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.

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.

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.

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.

Cobalt(III) Chaperone Complexes of Curcumin: Photoreduction, Cellular Accumulation and Light-Selective Toxicity towards Tumour Cells.

Light-activated prodrugs offer the potential for highly selective tumour targeting. However, the application of many photoactivated chemotherapeutics is limited by a requirement for oxygen, or for short activation wavelengths that can damage surrounding tissue. Herein, we present a series of cobalt(III)-curcumin prodrugs that can be activated by visible light under both oxygenated and hypoxic conditions. Furthermore, the photoproduct can be controlled by the activation wavelength: green light yields free curcumin, whereas blue light induces photolysis of curcumin to a phototoxic product. Confocal fluorescence microscopy and phototocytotoxicity studies in DLD-1 and MCF-7 tumour cells demonstrated that the cobalt(III) prodrugs are nontoxic in the dark but accumulate in significant concentrations in the cell membrane. When cells were treated with light for 15  min, the cytotoxicity of the cobalt complexes increased by up to 20-fold, whereas free curcumin exhibited only a two-fold increase in cytotoxicity. The nature of the ancillary ligand and cobalt reduction potential were found to strongly influence the stability and biological activity of the series.

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.

Facile preparation of mono-, di- and mixed-carboxylato platinum(IV) complexes for versatile anticancer prodrug design.

Facile strategies were developed for the versatile functionalization of platinum(IV) axial sites, allowing for easy accessibility to unsymmetric mono- and mixed-carboxylato, as well as symmetric di-substituted platinum(IV) complexes. The first method involves the direct oxidation and carboxylation of the platinum(II) center using an appropriate peroxide and the carboxylate of choice to firstly yield a monocarboxylato monohydroxido platinum(IV) complex. This platinum(IV) intermediate can undergo further carboxylation to give rise to a mixed-carboxylato platinum(IV) complex. The second method involves the activation of the carboxylate of choice by a common carbodiimide coupling reagent, and its reaction with a dihydroxido platinum(IV) precursor to give the monocarboxylato platinum(IV) complex. Uronium salts can be employed to promote efficient dicarboxylation of the dihydroxido platinum(IV) precursor. Lastly, an axial azide pendant group was demonstrated to be suitable for orthogonal "click" conjugation reactions.

Effects of enzymatic activation on the distribution of fluorescently tagged MMP-2 cleavable peptides in cancer cells and spheroids.

A peptide tagged at the N-terminus with FITC, at the C-terminus with coumarin-343, and incorporating a sequence selectively cleaved by the matrix metalloproteinase, MMP-2, was synthesized to investigate the effect of peptide cleavage on both cellular accumulation and distribution in cancer cell spheroids. The peptide was shown by HPLC and mass spectroscopy to be cleaved in the presence of MMP-2 at the expected site. The cellular and spheroid distribution of each of the fragments was monitored using confocal fluorescence microscopy. The intact peptide had minimal accumulation in 2D-cultured DLD-1 cells that do not express MMP-2 in these conditions. Following addition of serum containing MMP-2 to the cell media, the cleaved C-terminal fragment was seen to enter the cells, while the N-terminal fragment remained extracellular, evidently blocked by the presence of the FITC group. 3D culture of DLD-1 cells as spheroids resulted in measurable MMP-2 activity. Different distribution patterns of the two fluorophores were seen in spheroids treated with the intact peptide, consistent with cleavage occurring. Different rates of accumulation of each of the fragments were observed within the spheroid over time, which is attributed to the extent of accumulation and sequestration of the fragments by cells residing in the periphery of the spheroids. The outcomes suggest that tumor-associated enzymes have the potential to modify the distribution of peptides and peptide fragments in solid tumors by modifying the cellular uptake of those peptides.

Intracellular trafficking as a determinant of AS-DACA cytotoxicity in rhabdomyosarcoma cells.

BACKGROUND: Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma derived from skeletal muscle precursor cells, which accounts for 5-8% of all childhood malignancies. Disseminated RMS represents a major clinical obstacle, and the need for better treatment strategies for the clinically aggressive alveolar RMS subtype is particularly apparent. Previously, we have shown that the acridine-4-carboxamide derivative AS-DACA, a known topoisomerase II poison, is potently cytotoxic in the alveolar RMS cell line RH30, but is 190-fold less active in the embryonal RMS cell line RD. Here, we investigate the basis for this selectivity, and demonstrate in these RMS lines, and in an AS-DACA- resistant subclone of RH30, that AS-DACA-induced cytotoxicity correlates with the induction of DNA double strand breaks. RESULTS: We show that inhibition of the multidrug-resistance associated protein (MRP1) has no effect on AS-DACA sensitivity. By exploiting the pH-dependent fluorescence properties of AS-DACA, we have characterized its intracellular distribution, and show that it concentrates in the cell nucleus, as well as in acidic vesicles of the membrane trafficking system. We show that fluorescence microscopy can be used to determine the localization of AS-DACA to the nuclear and cytoplasmic compartments of RMS cells grown as spheroids, penetrance being much greater in RH30 than RD spheroids, and that the vesicular signal leads the way into the spheroid mass. EEA1 and Rab5 proteins, molecular markers expressed on early-endosomal vesicles, are reduced by >50% in the sensitive cell lines. CONCLUSION: Taking the evidence as a whole, suggests that endosomal vesicle trafficking influences the toxicity of AS-DACA in RMS cells.

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.

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.

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.

Accumulation of an anthraquinone and its platinum complexes in cancer cell spheroids: the effect of charge on drug distribution in solid tumour models.

The penetration of anthraquinones and their platinum complexes into cancer cell spheroids reveals that they model well the distribution of such compounds in solid tumours and that the proportion of the compound that accumulates deep in the spheroid is inversely related to the rate of cellular uptake which is affected by the charge of the compound.

Identification by NMR spectroscopy of the two stereoisomers of the platinum complex [PtCl2(S-ahaz)] (S-ahaz = 3(S)-aminohexahydroazepine) bound to a DNA 14-mer oligonucleotide. NMR evidence of structural alteration of a platinated A x T-rich 14-mer DNA duplex.

The enantiomers of the asymmetric, chiral platinum(II) complex [PtCl(2)(S-ahaz)] (S-ahaz = 3(S)-aminohexahydroazepine) each form two stereoisomers on binding to GpG sequences of DNA: one in which the primary amine is directed toward the 5' end of the DNA and one in which it is directed toward the 3' end. Previous binding studies have revealed that the S-enantiomer forms the two stereoisomers in a 7:1 ratio while the R-enantiomer forms them in close to a 1:1 ratio. In an attempt to elucidate the reasons behind the stereoselectivity displayed by the S-enantiomer and to establish which isomer is formed in the greater amount, we report here its reaction with a 14-mer oligodeoxyribonucleotide having a single GpG site. The two stereoisomers that formed were separated using HPLC methods, and their integrities were confirmed by electrospray ionization mass spectrometry. The DNA duplex was formed by combination of each of the purified reaction products with the complementary strand of DNA. Identification of both of the stereoisomers was achieved using 2D NMR spectroscopy, which is the first time this has been achieved for an unsymmetric platinum complex bound to DNA. The minor stereoisomer, with the bulk of the ahaz ring directed toward the 3' end of the platinated strand, induced considerable disruption to the 14-mer DNA duplex structure. The primary amine of the ahaz ligand was oriented toward the 3' side of the duplex in the major isomer, giving a DNA structure that was less disrupted and was more akin to the structure of the DNA on binding of cisplatin to the same sequence.

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.

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.

[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.

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.