amino acids; leu, met, thr, and ser are the carboxylate anions of l-leucine, l-methionine, l-threonine, and l-serine, respectively. Potassium salts of α-amino acids (leu (1a), met (2a), thr (3a), and ser (4a)) were prepared as precursors for complexes 1-4, respectively, by employing a nonaqueous synthesis route. The ligand exchange reaction of [Mo2O2(µ-S)2(DMF)6](I)2 with deprotonated α-amino acids afforded bis-α-amino acid complexes, [(L)2Mo2O2(µ-S)2] (6-8). A tris-α-amino acid complex, [(leu)2Mo2O2(µ-S)2(µ-leu + H)] (5; leu + H is the carboxylate anion of l-leucine with the amine protonated), formed in the reaction with leucine. 5 crystallized from methanol with a third weakly bonded leucine as a bridging bidentate carboxylate. An adduct of 8 with SCN- coordinated, 9, crystallized and was structurally characterized. Complexes 1-4 are air stable and highly water-soluble chiral molecules. Cytotoxicity studies in the A549 cell line gave IC50 values that range from 80 to 400 µM. Cyclic voltammetry traces of 1-8 show solvent-dependent irreversible electrochemical behavior. Complexes 1-4 demonstrated the ability to catalyze the reaction of thiosulfate and cyanide in vitro to exhaustively transform cyanide to thiocyanate in less than 1 h.

The effect of charge on the uptake and resistance to reduction of platinum(IV) complexes in human serum and whole blood models.

cis- and trans-Platinum(iv) complexes with diaminetetracarboxylate coordination spheres possess the highly desirable property of exhibiting unusual resistance to reduction by blood serum components and endogenous reductants such as ascorbate. At the same time they are rapidly reduced in the intracellular environment of cancer cells. Consequently, they can potentially be tuned to remain intact in vivo until arrival at the tumour target where they are rapidly reduced to yield the active platinum(ii) species. However, in order to achieve this, uptake must be largely restricted to tumour cells and therefore uptake by healthy cells including red blood cells must be prevented. In this proof of concept study, we report on the effect of net charge as a means of controlling the uptake by red blood cells. Using 1H NMR spectroscopy we found that modifying the net charge of the complex does not influence the rate of reduction of the complexes by an excess of ascorbate. Using XANES spectroscopy we found that modifying the net charge of the platinum(iv) complexes decreased the extent of reduction in whole blood, although probably not to the degree needed for the optimal delivery to tumours. Therefore, it is likely to be necessary to adopt higher charges and/or additional strategies to keep platinum(iv) prodrugs out of blood cells.

trans-Platinum(iv) pro-drugs that exhibit unusual resistance to reduction by endogenous reductants and blood serum but are rapidly activated inside cells: 1H NMR and XANES spectroscopy study.

Recent results have confirmed that protection of transplatin from reactions on the path to cancer cells substantially increases their activity, suggesting that such complexes have greater potential than previously thought. In this study we have investigated the use of the platinum(iv) oxidation state and the tetracarboxylate coordination sphere to determine whether these features could impart the same stability to trans-diammineplatinum complexes that they do to cis-diam(m)ineplatinum complexes. The cis complexes exhibit resistance to reduction by l-ascorbate and human blood serum, but are readily reduced inside cancer cells. Studies of reduction monitored by 1H NMR revealed that oxidation of trans-diammineplatinum(ii) complexes does not always result in significant stabilisation, but the complexes trans, trans, trans-[Pt(OAc)4(NH3)2] (OAc = acetate) and trans, trans, trans-[Pt(OPr)2(OAc)2(NH3)2] (OPr = propionate) exhibit second order half-lives of 33 h and 5.9 days respectively in the presence of a ten-fold excess of l-ascorbate. XANES spectroscopy studies of reduction in blood models showed that trans, trans, trans-[Pt(OAc)4(NH3)2] is stable in blood serum for at least 24 hours, but is reduced rapidly in whole blood and was observed to have a half-life of approximately 4 hours in DLD-1 colon cancer cells. Consequently, the tetracarboxylatoplatinum(iv) moiety has the properties required to enable the delivery of trans-diammine platinum complexes to cancer cells.

A Warburg effect targeting vector designed to increase the uptake of compounds by cancer cells demonstrates glucose and hypoxia dependent uptake.

Glycoconjugation to target the Warburg effect provides the potential to enhance selective uptake of anticancer or imaging agents by cancer cells. A Warburg effect targeting group, rationally designed to facilitate uptake by glucose transporters and promote cellular accumulation due to phosphorylation by hexokinase (HK), has been synthesised. This targeting group, the C2 modified glucose analogue 2-(2-[2-(2-aminoethoxy)ethoxy]ethoxy)-D-glucose, has been conjugated to the fluorophore nitrobenzoxadiazole to evaluate its effect on uptake and accumulation in cancer cells. The targeting vector has demonstrated inhibition of glucose phosphorylation by HK, indicating its interaction with the enzyme and thereby confirming the potential to facilitate an intracellular trapping mechanism for compounds it is conjugated with. The cellular uptake of the fluorescent analogue is dependent on the glucose concentration and is so to a greater extent than is that of the widely used fluorescent glucose analogue, 2-NBDG. It also demonstrates selective uptake in the hypoxic regions of 3D spheroid tumour models whereas 2-NBDG is distributed primarily through the normoxic regions of the spheroid. The increased selectivity is consistent with the blocking of alternative uptake pathways.

Transporter and protease mediated delivery of platinum complexes for precision oncology.

Precision approaches are rapidly becoming the norm in the treatment of cancer and this is already impacting on the way platinum complexes are used. In this commentary, we will argue that there is the potential for platinum complexes to make a much greater contribution to precision oncology, one that is complementary to many of the other approaches being used and developed. Our focus will be on two methods for targeting anticancer agents: ligand-targeted drug delivery and protease activation of prodrugs. We will describe work done to date and discuss the directions that appear to be showing most promise. We will also discuss the challenges involved in the testing of targeted prodrugs in biological models and the possible consequences of these difficulties.

The reduction of cis-platinum(iv) complexes by ascorbate and in whole human blood models using 1H NMR and XANES spectroscopy.

The efficacy of platinum(iv) prodrugs depends on their relative resistance to reduction in the extra- and intra-cellular environments. In the study reported here we investigated the influence of the nature of the axial and equatorial ligands on the pathway of reduction of the platinum(iv) complexes by the endogenous reductant, ascorbate, and their relative resistance to reduction in human blood serum and in a whole human blood model. The pathway of reduction of platinum(iv) complexes in the presence of excess ascorbate was found to be dependent on the nature of their axial and equatorial ligands in that complexes with chloride in the equatorial sites lost either both axial ligands or combinations of axial and equatorial ligands while those with oxalate occupying the equatorial sites lost both axial ligands only. Using XANES spectroscopy, complexes with axial hydroxide ligands were found to be highly resistant to reduction in blood serum and were only slowly and incompletely reduced in whole blood. The dihydroxide complex with an oxalate ligand occupying the equatorial leaving group sites was more resistant to reduction, both in serum and in whole blood, than the complex with chloride ligands in these sites. cis, trans-[PtCl2(OAc)2(en)] and trans-[Pt(OAc)2(ox)(en)] were observed to be reduced rapidly and almost completely in whole blood but the latter was substantially resistant to reduction in human blood serum, and consequently demonstrates many of the features of an optimal platinum(iv) anticancer agent.

Modulating the Cellular Uptake of Fluorescently Tagged Substrates of Prostate-Specific Antigen before and after Enzymatic Activation.

A series of peptides based on the prostate-specific antigen (PSA)-specific sequence histidine-serine-serine-lysine-leucine-glutamine were functionalized with an anthraquinone fluorophore at the C-terminal residue side chain using the copper(I)-catalyzed azide-alkyne cycloaddition reaction. The effect of incorporating a negatively charged N-terminal tetra-glutamic acid group into the substrate and the effect of masking the negatively charged C-terminal carboxylic acid functionality of the substrate were investigated using confocal fluorescence microscopy in two cell lines, DLD-1 and LnCaP. The addition of a tetra-glutamic acid group to the N-terminus of the intact sequence was shown to reduce cellular uptake of the intact substrate prior to activation by PSA. In contrast, masking the C-terminal carboxylic acid group of the substrate as a methyl ester was shown to improve cellular uptake of the peptide fragment after activation by PSA. The synthesized C-terminal methyl ester substrates with the anthraquinone attached to the side chain were confirmed to be cleaved by PSA in LC-MS analysis, and the cytotoxicity of the substrates was shown to increase in the presence of PSA, consistent with cleavage and uptake of the C-terminal fragment. The results indicate that C- and N-terminal functionalization of peptide substrates targeting PSA can be used to modulate the cellular uptake of peptides before and after enzymatic activation, which may thus be an important consideration in the design of tumor-activated prodrugs.

A ratiometric iron probe enables investigation of iron distribution within tumour spheroids.

Iron dysregulation is implicated in numerous diseases, and iron homeostasis is profoundly influenced by the labile iron pool (LIP). Tools to easily observe changes in the LIP are limited, with calcein AM-based assays most widely used. We describe here FlCFe1, a ratiometric analogue of calcein AM, which also provides the capacity for imaging iron in 3D cell models.

The influence of the ancillary ligand on the potential of cobalt(iii) complexes to act as chaperones for hydroxamic acid-based drugs.

Cobalt(iii) chaperones are a promising class of bioreductive prodrugs under investigation for the delivery of cytotoxic ligands to hypoxic solid tumours. Here we investigate a series of cobalt complexes as chaperones for hydroxamic acid ligands, comparing the properties of the cyclic cyclen (1,4,7,10-tetraazacyclododecane) ancillary ligand with the tripodal tpa (tris-(2-pyridylmethyl)amine) and tren (tris-(2-aminoethyl)amine). A small library of complexes containing several different hydroxamic acids, including the MMP inhibitor Marimistat and the fluorescent ligand C343haH2, were prepared and their pKa values, reduction potentials, and in some cases X-ray crystal structures, were determined. The antiproliferative actitivity of the series was evaluated against DLD-1 colon cancer cells and the cellular accumulation of the fluorescent C343haH2 complexes was monitored by ICPMS and confocal fluorescence microscopy, revealing that the nature of the ancillary ligand significantly influences the complexes' properties, cytotoxicity and cellular distribution.

The influence of the ethane-1,2-diamine ligand on the activity of a monofunctional platinum complex.

The continued use of platinum-based chemotherapeutic drugs in the clinic mandates the need for further investigation of the biological activity of structural analogues of the clinically approved complexes. Of interest are monofunctional platinum(II) complexes, which bear only one labile ligand, for which it is believed that each complex binds to DNA only once. Pyriplatin ([PtCl(NH3)2(py)]+) and enpyriplatin ([PtCl(en)(py)]+) are both monofunctional platinum(II) complexes that bear a pyridine ligand and a labile chlorido ligand, differing in their cis­ammine and ethane-1,2-diamine (en) ligands respectively. Despite their similar structure, the complexes exhibit dramatically different cytotoxicities. In this study, we synthesized and characterized both complexes in terms of their cytotoxicity, lipophilicity, DNA binding and cellular accumulation. There was no significant difference between the lipophilicities of the complexes and both complexes exhibited monofunctional type binding, but it was the temporal accumulation profiles of the two complexes which differed greatly. The complexes were further analyzed with size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICP-MS) to determine the platination state of the proteins. Consistent with the accumulation studies, pyriplatin bound to proteins in far greater amounts than enpyriplatin, and this study also revealed some different protein targets between the bifunctional cisplatin and monofunctional pyriplatin. This study highlights the need for more sophisticated techniques, such as SEC-ICP-MS, to determine not only how much of a platinum complex accumulates in cells, but also the speciation and metabolites of platinum anticancer drugs.

Interactions of cisplatin and the copper transporter CTR1 in human colon cancer cells.

There is much interest in understanding the mechanisms by which platinum-based anticancer agents enter cells, and the copper transporter CTR1 has been the focus of many recent studies. While there is a clinical correlation between CTR1 levels and platinum efficacy, cellular studies have provided conflicting evidence relating to the relationship between cisplatin and CTR1. We report here our studies of the relationship between cisplatin and copper homeostasis in human colon cancer cells. While the accumulation of copper and platinum do not appear to compete with each other, we did observe that cisplatin perturbs CTR1 distribution within 10 min, a far shorter incubation time than commonly employed in cellular studies of cisplatin. Furthermore, on these short time-scales, cisplatin caused an increase in the cytoplasmic labile copper pool. While the predominant focus of studies to date has been on CTR1, these studies highlight the importance of investigating the interaction of cisplatin with other copper proteins.

A ratiometric fluorescent sensor for the mitochondrial copper pool.

Copper plays a key role in the modulation of cellular function, defence, and growth. Here we present InCCu1, a ratiometric fluorescent sensor for mitochondrial copper, which changes from red to blue emission in the presence of Cu(i). Employing this probe in microscopy and flow cytometry, we show that cisplatin-treated cells have an impaired ability to accumulate copper in the mitochondria.

A novel class of copper(II)- and zinc(II)-bound non-steroidal anti-inflammatory drugs that inhibits acute inflammation in vivo.

BACKGROUND: The ability of Zn(II) and Cu(II) metal complexes of non-steroidal anti-inflammatory drugs (NSAIDs) to inhibit acute arterial inflammation in vivo has been studied. RESULTS: When acute vascular inflammation was induced in normocholesterolemic New Zealand White rabbits by inserting a non-occlusive silastic collar around the common carotid artery, a single oral dose of Cu(II)-indomethacin (Cu(II)Indo, 3 mg/kg) administered by laparotomy achieved a 67 % (8.2 ± 1.7 vs. 2.7 ± 0.4 image units, p < 0.05) reduction in endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) but did not inhibit endothelial intercellular adhesion molecule (ICAM-1) expression significantly. Treatment with Cu(II)-acemetacin (Cu(II)ACM, 3 mg/kg) led to a profound 88 % (8.2 ± 1.7 vs. 1.0 ± 0.5 image units, p < 0.01) reduction in endothelial VCAM-1 expression but did not inhibit ICAM-1 expression, while treatment with Zn(II)-acemetacin (Zn(II)ACM, 3 mg/kg) led to an 84 % (19.3 ± 1.0 vs. 3.1 ± 1.2 image units, p < 0.01) reduction in endothelial ICAM-1 expression and did not inhibit VCAM-1 expression. No adverse gastric, hepatic or renal effects were observed in treated animals. CONCLUSION: These findings provide the "proof of concept" that this novel class of drug, where there is complexation of NSAIDs with metal ions, has substantial anti-inflammatory effects in an animal model of acute vascular inflammation with the possibility of low rates of adverse effects.

Radiosynthesis and 'click' conjugation of ethynyl-4-[(18)F]fluorobenzene--an improved [(18)F]synthon for indirect radiolabeling.

Reproducible methods for [(18)F]radiolabeling of biological vectors are essential for the development of new [(18)F]radiopharmaceuticals. Molecules such as carbohydrates, peptides and proteins are challenging substrates that often require multi-step indirect radiolabeling methods. With the goal of developing more robust, time saving, and less expensive procedures for indirect [(18)F]radiolabeling of such molecules, our group has synthesized ethynyl-4-[(18)F]fluorobenzene ([(18)F]2, [(18)F]EYFB) in a single step (14 ± 2% non-decay corrected radiochemical yield (ndc RCY)) from a readily synthesized, shelf stable, inexpensive precursor. The alkyne-functionalized synthon [(18)F]2 was then conjugated to two azido-functionalized vector molecules via CuAAC reactions. The first 'proof of principle' conjugation of [(18)F]2 to 1-azido-1-deoxy-ß-D-glucopyranoside (3) gave the desired radiolabeled product [(18)F]4 in excellent radiochemical yield (76 ± 4% ndc RCY (11% overall)). As a second example, the conjugation of [(18)F]2 to matrix-metalloproteinase inhibitor (5), which has potential in tumor imaging, gave the radiolabeled product [(18)F]6 in very good radiochemical yield (56 ± 12% ndc RCY (8% overall)). Total preparation time for [(18)F]4 and [(18)F]6 including [(18)F]F(-) drying, two-step reaction (nucleophilic substitution and CuAAC conjugation), two HPLC purifications, and two solid phase extractions did not exceed 70 min. The radiochemical purity of synthon [(18)F]2 and the conjugated products, [(18)F]4 and [(18)F]6, were all greater than 98%. The specific activities of [(18)F]2 and [(18)F]6 were low, 5.97 and 0.17 MBq nmol(-1), respectively.

Fluorescent sensing of monofunctional platinum species.

We report here FDCPt1, a novel selective fluorescent sensor for monofunctional platinum species. In the presence of such species, FDCPt1 exhibits a 70-fold increase in fluorescence emission, and can be used to monitor the metabolism of Pt(II)-based complexes in colorectal cancer cells. This probe is therefore expected to be valuable in studying changes in Pt coordination and distribution during chemotherapy.

Mechanisms of cell uptake and toxicity of the anticancer drug cisplatin.

Two major issues which hamper the use of the anticancer drug cisplatin are the development of cancer cell resistance and its nephrotoxicity. One possible mechanism by which resistance is reported to develop is a reduction in drug uptake across the cell membrane. While the passive uptake of cisplatin has long been cited as an important contribution, far greater attention has been given to active modes of uptake, particularly in recent research. Using unilamellar lipid vesicles together with the stopped-flow kinetic method we show here that the permeability coefficient of cisplatin increases significantly with the chloride concentration of the medium. This supports the hypothesis that cisplatin can enter cells via passive permeation through the lipid phase of the membrane, but becomes trapped within the cytoplasm because dissociation of chloride ligands yields a membrane-impermeant positively-charged aqua derivative. This is important evidence for a major role of passive membrane diffusion in the uptake of cisplatin, and suggests that reduced cell uptake is unlikely to be a significant mechanism leading to the development of drug resistance. Studies of rubidium ion uptake into the cytoplasm of Xenopus oocytes via the Na(+),K(+)-ATPase show significant inhibition of this ion pump when cisplatin is present in the cytoplasm. Because Na(+),K(+)-ATPase activity is essential to the survival of all animal cells, e.g. via maintenance of cell volume, and the Na(+),K(+)-ATPase is expressed at particularly high levels within the membranes of kidney tubules where it plays a crucial role in nutrient reabsorption, these results suggest that cisplatin-induced inhibition of the Na(+),K(+)-ATPase is a likely contributing cause for the nephrotoxicity of cisplatin.

Influence of equatorial and axial carboxylato ligands on the kinetic inertness of platinum(IV) complexes in the presence of ascorbate and cysteine and within DLD-1 cancer cells.

The rapid and premature reduction of platinum(IV) complexes in vivo is a significant impediment to these complexes being successfully employed as anticancer prodrugs. This study investigates the influence of the platinum(IV) coordination sphere on the ease of reduction of the platinum center in various biological contexts. In the presence of the biological reductants, ascorbate and cysteine, platinum(IV) complexes with dicarboxylato equatorial ligands were observed to exhibit lower reduction potentials and slower reduction rates than analogous platinum(IV) complexes with dichlorido equatorial ligands. Diaminetetracarboxylatoplatinum(IV) complexes exhibited unusually long half-lives in the presence of excess reductants; however, the complexes exhibited moderate potency in vitro, indicative of rapid reduction within the intracellular environment. By use of XANES spectroscopy, trans-[Pt(OAc)2(ox)(en)] and trans-[PtCl2(OAc)2(en)] were observed to be reduced at a similar rate within DLD-1 cancer cells. This large variability in kinetic inertness of diaminetetracarboxylatoplatinum(IV) complexes in different biological contexts has significant implications for the design of platinum(IV) prodrugs.