Patricia Marqués-Gallego

Hans Den Dulk

Jaap Brouwer

Stefania Tanase

Ilpo Mutikainen

Urho Turpeinen

Jan Reedijk
email: reedijk@chem.leidenuniv.nl

Cytotoxic activity

Keywords: Cytotoxic activity, Fluorescence microscopy, Nucleobases, Platinum resistance, Glutathione Abbreviations

teaching and research institutions in France or abroad, or from public or private research centers.

A c c e p t e d M a n u s c r i p t 3

Introduction

In the last three decades many efforts have been dedicated to establish the factors related to the antitumor activity of different platinum compounds. Even though the molecular mechanistic studies of cisplatin and analogues have been largely developed [START_REF] Boulikas | Cisplatin and platinum drugs at the molecular level (review)[END_REF][START_REF] Reedijk | Metal-Ligand Exchange kinetics in Platinum and Ruthenium Complexes[END_REF], the cellular response to these compounds is still poorly understood. Much of the current understanding of the mechanism of action of platinum-based drugs comes from studies with cisplatin. One interesting approach proposed recently, based on a fluorescein-labeled cisplatin derivative [START_REF] Molenaar | New insights in the cellular processing of platinum antitumor compounds, using fluorophore-labeled platinum complexes and digital fluorescence microscopy[END_REF], has brought new insights on the cellular response against cisplatin antitumor agent with the use of fluorescence microscopy [START_REF] Kalayda | Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells[END_REF][START_REF] Safaei | Intracellular localization and trafficking of fluorescein-labeled cisplatin in human ovarian carcinoma cells[END_REF]. Thus, studies concerning antitumor drugs and cellular response open new options towards the design and development of new platinum compounds [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF][START_REF] Kalayda | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780 cisplatin-resistant human ovarian carcinoma cells: new insights into the mechanism of resistance[END_REF][START_REF] Kalayda | Dinuclear platinum anticancer complexes with fluorescent N,N'-bis(aminoalkyl)-1,4-diaminoanthraquinones: cellular processing in two cisplatin-resistant cell lines reflects the differences in their resistance profiles[END_REF][START_REF] Kalayda | Application of fluorescence microscopy for investigation of cellular distribution of dinuclear platinum anticancer drugs[END_REF].

The strategy of the present study deals with the coordination of a platinum(II) moiety to the fluorescent carrier ligand N,N'-bis(anthracen-9-ylmethyl)propane-1,3-diamine (abbreviated as bapda). It has been shown that the coordination of platinum(II) to fluorescent ligands is a successful approach to synthesize fluorescent platinum drugs [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF][START_REF] Alderden | Cytotoxic efficacy of an anthraquinone linked platinum anticancer drug[END_REF]. Moreover, coordination compounds containing propane-1,3-diamine have been used as model anticancer-agents to study the interaction with DNA [START_REF] Akdi | Effects of Tris and Hepes buffers on the interaction of palladium-diaminopropane complexes with DNA[END_REF]. The N-methyl substituted trimethylenediamines, N,N'-dimethylpropane-1,3diamine, and derivates have been used in the synthesis of platinum(II) complexes giving a sixmembered chelating ring with the platinum(II) ion [START_REF] Appleton | Complexes With 6 Membered Chelate Rings.VI. Proton Magnetic-Resonance Study Of Some Platinum(II) Complexes Of N,N'-Dimethylpropane-1[END_REF][START_REF] Appleton | Complexes With 6-Membered Chelate Rings.I. Preparation Of Platinum(II) And Palladium(II) Complexes Of Trimethylenediamine And Some Methyl-Substituted Derivatives[END_REF]. Thus, chelation of the bapda carrier ligand to the platinum(II) ion through the aliphatic diamines moiety would be expected.

The present contribution describes synthesis and detailed structural characterization of a new fluorescent platinum(II) compound, the cis-[Pt(bapda)Cl 2 ]. In addition, cytotoxic studies of this compound have been performed and compared to cisplatin, as well as to the free bapda, in several human cancer cell lines. The possible interaction of the cis-[Pt(bapda)Cl 2 ] compound with nucleobases has been explored with the use of the 9-ethylguanine as a DNA model base. Moreover, the interaction of cis-[Pt(bapda)Cl 2 ] with calf thymus DNA, as determined by fluorescence titration, has been explored. The cellular processing of cis-[Pt(bapda)Cl 2 ] and the ligand bapda using A c c e p t e d M a n u s c r i p t 4 fluorescent microscopy is also reported. The implication of the GSH in the cellular processing of cis-[Pt(bapda)Cl 2 ] in human ovarian carcinoma cisplatin-resistant (A2780R) cells has been explored, as a potential resistance mechanism.

Experimental Section

Physical Measurements

C, H and N analyses were performed with a Perkin-Elmer 2400 series II analyzer. Infrared spectra (4000-300 cm -1 ) were recorded on a Perkin-Elmer Paragon 1000 FTIR spectrometer equipped with a Golden Gate ATR device, using the reflectance technique (resolution 4 cm -1 ). All NMR spectra were recorded with a 300-MHz Bruker DPX 300 spectrometer with a 5-mm multinucleus probe. The temperature was kept constant at 298 K for standard spectra, while the temperature was changed to 310 K to mimic in vitro studies, by a variable-temperature unit. 195 Pt chemical shifts was referenced to Na 2 [PtCl 6 ] (! = 0 ppm).

Materials and Reagents

The compound K 2 PtCl 4 was obtained from Johnson & Matthey (Reading, U.K). All other chemicals and solvents were reagent-grade commercial materials and were used as received.

Synthesis of ligand and platinum compound

Synthesis of bapda

The ligand N,N'-bis(anthracen-9-ylmethyl)propane-1,3-diamine (abbreviated as bapda) was prepared by reported methods [START_REF] Zhang | A highly fluorescent anthracenecontaining hybrid material exhibiting tunable blue-green emission based on the formation of an unusual "T-shaped" excimer[END_REF]. O: % found (calculated) C 85.9(85.5), H 6.4 (6.7), N 6.1 (6.0).

2.3.2.Synthesis of cis-[Pt(bapda)Cl 2 ]

To a stirred solution of bapda (109 mg, 0.24 mmol) in 2.5 ml DMF, a filtered aqueous solution of K 2 PtCl 4 (100 mg, 0.24 mmol) was added. The reaction mixture was stirred two days in dark at room temperature to afford an orange solution. The resulting pale-yellow precipitate was

X-ray Crystallographic Analysis and Data collection

Crystal data, data collection parameters, and structure refinement details are given in Table S-1, Supporting Information. A crystal was selected for the X-ray measurement and mounted to the glass fiber using the oil drop method, and data were collected at 173 K on a Nonius Kappa CCD diffractometer (Mo K! radiation, graphite monochromator, ! = 0.71073 Å). The intensity data were corrected for Lorentz and polarization effects and for absorption. The programs COLLECT [15], SHELXS-97, and SHELXL-97 [16] were used for data reduction, structure solution and structure refinement respectively. The 3D coordinates have been submitted to the CSD as a CIF file (CCDC 711124); these data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Cell culture conditions and drug cytotoxicity assays

The human ovarian carcinoma cell line A2780 and its cisplatin resistant counterpart A2780R were grown as monolayers at 37 °C in a 7% CO 2 atmosphere, and were maintained in continuous logarithmic culture in Dulbecco´s modified Eagle´s Medium (DMEM) (Gibco BRL TM , Invitrogen Corporation, The Netherlands) supplemented with 10% heat inactivated fetal calf serum (Hyclone, Perbio Science, The Netherlands), penicillinG Sodium (100 units/ml: Dufecha, Biochemie BV, The Netherlands), streptomycin (100 µg/ml: Dufecha, Biochemie BV, The Netherlands) and Glutammax 100x (Gibco BRL TM , The Netherlands).

The cytotoxicity assays in A2780 and A2780R cells have been performed as previously described [START_REF] Marqués-Gallego | Cellular accumulation and DNA platination of two new platinum(II) anticancer compounds based on anthracene derivatives as carrier ligands[END_REF]. For GSH-depleted A2780R cytotoxicity assays the cells were seeded in 96-wells flat A c c e p t e d M a n u s c r i p t 6 bottom microtiter plates using medium containing 50 μM L-BSO, as previously described [START_REF] Jansen | Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs[END_REF]. The cytotoxicity assays in A2780, A2780R and GSH-depleted A2780R cells were performed in parallel and all the cytotoxic assays were repeated five times to confirm reproducibility.

The results were analysed and the pEC 50 values (EC 50 is the drug concentration that produces 50% of the maximum possible response) were determined with the GraphPad Prism™ analysis software package (Graph-Pad Software, San Diego, USA) using non-linear regression (sigmoidal dose response, variable slope). The resistance factor (RF) was calculated by dividing EC 50 in the resistant variant by the EC 50 in the respective sensitive cell line.

In addition, the cytotoxicity of cis-[Pt(bapda)Cl 2 ], and its free ligand, were studied (PCN (Tavapharmachemie) Nederland) by means of a colorimetric microculture assay (SRB assay) [START_REF] Keepers | Comparison Of The Sulforhodamine-B Protein And Tetrazolium (MTT) Assays For In vitro Chemosensitivity Testing[END_REF] in seven well-characterized human tumor cell lines containing examples of breast (MCF7 and EVSA-T), colon (WIDR), ovarian (IGROV), melanoma (M19 MEL), renal (A498), and non-small cell lung cancer (H226) as previously described [START_REF] Marqués-Gallego | Synthesis, crystal structure, studies in solution and cytotoxicity of two new fluorescent platinum(II) compounds containing anthracene derivatives as a carrier ligand[END_REF]. The IC 50 values in the ! M range are summarized in Table S-2 (see Supporting Information). The cell lines WIDR, M19, A498, IGROV and H226 belong to the currently used anticancer screening panel of the National Cancer Institute, USA. The MCF7 cell line is estrogen (ER)+/progesterone (pgR)+ and the cell line EVSA-T is (ER)-/(pgR).

Interaction of cis-[Pt(bapda)Cl 2 ] with GSH

To establish the possible reactivity of cis-[Pt(bapda)Cl 2 ] towards glutathione, cis-[Pt(bapda)Cl 2 ] and GSH were allowed to react in a 1:4 molar ratio in DMF/PBS at 310 K, and the reaction was monitored by 195 Pt NMR spectroscopy for 24 h.

Reaction of cis-[Pt(bapda)Cl 2 ] with 9-Ethylguanine

The cis-[Pt(bapda)Cl 2 ] (2.44 mg, 3.4 mmol) was allowed to react with 9-ethylguanine (9-EtG, 1.2 mg, 6.7 mmol) in DMF-d 7 at 310 K to investigate how this compound interact with nucleobases. Cis-[Pt(bapda)Cl 2 ] and 9-EtG were both dissolved in DMF-d 7 and 1 H NMR spectra A c c e p t e d M a n u s c r i p t 7 were recorded at 310 K and followed over time. After 24 h of reaction the 195 Pt NMR spectrum at 310 K was recorded.

DNA fluorescence titration

Fluorescence titration of cis-[Pt(bapda)Cl 2 ] was performed as described previously [START_REF] Alderden | Cytotoxic efficacy of an anthraquinone linked platinum anticancer drug[END_REF].

Small aliquots of a concentrated calf thymus DNA solution were added to 50 ! M solutions of cis-[Pt(bapda)Cl 2 ] ranged from 0:1 to 4:1 base pairs:cis-[Pt(bapda)Cl 2 ]. Fresh solutions of cis-[Pt(bapda)Cl 2 ] in DMF diluted with phosphate buffer were prepare fresh and the titration spectra were recorded after mixing.

Digital Fluorescence Microscopy

Cellular processing studies

Fluorescence microscopy experiments in living cells were performed in the pair of human ovarian carcinoma A2780 and A2780R cell lines. For the living cell observations the cells were grown in 35 mm culture dish to 30-50% confluence in red phenol-free Dulbecco´s modified Eagle´s Medium (DMEM) (Gibco BRL TM , Invitrogen Corporation, The Netherlands) supplemented with 10% heat inactivated fetal calf serum (Hyclone, Perbio Science, The Netherlands), penicillinG Sodium (100 units/ml: Dufecha, Biochemie BV, The Netherlands), streptomycin (100 µg/ml: Dufecha, Biochemie BV, The Netherlands) and Glutammax 100x (Gibco BRL TM , The Netherlands). Before the incubation with the compounds, the cells were washed twice with PBS.

Subsequently, the compounds were added to the cells at a final concentration of 10 μM in serumfree medium for 15 min at 37 °C and 7% CO 2 atmosphere. After incubation the cells were washed twice with PBS, and drug-free red phenol-free complete medium is added to the cells. Phase contrast and the corresponding fluorescence images were taken at different time points after incubation at 37 °C and 5.2% CO 2 atmosphere. After imaging the medium was refreshed, and the cells were kept at 37 °C in a 7% CO 2 incubator to study the accumulation 24 h after incubation with bapda or cis-[Pt(bapda)Cl 2 ] compounds.

A c c e p t e d M a n u s c r i p t 8 Pictures were taken using a microscope (IX81; Olympus The Netherlands) with ×60 objective (Olympus, The Netherlands). The temperature of the culture medium was controlled between 36 and 37 °C by an objective heater and a heated ring surrounding the culture chamber.

The CO 2 atmosphere was kept at 5.2% during imaging. To detect fluorescence signal of bapda ligand and cis-[Pt(bapda)Cl 2 ] a filter for λ ex 377 nm; time exposure = 10 ms was used. Digital images were taken with cooled CCD camera (F-View, Olympus The Netherlands). Images were processed by Cell M software.

Staining of lysosomes

Lyso Tracker Red DND-99 (Molecular Probes, Leiden, The Netherlands) was used to stain the vesicles observed in the cytosol of both A2780 and A2780R cells. The staining was performed 24 h after incubation with the corresponding compound. Lyso Tracker Red DND-99 was added to the culture medium in a final concentration of 50 nM. After 15 min of incubation with the dye, the cells were washed twice with PBS, and complete phenol red-free medium was added before imaging. For detecting fluorescence of Lyso Tracker localized in lysosomes TRITC-filter (! ex 573 nm; time exposure = 10 ms) was used.

Statistical analysis

For statistical comparison in the cytotoxic activity against A2780 and A2780R cell lines, the Student's t-test was performed. A p-value < 0.05 was considered statistically significant.

Results

General characterization

The cis-[Pt(bapda)Cl 2 ] was characterized by several physical methods, such as IR, 1 H and 195 Pt NMR spectroscopy. In addition, single crystals suitable for X-Ray diffraction were obtained and the crystal structure of the platinum(II) compound has been determined and is described below.

The IR spectrum of cis-[Pt(bapda)Cl 2 ] shows the two bands expected for ! as (Pt-Cl) and ! s (Pt-Cl) in a cis conformation at 322 and 328 cm -1 . In addition, the N-H and C-H stretching region Next to this peak, a broad singlet at 6.18 ppm corresponding to the NH protons can be observed. It is worth to note that both anthryl moieties appear as identical in the 1 H NMR spectrum, in agreement with the symmetry of the platinum(II) compound. In addition, different diastereoisomers from the chiral nitrogen formed upon coordination of bapda to platinum(II) ion were not observed in a DMF-d 7 solution of cis-[Pt(bapda)Cl 2 ] at 298 K. The coordination of the platinum(II) ion to bapda is also visible from the 195 Pt NMR spectrum, which displays a single peak at -2186 ppm corresponding to a [N 2 Cl 2 ] environment [START_REF] Pregosin | Pt-195 Nuclear Magnetic-Resonance[END_REF].

The stability of cis-[Pt(bapda)Cl 2 ] compound in solution has been followed in time in DMFd 7 by 1 H NMR and 195 Pt NMR spectroscopy. Changes over time were not observed (spectra not shown); therefore, the biological studies reported below were performed using a concentrated stock solution of cis-[Pt(bapda)Cl 2 ] compound in DMF and diluting it in cell medium at the desired concentration when needed. are listed in Table 1.

X-ray Analysis and Structure description

Single crystals of cis-[Pt(bapda)Cl

The X-ray crystallographic analysis of a single crystal shows two crystallographically independent, but almost identical molecules in the asymmetric unit and 1.5 tetrahydrofurane A c c e p t e d M a n u s c r i p t 10 molecules (one ordered THF and one disordered THF, refined with occupancy factor 0.5), with a final composition of {[Pt(bapda)Cl 2 )} 2 ](THF) 1.5 , which crystallizes in a monoclinic P2 1 /n space group (Fig. 1). The solid-state crystal structure of cis-[Pt(bapda)Cl 2 ] shows the nitrogen atoms of the coordinated (now) chiral diamines to have the R and S configuration, respectively. The plane of the platinum(II) coordination is defined by the two aliphatic nitrogens atoms and the two chloride ligands. Both mononuclear units differ only slightly in the angles formed between the plane of each anthryl ring and the metal plane. The angle N1-Pt1-N5 of 95.29 [START_REF] Jansen | Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs[END_REF]° was found to be the largest distortion for a square-planar configuration. The platinum(II)-amine chelate forms a six-membered chelate ring, which adopts a chair conformation (Fig. 1). The dihedral angles of the Pt1-N1-N5 and C2-C3-C4 planes with respect to the N1-C2-N5-C4 plane are 32.5(2) and 62.3(2)°, respectively. These angles and the Pt-Cl and Pt-N bond lengths (Table 1) in cis-[Pt(bapda)Cl 2 ]can be compared to the ones found by Odoko et al. [START_REF] Odoko | Dichloro(propane-1,3-diamine-! 2 N,N')platinum(II), dichloro(propane-1,3-diamine-! 2 N,N')palladium(II) and ! -4,9-diazadodecane-1[END_REF]; for the cis-[Pt(tn)Cl 2 ] (tn stands for propane-1,3-diamine).

Table 1. Selected bond lengths [Å] and angles [º] for the different units cis-[Pt(bapda)Cl

Bond lengths

Bond angles

Pt1 -Cl1 Pt1 -Cl2 Pt1 -N1 Pt1 -N5 N1 -C2 N5 -C4 Pt2 -Cl3 Pt2 -Cl4 Pt2 -N21 Pt2 -N25 N25 -C24 N21 -C22 2
N1 -Pt1 -N5 N1 -Pt1 -Cl1 N5 -Pt 1 -Cl2 Cl1 -Pt1 -Cl2 N21 -Pt2 -N25 N21 -Pt2 -Cl3 N25 -Pt2 -Cl4 Cl4 -Pt2 -

Cytotoxicity in human ovarian carcinoma cell lines

The cytotoxic activity of cis-[Pt(bapda)Cl 2 ], the platinum-free ligand bapda, and cisplatin as reference compound against A2780 and A2780R cell lines were studied. The EC 50 (mM) values after 48 h incubation are listed in Table 2. 2), however, significant differences (p < 0.001) between A2780 and A2780R cytotoxic activity of cis-[Pt(bapda)Cl 2 ] were observed, suggesting crossresistance to platinum of cis-[Pt(bapda)Cl 2 ] in the A2780R cells, which is indicated also by the resistance factor (RF > 2) listed in Table 2. Elevated levels of GSH provide some cancer cells resistance against platinum cancer drugs such as cisplatin [START_REF] Kalayda | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780 cisplatin-resistant human ovarian carcinoma cells: new insights into the mechanism of resistance[END_REF], and the binding to such molecules, prior the binding to DNA, has been 2). Not significant differences were found (p >0.05) when the cytotoxic activity of cis-[Pt(bapda)Cl 2 ] in normal A2780R and GSH-depleted A2780R cells are compared.

Surprisingly, the depletion of GSH levels in A2780R cells influences the ligand bapda cytotoxic activity, with significant differences between normal A2780R cells and L-BSO treated A2780R cells. Moreover, it is important to note that the cytotoxic activity of cisplatin against GSHdepleted A2780R is significantly different from that of non-L-BSO treated cells (p < 0.05). This suggests that, under the studied conditions, the GSH levels in A2780R cells contribute to the cisplatin resistance during the cisplatin incubation. The modulation factor (MF = EC 50 (A2780R)/ EC 50 (A2780R-L-BSO)) of 2.6 is in agreement with literature data [START_REF] Jansen | Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs[END_REF].

In addition to the cytotoxic activity against human ovarian carcinoma cell lines, it has been also investigated whether cis-[Pt(bapda)Cl The platinum(II) compound was reacted with an excess of GSH as described above, and the reaction was followed with 195 Pt-NMR spectroscopy. No interaction between cis-[Pt(bapda)Cl 2 ] and GSH was detected, finding only a constant single peak at -2182 ppm corresponding to unreacted cis-[Pt(bapda)Cl 2 ] up to 24 h (serial spectra not shown).

Reaction with 9-Ethylguanine

The As mentioned above, the cytotoxic activity of ligand bapda as compared to cis- S2 (see Supporting Information) shows that the coordination of the carrier ligand to platinum improves slightly the biological activity. Ligand bapda was found to intercalate to the DNA, as well as its corresponding platinum(II) compound. Nevertheless, cis-[Pt(bapda)Cl 2 ] presents also the possibility of interacting with the N7 position of the guanine bases (Fig. 2), although this interaction appears to be strongly hindered.

[Pt(bapda)Cl 2 ] summarized in Table

Digital fluorescence microscopy

The cellular response of cis-[Pt(bapda)Cl 2 ] in A2780 and A2780R cells has been investigated and is compared to that of the ligand bapda. Incubation with both compounds,i.e. bapda and cis-[Pt(bapda)Cl 2 ] has been relatively short (15 min) due to the high cytotoxic activity in A2780 and A2780R cells; nevertheless, both compounds are able to enter into the cells after 15 min of incubation, and after 24 h the compounds are still visible inside the cells, with retention within large vesicles.

According to the cellular processing images of cis-[Pt(bapda)Cl 2 ] shown in Fig. 4, the distribution over time of the platinum(II) compound in A2780 and A2780R is slightly different. E, andF). This accumulation remains constant over time up to 24 h after incubation (Fig. 5; A, B, C and D). In contrast, the cellular distribution of cis-[Pt(bapda)Cl 2 ] within the A2780 cell line changes over time (Fig. 4; A,B and C). After incubation with the compound, a fluorescence signal is visible in some vesicles close to the cell nucleus. However, three hours after the incubation cis-[Pt(bapda)Cl 2 ] is accumulated in vesicles extended over the cytosol. This accumulation remains visible 24 h after the incubation (Fig. 5; E, F, G and H). In both cell lines the large vesicles were identified as acidic lysosomes (Fig. 5) with the use of a specific stain (Lyso Tracker Red DND-99). In addition, as it is clear from the phase-contrast images, the number of vesicles around the nucleus present in the cell increases over time. 6, the accumulation of bapda is less localized when imaging for a few minutes, 1 or 3 h after the incubation, while for its platinum(II) compound the accumulation is very specific (Fig. 4; A, B, andC)) after incubation. However, similar cellular processing 24 h after incubation with ligand bapda, compared to that of cis-[Pt(bapda)Cl 2 ] (Fig. 5) is observed in A2780 cells (Fig. S5, supporting Information). Both compounds are accumulated in acidic lysosomes, as shown from co-localization with Lyso Tracker Red DND-99, however, sequestration in additional vesicles in the A2780 cells is also observed. To investigate whether glutathione facilitates the sequestration of these compounds into the lysosomes as a detoxification cell-mechanism, cellular processing of cis-[Pt(bapda)Cl 2 ] and bapda in GSH-depleted A2780R cells was followed over time. In correlation to the cytotoxic data, no changes in the cellular processing have been found related to cis-[Pt(bapda)Cl 2 ] treatment, where similar lysosomal accumulation could be observed in L-BSO treated or untreated A2780R cells (pictures not shown). However, differences in the cellular processing of bapda in L-BSO treated and untreated A2780R cells have been found (Fig. 7). This observation is also in agreement with the cytotoxic activity data (Table 2), where significant differences in the cytotoxic activity in A2780R and GSH-depleted A2780R cells were observed. The sequestration of the ligand bapda is visible over the cell in some vesicles different than acidic lysosomes (Fig. 7), as well as in the lysosomes.

Discussion

A new fluorescent platinum(II) compound containing bapda as carrier ligand has been synthesized and characterized by several physical methods. The cytotoxic activities of both free ligand and cis-[Pt(bapda)Cl 2 ] a high antiproliferative activity in A2780 and A2780R cells.

The biological activity of cis-[Pt(bapda)Cl 2 ] is significantly different from that found for ligand bapda, suggesting that the coordination to platinum(II) ion improves the activity of bapda.

However, cross-resistance to platinum has been found for cis-[Pt(bapda)Cl 2 ] against A2780R cells.

As it is well known, the inactivation of platinum(II) compounds by GSH or MT molecules is an important resistance mechanism [START_REF] Rabik | Molecular mechanisms of resistance and toxicity associated with platinating agents[END_REF]. However, the GSH inactivation does not seem to play an important role towards cross-resistance to platinum found for cis-[Pt(bapda)Cl 2 ] during the in vitro studies presented herein, since no significant changes have been observed between GSH-depleted A2780R cells and normal A2780R cells. In addition, the reaction between GSH and cis- compounds are reported in the literature [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF][START_REF] Kalayda | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part II. Cellular processing in A2780 cisplatin-resistant human ovarian carcinoma cells: new insights into the mechanism of resistance[END_REF][START_REF] Kalayda | Dinuclear platinum anticancer complexes with fluorescent N,N'-bis(aminoalkyl)-1,4-diaminoanthraquinones: cellular processing in two cisplatin-resistant cell lines reflects the differences in their resistance profiles[END_REF][START_REF] Kalayda | Application of fluorescence microscopy for investigation of cellular distribution of dinuclear platinum anticancer drugs[END_REF][START_REF] Alderden | Cytotoxic efficacy of an anthraquinone linked platinum anticancer drug[END_REF]. Interestingly, dinuclear platinum(II) compounds containing anthraquinone ligands trapped in lysosomal vesicles were previously reported [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF], and accumulation in A2780R cells was found constant over time, which was not observed for the sensitive cell line A2780. This observation suggested that the vesicular sequestration was due to a resistance mechanism present in the A2780R cells. However, in the same studies, after 24 h of incubation with the corresponding ligand no vesicles containing the compounds were observed [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF],

suggesting that the sequestration in acidic vesicles in A2780R cell line is a specific response to the corresponding platinum compound [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF]. In the case of cellular processing of cis-[Pt(bapda)Cl 2 ] and bapda in A2780R cells, fast sequestration after short incubation in acidic lysosomes is observed, which suggests that it is not a specific cellular response to the platinum compound. Moreover, accumulation of cis-[Pt(bapda)Cl 2 ] in acidic lysosomes in A2780 cells has been observed as well, indicating that this sequestration might not be related to resistance mechanisms. Putting all together, these observations suggest that the lysosomal sequestration is a detoxification mechanisms of the cells to cis-[Pt(bapda)Cl 2 ] and bapda. Organic drugs, such as daunorubicin and mitoxantrone were found to be accumulated in acidic vesicles [START_REF] Merlin | Modulation of daunorubicin cellular resistance by combination of P-glycoprotein blockers acting on drug efflux and intracellular drug sequestration in golgi vesicles[END_REF], which indicates that this might be a protective As it has been reported previously, the glutathione is involved in the excretion and/or sequestration of toxic compounds as a cellular protection system [START_REF] Ishikawa | The GS-X pump in plant, yeast, and animal cells: Structure, function, and gene expression[END_REF]. To investigate whether glutathione facilitates sequestration of cis-[Pt(bapda)Cl 2 ] in lysosomes, the fluorescent microscopy in GSH-depleted A2780R cells has been investigated and compared to that in normal A2780R cells.

The cellular processing of cis-[Pt(bapda)Cl 2 ] in GSH-depleted A2780R cells shown no significant differences as compared to the same studies in normal A2780R cells. This observation correlates with the cytotoxic activity data of cis-[Pt(bapda)Cl 2 ] in normal A2780R cells and in GSH -depleted A2780R cells, where no significant differences were found between the EC 50 values. Therefore, the sequestration of cis-[Pt(bapda)Cl 2 ] after short-time incubation in lysosomes seems to be not related to the GSH levels, and might indicate a specific drug uptake pathway, such as endocytosis. To elucidate this behavior in detail, further studies are in progress and will be published elsewhere. In contrast, a quite different cellular processing has been observed with the ligand bapda in GSHdepleted A2780R cells, where an increased number of different vesicles are visible in the cells after incubation with bapda (Fig. 6). This suggests that the cellular processing of the ligand bapda is influenced by the concentration of GSH in A2780R cells. This is also correlated with the cytotoxic activity data, where significant differences between cytotoxic activity of bapda in normal A2780R cells and GSH-depleted A2780R cells.

In addition to the in vitro studies and because it is believed that the main target of platinum Moreover, related platinum compounds with ethyl substituents on the amine groups have been described in the literature [START_REF] Vickery | Preparation, Characterization, Cytotoxicity, And Mutagenicity Of A Pair Of Enantiomeric Platinum(II) Complexes With The Potential To Bind Enantioselectively To DNA[END_REF], and were found to be cytotoxic against several cell lines in the absence of the intercalating groups, compared to cis-[Pt(bapda)Cl 2 ]. These results, together with the low reactivity of cis-[Pt(bapda)Cl 2 ] towards 9-EtG and low cross-resistance with cisplatin, suggest that the biological activity of this new platinum(II) compound stems primarily from the anthracene groups. In the literature interesting classes of organic molecules, so-called bisintercalators [START_REF] Dawson | Bisintercalator natural products with potential therapeutic applications: isolation, structure determination, synthetic and biological studies[END_REF],

have been synthesized by linking two heterocycles with chains of varying lengths between the intercalator moieties [START_REF] Becker | Molecular Recognition Of Nucleic-Acid By Small Molecules -Binding-Affinity And Structural Specificity Of Bis-(Methidium)Spermine[END_REF]. Such special molecules have been investigated successfully as carrier ligands in the synthesis of new platinum(II) antitumor agents [START_REF] Perez | Combined effect of platination and intercalation upon DNA binding of novel cytotoxic Ptbis(naphthalimide) complexes[END_REF]. Thus, the cis-[Pt(bapda)Cl 2 ] may intercalate with the anthracene ring between the DNA base pairs. Moreover, it has been previously reported fluorescence quenching experiment, where small amounts of a concentrated stock solution of pBR322 plasmid DNA [START_REF] Jansen | Dinuclear platinum complexes with N,N '-bis(aminoalkyl)-1,4-diaminoanthraquinones as linking ligands. Part I. Synthesis, cytotoxicity, and cellular studies in A2780 human ovarian carcinoma cells[END_REF] or calf thymus DNA [START_REF] Alderden | Cytotoxic efficacy of an anthraquinone linked platinum anticancer drug[END_REF] were added to the solutions of fluorescent

A c c e p t e d M a n u s c r i p t 5 washed

 5 with water, ethanol, and diethyl ether and dried in vacuum. Crystalline product from diffusion of n-hexane into a THF solution of the compound was obtained. Yield 46% (79 mg). 1 H NMR (DMF-d 7 ): ! (ppm) = 9.08 (4H, d), 8.82 (2H, s), 8.22 (4H, d), 7.79 (4H, t), 7.64 (4H, t), 6.18 (2H, s), 5.71 (2H, d), 2.15 (2H, d), 1.43 (2H, d) 1.12 (2H, t); 195 Pt NMR (DMF-d 7 ):! (ppm) = -2186; anal. for [C 33 H 30 N 2 PtCl 2 ]!1.5 THF : % found (calculated) C 55.5 (56.5), H 4.9 (5.1), N 3.7 (3.4).

A c c e p t e d M a n u s c r i p t 9 contains

 9 three peaks (3200-3000 cm -1 )[START_REF] Appleton | Complexes With 6-Membered Chelate Rings.I. Preparation Of Platinum(II) And Palladium(II) Complexes Of Trimethylenediamine And Some Methyl-Substituted Derivatives[END_REF]. The 1 H NMR spectra of the carrier ligand and the cis-[Pt(bapda)Cl 2 ] in DMF-d 7 were recorded and compared. Significant changes upon coordination of the ligand bapda to the platinum(II) ion are observed. The H 2 and H 4 protons appear at 2.98 ppm (triplet) in the ligand spectrum, while the spectrum of cis-[Pt(bapda)Cl 2 ] shows two doublets at 2.15 and 1.43 ppm, respectively. In addition the H 3 peak is shifted from 1.84 to 1.09 in the platinum(II) compound. Moreover, the singlet at 4.69 ppm corresponding to H 6 and H 6' protons in the ligand displays a clear shift to 5.71 ppm, which indicates clearly the coordination to the platinum(II) ion.

  2 ] were obtained by slow diffusion of n-hexane into a THF solution containing cis-[Pt(bapda)Cl 2 ]. Selected bond lengths and angles for cis-[Pt(bapda)Cl 2 ]

  2 ] and relevant hydrogen bonding parameters of cis-[Pt(bapda)Cl 2 ].

Du s c r i p t 11 Fig. 1 .

 111 Fig. 1. PLATON/POVRAY view of one of the two cis-[Pt(bapda)Cl 2 ] entities. Hydrogen atoms and

The

  cis-[Pt(bapda)Cl 2 ] displays a high cytotoxic activity against both the human ovarian carcinoma sensitive and cisplatin-resistant cell lines. Moreover, significant difference (p < 0.05) between the cytotoxic activity in A2780 cell line of cis-[Pt(bapda)Cl 2 ] and the ligand bapda was found, indicating that the coordination compound is significantly more active against the cisplatin-s c r i p t sensitive human ovarian carcinoma A2780 cells. In addition, cis-[Pt(bapda)Cl 2 ] displays the same level of cytotoxic activity as cisplatin in A2780 cells, where significant differences (p > 0.05) were not observed. Both bapda and cis-[Pt(bapda)Cl 2 ] improve the cytotoxic activity against A2780R as compared to cisplatin treatment (Table

Table 2 .- 3 *

 23 pEC 50 and EC 50 values after 48 h incubation and with cis-[Pt(bapda)Cl 2 ], bapda, and cisplatin as a reference compound in human ovarian carcinoma cell lines A2780 and A2780R (pEC 50 , mean ± SD, n = 4 -5). RF = EC 50 (A2780R)/ EC 50 (A2780) A2780R-L-BSO = A2780R cells pre-and co-incubated with L-BSO during cytotoxic studies

A c c e p t e d M a n u s c r i p t 13 presented

 13 earlier from our laboratory[START_REF] Lempers | Reactions Of [PtCl(Dien)]Cl With Glutathione, Oxidized Glutathione And S-Methyl Glutathione -Formation Of An S-Bridged Dinuclear Unit[END_REF][START_REF] Reedijk | Why does cisplatin reach guanine-N7 with competing S-donor ligands available in the cell?[END_REF][START_REF] Vanboom | Unprecedented Migration Of [Pt(dien)] 2+ (Dien = 1,5-Diamino-3-Azapentane) From Sulfur To Guanosine-N7 In S-Guanosyl-L-Homocysteine (sgh)[END_REF]. As mentioned above, cis-[Pt(bapda)Cl 2 ] shows crossresistance to platinum after 48 h incubation in A2780R cells. It is well known that the resistance mechanisms against platinum drugs are multifactorial, and four mechanisms for drug resistance have been proposed[START_REF] Rabik | Molecular mechanisms of resistance and toxicity associated with platinating agents[END_REF]: (i) inhibition of drug uptake or increased drug efflux, (ii) inactivation of the drug by thiol-containing molecules (metallothionein and/or glutathione) within the cells, (iii) accelerated repair of chemotherapy-induced cellular damage, and (iv) inhibition of cell death pathways. To investigate the implication of the GSH in our results, depletion of GSH levels by Lbuthionine-S,R-sulfoximine (L-BSO) in A2780R was investigated towards cytotoxic activity of cis-[Pt(bapda)Cl 2 ] (Table

2 ]A c c e p t e d M a n u s c r i p t 15 3. 4 .

 2154 is complementary to cisplatin against a wider range of cancer cell lines. Therefore, the cytotoxic activity of cis-[Pt(bapda)Cl 2 ], the ligand bapda, and cisplatin were tested in a larger panel of human cancer cell lines (PCN Tavapharmachemie, The Netherlands). The IC 50 values are listed in Table S-2 (see Supporting Information). Cis-[Pt(bapda)Cl 2 ] indeed shows a higher activity than cisplatin in all the cell lines tested, with the only exception of the IGROV cell line. Additionally, ligand bapda shows lower cytotoxicity compared to that of cis-[Pt(bapda)Cl 2 ] in most of the cell lines, with the exception of the H226 (non-small lung A c c e p t e d M a n u s c r i p t 14 cancer) cell line. The biological activity of cis-[Pt(bapda)Cl 2 ] and ligand bapda may stem from the interaction with the nuclear DNA; however, the differences in the cytotoxicity are small. Interaction of cis-[Pt(bapda)Cl 2 ] with GSH

reaction of 9 -

 9 EtG with cis-[Pt(bapda)Cl 2 ] results in the slow formation of small amounts of [Pt(bapda)(9-EtG)Cl]Cl after 10 h of reaction; nevertheless, the original cis-[Pt(bapda)Cl 2 ] remains the major species in solution (Fig. 2), even after 12 days of reaction, and only 20% of a Pt-9EtG species is obtained over this time. The 195 Pt NMR spectrum after 24 h of reaction was recorded, giving two peaks: one at -2181 ppm corresponding to the unreacted cis-[Pt(bapda)Cl 2 ], and a new small peak at -2335 ppm; which corresponds to the platinum-9-EtG adduct product of the reaction (as expected for a [N 3 Cl] environment; See Fig. S2, supporting Information) [21]. In addition to the NMR spectroscopy, ESI-MS of the DMF-d 7 solution after 24 h of reaction shows the formation of [Pt(bapda)(9-EtG)Cl] + species, as confirmed by a m/z with platinum isotopic pattern at 864. Moreover, a peak at m/z = 686 corresponding to [Pt(bapda)Cl] + is observed as well (Fig. S3, Supporting Information).

Fig. 2 . 3 . 6 .

 236 Fig. 2. Aromatic region of the 1 H NMR spectrum (300 MHz) of (a) cis-[Pt(bapda)Cl 2 ] in DMF-d 7 at 310 K (b) reacted with 2 eq. 9-EtG 24 h after mixed, and (c) 48 h after mixed. (↑) reaction product. (*)H 6 and H 6' modification upon 9-EtG addition.

A c c e p t e d M a n u s c r i p t 17 Fig. 3 .

 173 Fig. 3. Quenching of cis-[Pt(bapda)Cl 2 ] fluorescence emission upon titration with calf thymus DNA

s c r i p t 18 Quickly

 18 after incubation with cis-[Pt(bapda)Cl 2 ] accumulation in A2780R cells in large vesicles is observed (Fig.4; D,

Fig. 4 .s c r i p t 19 Similar

 419 Fig. 4. Cellular distribution of cis-[Pt(bapda)Cl 2 ] in the A2780 cells (A, B and C) and in in the

Fig. 5 . 20 Fig. 6 .

 5206 Fig. 5. Cellular distribution of cis-[Pt(bapda)Cl 2 ] in the A2780R cells (images A,B,C and D) and in

21 Fig. 7 .

 217 Fig. 7. A: Cellular distribution of bapda in depleted GSH A2780R cells 24 h after incubation time.

u s c r i p t 22 [

 22 Pt(bapda)Cl 2 ] (1:4) was investigated by195 Pt NMR spectroscopy for the first 24 h, and no reaction was observed. This observation confirm that GSH cannot react with cis-[Pt(bapda)Cl 2 ]. Moreover, cationic 1,1/t,t type platinum(II) compounds that reacted with GSH by the displacement of the chlorides and subsequent degradation of the compound by the replacement of the diaminealkyl linker[START_REF] Jansen | Glutathione induces cellular resistance against cationic dinuclear platinum anticancer drugs[END_REF]. However, in the case of cis-[Pt(bapda)Cl 2 ], the carrier ligand is coordinated in a chelating fashion way. Thus, the stability of this new platinum(II) compound is expected to be higher against GSH inactivation.Cellular processing studies of cis-[Pt(bapda)Cl2 ] in A2780R cells with fluorescence microscopy show accumulation in acidic lysosomes after short incubation, which could be attributed to the resistant mechanisms. Several examples of such accumulation of platinum(II)

A c c e p t e d M a n u s c r i p t 23 response

 23 of the cells to different external agents. Moreover, the cellular distribution of cis-[Pt(bapda)Cl 2 ] and bapda in A2780 cells appears to be slightly different. Three hours after incubation with cis-[Pt(bapda)Cl 2 ] lysosomal sequestration of this platinum(II) compound was observed. The free ligand is sequestered within lysosomes as well; however, this was not observed within the first 3 h after the incubation, suggesting that the detoxification mechanisms of A2780 cells respond differently to the platinum(II) compound.

  compounds is the DNA, two different approaches to elucidate the reactivity of cis-[Pt(bapda)Cl 2 ] A c c e p t e d M a n u s c r i p t 24 with the DNA have been performed. The interaction of cis-[Pt(bapda)Cl 2 ] with the 9-EtG was investigated by 1 H and 195 Pt-NMR spectroscopy. After 12 days reaction it can be concluded that the steric protection around the platinum(II) ion most likely hampers the 9-EtG approaches to the metal ion. Nevertheless, it is clear that small amounts of [Pt(bapda)(9-EtG)Cl] species are formed.

  platinum(II) compounds to investigate the possible intercalative properties of such molecules.Similar studies has been performed with cis-[Pt(bapda)Cl 2 ], with a significant decrease in the emission spectrum after the addition of calf thymus DNA. This observation suggests that the anthracene rings of cis-[Pt(bapda)Cl 2 ] intercalate between the DNA base pairs. The data reported herein is a useful baseline for future studies with cis-[Pt(bapda)Cl 2 ]. The cellular accumulation and DNA platination of cis-[Pt(bapda)Cl 2 ] are of high interest, since decreased uptake and increased tolerance to DNA damage have been found to be involved in the resistance mechanisms of several platinum-based drugs. These studies are in progress and will be reported elsewhere.

A c c e p t e d M a n u s c r i p t 25 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 27 The

 2527 CIF file for compound cis-[Pt(bapda)Cl 2 ] (CCDC 711124) giving full X-ray crystallographic data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. A c c e p t e d M a n u s c r i p t

Acknowledgments

This work has been performed under the auspices of the Graduate Research School HRSMC, a joint activity of Leiden University and the two Universities in Amsterdam. This research has been financially supported by the Council for Chemical Sciences of The Netherlands Organization for Scientific Research (CW-NWO). The support and sponsorship concerted by COST Actions D39/0006/02 and D39/0005/11 is kindly acknowledged. The authors thank Johnson & Matthey (Reading, UK) for their generous loan of K 2 PtCl 4 . The cytotoxicity test of the compounds was generously supported by PCN (Tavapharmachemie) The Netherlands. Fons Lefeber, Jos van Brussel, John A.P.P. van Dijk and Jopie A. Erkelens-Duijndam are acknowledged for assistance with the NMR techniques, for the C, H and N analyses, and for the ESI-MS determinations, respectively. The authors would like to acknowledge Simon Contaldi for useful discussions. Both A2780 cell lines were kindly provided by Prof. Dr. Carmen Navarro-Ranninger (Universidad Autonoma de Madrid, Spain).

Supporting Information Available: Table S1. Crystal data and structure refinement details for cis-Pt(bapda)Cl 2 , Table S2. IC 50 values in ! M of compound cis-[Pt(bapda)Cl


Maria A Günther Sillero

Anabel De Diego

Janeth E F Tavares

Joana A D Catanho

Da Silva

Francisco J Pérez-Zúñiga

Antonio Sillero
email: antonio.sillero@uam.es

Joana A D Catanho Da Silva

geranyl di-and triphosphate, farnesyl di-and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases. Potential relationship with the effect of bisphosphonates on osteoclasts

Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di-and triphosphate;

geranyl di-and triphosphate, farnesyl di-and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases. Potential relationship with the effect of bisphosphonates on osteoclasts

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A c c e p t e d M a n u s c r i p t 2 ABSTRACT Compounds of the mevalonate pathway containing a terminal di-or triphosphate (mev-PP or mev-PPP) were tested as substrates of several enzyme ligases (T4 RNA ligase, T4 DNA ligase, firefly luciferase and other ligases) for the synthesis of ATP derivatives of the mev-pppA or mev-ppppA type. T4 RNA ligase, in the presence of ATP and the substrates: geranyl, farnesyl or isopentenyl triphosphates, and geranyl, farnesyl, dimethylallyl or isopentenyl diphosphates, all at 0.3 mM concentration, catalyzed the synthesis of the corresponding ATP derivatives at a relative rate of activity of: 7.6 ± 1.4 mU/mg or 100 %; 39%; 42%; 24%; 18%; 12% and 6%, respectively. Inhibition (%) of the synthesis by excess of substrate (0.8 mM vs. 0.3 mM) was observed with farnesyl diphosphate (99%); farnesyl triphosphate (96%) and geranyl triphosphate (32%). V max , K m , K cat and K cat /K m values were also determined. The K cat /K m values calculated were for: farnesyl triphosphate, 166; geranyl triphosphate, 52.2; farnesyl diphosphate, 12.1; geranyl diphosphate, 8.6; isopentenyl triphosphate, 6.7; dimethylallyl diphosphate, 3.1 and isopentenyl diphosphate, 0.9. Similar results were obtained with T4 DNA ligase. The above-mentioned compounds were also substrates of firefly luciferase synthesizing the mev-pppA or mev-ppppA derivatives. In our hands, neither the acyl-or acetyl-CoA synthetases nor the ubiquiting activating enzyme (E1) catalyzed the synthesis of ATP derivatives of these compounds. The results here presented could be related with the mechanism of action of bisphosphonates on osteoclasts or tumor cells. Introduction Bisphosphonates (P-C(R1)(R2)-P) (BPs) are widely used in the treatment of osteoporosis and other bone diseases [START_REF] Bartl | Bisphosphonates in Medical Practice[END_REF]. After being fixed to bone tissues [START_REF] Lin | Bisphosphonates: a review of their pharmacokinetic properties[END_REF][START_REF] Chen | Pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with bone metastases[END_REF][START_REF] Skerjanec | The pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with varying degrees of renal function[END_REF], they exert a damaging effect on osteoclasts through two classically described mechanisms: a) as substrates of different ligases generating BP derivatives of ATP through reactions 1, 5; 1a, 5a (see Table 1); this effect is supposed to be carried out by the first generation, or not containing nitrogen, bisphosphonates (non-N-BPs) [START_REF] Klein | Methylenediphosphonates, a metabolite poison in Dictyostelium discoideum. 31 P NMR evidence for accumulation of adenosine 5´-(,Methylenetriphosphate) and diadenosine 5´-5´´´-(P 2 ,P 3 -methylenetetraphosphate)[END_REF][START_REF] Rogers | Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-tRNA synthetase enzymes[END_REF][START_REF] Rogers | New insights into the molecular mechanisms of action of bisphosphonates[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases[END_REF]; b) as inhibitors of the mevalonate pathway (Fig. 1) by causing several pathological consequences such as: inhibition of the synthesis of cholesterol and its derivatives and, secondarily, inhibition of the post-translational modifications (isoprenylation, farnesylation) of important regulatory proteins [START_REF] Reszka | Bisphosphonate mechanism of action[END_REF][START_REF] Bergstrom | Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase[END_REF][START_REF] Rogers | New insights into the molecular actions of nitrogencontaining bisphosphonates[END_REF][START_REF] Amin | Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis[END_REF][START_REF] Fisher | Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro[END_REF][START_REF] Roelofs | Molecular mechanisms of action of bisphosphonates: current status[END_REF].

The stimulation of the synthesis of ApppI (here named iso-pppA, see nomenclature below) in osteoclasts and J774 macrophages when treated with nitrogen containing bisphosphonates (N-BPs) (zoledronate, risedronate, ibandronate, alendronate) has been recently reported [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF].

ApppI is the compound resulting from the union of the AMP moiety of ATP with isopentenyl pyrophosphate, a component of the mevalonate pathway.

The synthesis of iso-pppA correlated well with the capacity of those BPs to inhibit the mevalonate pathway. Moreover, iso-pppA, as well as the ATP derivative of clodronate (AppCCl 2 p), inhibited the mitochondrial ADP/ATP translocase and caused apoptosis in osteoclasts [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF][START_REF] Lehenkari | Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite[END_REF]. ApppI (iso-pppA) can then be an interesting compound to be considered in the mechanism of action of bisphosphonates. Mönkkönen et al. [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF] suggested that the A c c e p t e d M a n u s c r i p t 4 synthesis of iso-pppA could be catalyzed by aminoacyl tRNA synthetases through reactions 1 and 6 (Table 1).

The work here presented started by exploring the synthesis of iso-pppA by different enzyme ligases, and has been further extended to explore the synthesis of similar derivatives (mev-p n A) of other compounds of the mevalonate pathway, based on the following grounds.

A. In our experience with enzyme ligases [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF], the formation of the complex E-X-AMP (or E-AMP) is rather specific for each enzyme (Table 1, reactions 1 or 1a). Once the E-X-AMP (or E-AMP) complex is formed, its AMP moiety reacts forming a phosphodiester bond with nucleotides containing 2, 3, or more terminal phosphates or even with polyphosphate chains of variable length (Table 1, reactions 2, 3 and 4). However, each ligase behaves differently concerning the synthesis of A(p) n N or (p) n A [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF]; as an example, the synthesis of compounds as bizarre as p 20 A or Ap 16 A are catalyzed by luciferase in the presence of luciferin, ATP and a polyphosphate chain [START_REF] Ortiz | Specific synthesis of adenosine(5')tetraphospho(5')nucleoside and adenosine(5')oligophospho(5')adenosine (n > 4) catalyzed by firefly luciferase[END_REF].

B. Several ligases catalyze the transfer of AMP from the E-X-AMP or E-AMP complexes to a bisphosphonate, with formation of the corresponding derivative (reactions 5, 5a, Table 1) [START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases[END_REF].

C. The interesting finding of the occurrence of ApppI (iso-pppA) in osteoclasts incubated with zoledronate [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF] prompted us to investigate the possible synthesis by ligases of compounds of the mev-pppA type (where mev represents any compound of the mevalonate pathway ending with a terminal pyrophosphate, (Fig. 1)). As shown here, this synthesis can be catalyzed by a variety of AMP forming ligases. This finding, apart from being of potential basic interest, may help to unravel the mechanisms of

Materials and methods

1. Materials

Mevalonate pathway compounds: ,dimethylallyl pyrophosphate, Cat. Other enzymes. acyl-CoA synthetase [START_REF] Fontes | Acyl coenzyme A synthetase from Pseudomonas fragi catalyzes the synthesis of adenosine 5'-polyphosphates and dinucleoside polyphosphates[END_REF], acetyl-CoA synthetase [START_REF] Guranowski | Adenosine 5'tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase[END_REF] and the ubiquitin activating enzyme (E1) [START_REF] Sillero | Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1[END_REF] were assayed as previously described.

Enzyme units (U). One unit (U) is the amount of enzyme transforming one mol of substrate per min.

Analysis by TLC. Aliquots of 1.5 l of the reaction mixtures were spotted on silica gel plates and developed for 2 h in dioxane:ammonium hydroxide:water (6:1:6 by volume).

Nomenclature.

The compounds here synthesized are structurally related to dinucleoside polyphosphates (Np n N´) or nucleoside(5´)oligophospho(5´)nucleoside or N(5´)p n (5´)N´. There are a plethora of such compounds, some of them of physiological relevance [START_REF] Mclennan | Ap 4 A and other dinucleoside polyphosphates[END_REF][START_REF] Mclennan | Recent progress in the study of the intracellular functions of diadenosine polyphosphates[END_REF][START_REF] Delicado | Dinucleoside polyphosphates and their interaction with other nucleotide signaling pathways[END_REF]. Most of their abbreviations start and end with a capital letter representing a nitrogenous base, for instance Ap 4 A or AppppA or diadenosine tetraphosphate. For a review on the chemistry and nomenclature of dinucleoside polyphosphates see [START_REF] Holler | The chemistry of dinucleoside polyphosphates[END_REF].

The recently discovered ATP derivative of isopentenyl diphosphate has been abbreviated as ApppI (I standing for isopentenyl) and named as 1adenosin-5´-yl ester 3-(3-methylbut-3-enyl) ester [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF]. In this report, to avoid confusion with the already well-established nomenclature for the dinucleoside polyphosphates, the ATP derivatives of the compounds of the mevalonate pathway have been abbreviated using the first three letters of A c c e p t e d M a n u s c r i p t 8 its name, with a dash followed by the number of inner phosphates and the suffix adenosine. So, the ATP derivatives of isopentenyl diphosphate or of farnesyl triphosphate have been abbreviated as iso-pppA or far-ppppA, respectively. In general, the compounds of the mevalonate pathway covalently bound to AMP are named as mev-(p) n A. (Table 1).

Results

Synthesis and characterization of isopentenyl triphospho adenosine

(iso-pppA) by T4 RNA ligase. Analysis by TLC.

To investigate whether the ligases capable to synthesize bisphosphonate derivatives of ATP, as already shown in our laboratory [START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases[END_REF], were able to synthesize as well, the ATP derivative of isopentenyl pyrophosphate (ApppI [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF], here iso-pppA), the enzyme T4 RNA ligase was firstly tested. The reaction mixture contained of 0.02 mM [-32 P]ATP and 1 mM iso-PP.(Fig. 2, lanes 3,5,and 7). At different times of incubation samples were taken and analyzed by TLC as described in Materials and methods. Two control reactions (with no added enzyme (Fig. 2, lane 1) and with no added iso-PP (Fig. 2, lanes 2, 4 and 6), were run in parallel. In the reaction mixture containing iso-PP (Fig. 2, lanes 3, 5 and 7) a new radioactive spot migrating ahead of AMP and increasing with incubation time was formed. This spot, the presumptive iso-pppA derivative, was not degraded by alkaline phosphatase (lane 9) and, after inactivation of the phosphatase, treatment with snake venom phosphodiesterase (SVP) rendered labeled AMP. In our RNA ligase takes place through reactions 1a and 6a (Table 1).

In the control reactions (lanes 2, 4 and 6), containing T4 RNA ligase but not iso-PP, the formation of radioactive spots that increased with incubation time and co-migrated with standards of AMP and Ap 4 A, were also observed.

Treatment with alkaline phosphatase, resulted in the appearance of a new spot (that eluted slightly above a standard of ADP) and was resistant to phosphodiesterase. This product, unrelated to the presence or absence of isopentenyl-PP, could not be identified.

Synthesis and characterization of isopentenyl tetraphospho adenosine (iso-ppppA) by T4 RNA ligase. Analysis by HPLC

Reaction mixtures (0.08 ml) containing 60 U T4 RNA ligase, 0.3 mM ATP and 0.4 mM Iso-PPP (other components as indicated in Material and methods), were incubated for 6 and 20 h, and aliquots analyzed by HPLC (Fig. 3, b andc). The rest of the reaction mixture was treated with 1 l (1U) of shrimp alkaline phosphatase (AP) (for 2 h at 37ºC) (Fig. 3,d) and, after inactivation of the phosphatase by heating, treated with snake venom phosphodiesterase (SVP) (Fig. 3,e). The control reaction with no added enzyme is shown in (Fig. 3,a). As shown here, the presumptive iso-ppppA, (with a retention time (t R ) of around 54 min), was resistant to the phosphatase and yielded AMP when incubated with phosphodiesterase (Fig. 3,e). In the course of the reaction, the synthesis of a compound (t R of 34 min) that co-migrated with a standard of Ap 4 A (not shown) was also observed (Figs. 3, b, c andd).

A c c e p t e d M a n u s c r i p t 10

A comparative study of several compounds of the mevalonate pathway as substrates of T4 RNA ligase

On the accounts presented in the Introduction, the following compounds of the mevalonate pathway were assayed comparatively as substrates of T4

RNA ligase, at two different concentrations (0. was observed with far-PP, far-PPP and ger-PPP; the enzyme activities determined with these substrates at 0.8 mM were <1%, 7% and 68%, respectively, of those obtained at 0.3 mM (Figs. 4 and5). The inhibition was also followed at different concentrations of substrate (far-PP and far-PPP) as shown in Fig. 6a,b, and Fig. 6c,d, respectively.

The V max , K m , K cat and K cat /K m values of T4 RNA ligase determined for each one the substrates tested are compiled in Table 2. The V max values were higher for the substrates with a terminal triphosphate. Regarding K m values, iso-PP and iso-PPP were the substrates with the lowest affinity for the

Synthesis of ATP derivatives of compounds of the mevalonate pathway catalyzed by T4 DNA ligase

This point started with the synthesis and characterization of iso-pppA using iso-PP as substrate. The reaction mixtures contained 0.02 mM [-32 P]ATP, 1 mM iso-PP, and other components as indicated in Material and methods (Fig. 7). Samples were taken from the reaction mixture at different times of incubation and analyzed by TLC. As performed with T4 RNA ligase (Fig. 2), two control reactions (with no added enzyme (Fig. 7, lane 1) and with no added iso-PP (Fig. 7, lanes 2, 4 and 6), were run in parallel. In the reaction mixture containing iso-PP (Fig. 7, lanes 3, 5 and 7) a new spot migrating ahead of AMP and increasing with incubation time was formed. This spot (presumptive iso-pppA), as with T4 RNA ligase (Fig. 2), was not degraded by alkaline phosphatase (AP) (lane 9), and treatment with snake venom phosphodiesterase (SVP) rendered AMP, demonstrating the synthesis of iso-pppA from ATP and iso-PP. As occurred with T4 RNA ligase (Fig. 2), the control reactions with added enzyme but not iso-PP (Fig. 7, lanes 2, 4 and 6), showed also spots that co-migrated with standards of AMP and Ap 4 A. After alkaline phosphatase treatment, a spot resistant to phosphodiesterase, eluting slightly above a standard of ADP, was also observed.

Before comparing the results obtained with T4 RNA ligase (Fig. 2) and T4

DNA ligase (Fig. 7), it should be remind that the T4 RNA ligase assays A c c e p t e d M a n u s c r i p t

12 include an ATP regenerating system, due to the ATPase activity present in the commercial preparation of T4 RNA ligase [START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase[END_REF]. Therefore, ADP (an usual contaminant of the [-32 P]ATP preparation) is not observed, while it is clearly appreciated in the T4 DNA ligase assays that do not include the ATP regenerating system. Since both enzymes are able to catalyze the synthesis of Ap 4 A from ATP [START_REF] Madrid | T4 DNA ligase synthesizes dinucleoside polyphosphates[END_REF][START_REF] Atencia | T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates[END_REF], a small amount of Ap 4 A used to be formed in the absence or presence of iso-PP. However, in the context of this work the important points are: i) the appearance of a product, present exclusively in the reaction mixtures containing iso-PP; ii) its resistant to alkaline phosphatase and iii) the formation of AMP following treatment with phosphodiesterase.

All the compounds tested as substrates of T4 RNA ligase (see above) were also assayed as substrates of T4 DNA ligase, at a concentration of 0.3 and 0.8 mM, and the initial velocities determined (Fig. 8). At 0.3 mM the relative velocities (%) to synthesize the corresponding mev-p n A derivatives were for: far-PPP, 100 % or 25.1±0.9 mU/mg); iso-PPP, 89%; ger-PPP, 78 %; iso-PP, 17%); dim-PP, 14 %; far-PP, 7 % and ger-PP, 2%. Inhibition (%) of the activity by 0.8 mM substrate vs. 0.3 mM was also observed with far-PPP (81% ) and far-PP (50%) (Fig. 8).

For the V max , K m , K cat and K cat /K m values calculated for T4 DNA ligase and the mevalonate compounds see Table 3. Again, the maximum catalytic efficiency (K cat /K m ) was obtained for far-PPP as a substrate. In general, the efficacy of T4 DNA ligase to synthesize mev-p n A derivatives is better than with T4 RNA ligase (compare the K cat /K m values for both enzymes in Tables 2 and3.

A c c e p t e d M a n u s c r i p t 13

Synthesis of mevalonate derivatives of ATP by other ligases

Luciferase, acetyl-and acyl-CoA synthetases, and the ubiquitin activating enzyme (E1), previously assayed in the synthesis of both (di)nucleoside polyphosphates and ATP derivatives of bisphosphonates [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF][START_REF] Sillero | Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1[END_REF], were also tested for the synthesis of mev-p n A derivatives. From these enzymes, luciferase was the only one able to catalyze the synthesis of these derivatives. As previously known, luciferase (E), in the presence of Dluciferin and ATP, forms the complex E-luciferin-AMP, the AMP moiety may be donated to nucleosides containing at least a tripolyphosphate chain, and the synthesis of dinucleoside polyphosphates occurs (Table 1, reactions 1,

2) [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF]. However when the ATP concentration in the assay is very low (M), the enzyme behaves as an ATPase liberating AMP [START_REF] Deluca | The hydrolase properties of firefly luciferase[END_REF][START_REF] Garrido | Labeled adenosine(5')tetraphospho(5')adenosine (Ap 4 A) and adenosine(5')tetraphospho(5')nucleoside (Ap 4 N). Synthesis with firefly luciferase[END_REF]. The synthesis of ATP mevalonate derivatives was approached here, incubating luciferase in the presence of 0.02 mM [-32 P]ATP and iso-PPP, far-PPP or ger-PPP at 0.3 mM each (Fig. 9, A); a control in the absence of added substrate was run in parallel. After 20 min incubation, the reaction mixtures were treated with alkaline phosphatase. In addition to an important amount of AMP and a minor amount of Ap 4 A, the synthesis of the corresponding ATP derivatives was also observed. Formation of adenosine 5´-tetraphosphate (p 4 A) was detected with far-PPP as substrate, revealing the occurrence of free tripolyphosphate in this commercial preparation. The radioactive spots corresponding to the presumptive mev-ppppA were not sensitive to alkaline phosphatase as shown in Fig. 9, B and sensitive to phosphodiesterase, yielding AMP as one of the products (results not shown). A similar experiment using iso-PP as substrate is shown Fig. 9, C. The iso-pppA A c c e p t e d M a n u s c r i p t 14 synthesized was not affected by treatment with alkaline phosphatase. The initial velocities calculated for the synthesis of the ATP derivatives shown in Fig. 9 were for: iso-PPP (2.0 mU/mg); far-PPP (1.8 mU/mg); ger-PPP (3.1 mU/mg) and iso-PP (0.16 mU/mg). Luciferase prefers the triphosphate derivatives of the compounds of mevalonate pathway, equally as it prefers nucleosides with a terminal P 3 over those with a terminal P 2 , as substrates for the synthesis of dinucleoside polyphosphates [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF].

In our hands, the other ligases tested, i.e. acetyl-CoA synthetase, acyl-CoA synthetase, and the ubiquitin activating enzyme E1 did not catalyze the synthesis of ATP derivatives with any one of the mevalonate compounds here mentioned.

Discussion

Our previous work on the synthesis of dinucleoside polyphosphates [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF] and on ATP derivatives of bisphosphonates catalyzed by ligases [START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases[END_REF], and the finding by Mönkkönen of the occurrence of isopentenyltriphosphoadenosine (ApppI, here iso-pppA) in osteoclasts cells treated with bisphosphonates [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF], prompted us to study the synthesis of ATP derivatives (mev-pppA and mev-ppppA) by several ligases. About the potential relationship between the effect of bisphosphonates both on the synthesis of these compounds in particular and on osteoclasts in general, two main possibilities can be envisaged.

A. Bisphosphonates could stimulate the synthesis of mev-pppA considering simply that, if BPs accumulate in bone tissues inhibiting some of the A c c e p t e d M a n u s c r i p t enzymes of the mevalonate pathway (Fig. 1), an increase in the concentration of metabolites upstream the inhibited step would take place, and consequently, an increase of the synthesis of the mevalonate derivatives (mev-pppA) catalyzed by ligases, could occur (Table 1).

Recently, several laboratories have studied the interaction of alendronate, ibandronate, risedronate and zoledronate (bisphosphonates of the second and third generation, all N-BPs), with crystallized human farnesyl pyrophosphate synthetase (Fig. 1) with similar and complementary results [START_REF] Hosfield | Structural basis for bisphosphonate-mediated inhibition of isoprenoid biosynthesis[END_REF][START_REF] Kavanagh | The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs[END_REF][START_REF] Rondeau | Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs[END_REF]. The nitrogen atom of N-BPs interacts with the Thr-201 and Lys-200 of the enzyme, located in its carbocation-binding site favoring the position of BPs in the dimethylallyl/geranyl diphosphate ligand pocket [START_REF] Kavanagh | The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs[END_REF]. Those N-BPs bind to this site through a trinuclear metal center similar to that formed with the terminal pyrophosphate of some compounds of the mevalonate pathway [START_REF] Rondeau | Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs[END_REF]. In addition, the isopentenyl diphosphate accumulated (due to the inhibition of the enzyme), rather than competing for and displacing the BP, binds and stabilizes the enzyme-N-BP complex, forming a ternary complex. This fact contributes to the exceptional in vivo efficacy of the N-BP drugs [START_REF] Rondeau | Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs[END_REF], and could explain the preferential synthesis of iso-pppA reported by Mönkkönen [START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF]. At any rate, both the N-atom and the bisphosphonate chain itself are critical for the inhibitory effect of N-BP on this enzyme. In this context, we may recall that the components of the mevalonate pathway comprise the highest gathering of consecutive compounds with a terminal pyrophosphate in any known metabolic pathway in mammals, what could be another characteristics for the preferential action of bisphosphonates in this pathway (Fig. 1). It may also be recalled that some BPs are substrates by ligases [START_REF] Klein | Methylenediphosphonates, a metabolite poison in Dictyostelium discoideum. 31 P NMR evidence for accumulation of adenosine 5´-(,Methylenetriphosphate) and diadenosine 5´-5´´´-(P 2 ,P 3 -methylenetetraphosphate)[END_REF][START_REF] Rogers | Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-tRNA synthetase enzymes[END_REF][START_REF] Rogers | New insights into the molecular mechanisms of action of bisphosphonates[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase[END_REF][START_REF] Sillero | Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases[END_REF]; in principle, these ATP derivatives could interfere with the activity of enzymes using ATP as substrate. To this group belong at least 150 enzymes with transferase activity and more than 90 with ligase activity (see Enzyme Nomenclature). Probably most of these ATP derivatives could act on these enzymes as competitive inhibitors (although other type of inhibition could also be contemplated) with variable efficacy. In this regard it is worthy noting that, the mevalonate pathway presents the exceptional characteristic of holding three enzymes (2 kinases and 1 decarboxilase)

requiring ATP and operating in sequence (Fig. 1). The concatenation of these steps could make the mevalonate pathway specially exposed to the effect of the BP derivatives of ATP.

B. The synthesis of mev-pppA is partially or totally unrelated to the effect of bisphosphonates. The occurrence of mev-pppA in liver and other tissues cannot be disregarded in view of the quantity of ligases potentially able to transfer the AMP moiety of the E-AMP or EX-AMP complexes to compounds with a terminal di or triphosphate [START_REF] Sillero | Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase and several ligases[END_REF]. Examples of this possibility are here reported for the case of T4 RNA ligase, T4 DNA ligase and luciferase. Moreover, the synthesis of compounds with inner phosphate(s) and containing at least a terminal nucleotide (AMP, GMP, UMP or CMP, depending on the ligase involved) can be equally pondered.

These compounds can be considered as metabolic by-products, with no metabolic significance, whose natural destiny is to be eliminated by specific or unspecific enzymes and, because of the low concentration reached in the cell, have passed unnoticed. Experiments performed years ago in our 1) E + ATP + X <----> E-X-AMP + PPi 1a) E + ATP <-----> E-AMP + PPi

2) E-X-AMP + NTP ---->E + X+ AppppN

3) E-X-AMP + (p) n A -----> E+ X+ A(p) n+1 A

4) E-X-AMP + (P) n -----> E + X + (p) n+1 A 5) E-X-AMP + P-C(R1)(R2)-P -----> E + X + AppC(R1)(R2)p 5a) E-AMP + P-C(R1)(R2)-P -----> E+ AppC(R1)(R2)p 6) E-X-AMP + iso-PP -----> E + X + iso-pppA 6a) E-AMP + iso-PP -----> E + iso-pppA 7) E-X-AMP +mev-PP -----> E + X+ mev-pppA 8) E-X-AMP + mev-PPP -----> E + X + mev-ppppA

A c c e p t e d M a n u s c r i p t 5 action

 5 of bisphosphonates, or even may have metabolic or therapeutic implications.

32 PA c c e p t e d M a n u s c r i p t 7

 327 ]ATP (0.4 Ci), 0.02 mM D-luciferin, compounds of the mevalonate pathway and enzyme as indicated. The enzyme was diluted in 0.5 M Hepes/KOH (pH 7.5)/10% glycerol/1 mM DTT. After incubation at 30ºC the reaction mixtures were analysed by TLC.

A c c e p t e d M a n u s c r i p t 9 view

 9 , these results indicate that the synthesis of iso-pppA catalyzed by T4

A c c e p t e d M a n u s c r i p t 11 enzyme

 11 (K m values around 4 mM), while the higher efficacy (K cat /K m ) was found for far-PPP, a compound whose derivative formation is inhibited by an excess of substrate (see Figs.4, 5 and 6c, d).

A c c e p t e d M a n u s c r i p t 16 for

 16 the synthesis of ATP derivatives of the type AppC(R1)(R2)p catalyzed

A c c e p t e d M a n u

  s c r i p t laboratory showed the existence of a number of still unknown nucleotides with these peculiarities in rat liver[34]. However, in our view, compounds present at low concentration in tissues and containing inner phosphate(s) are also candidates to be regulatory molecules[35]. In the particular case of the mevalonate derivative, iso-pppA, this compound inhibited the ADP/ATP translocase and caused apoptosis in osteoclasts[START_REF] Mönkkönen | A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates[END_REF]. The possibility that other mev-pppA derivatives could also have regulatory effects remains an open question.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 20 [Fig. 1 .

 201 Fig. 1. Components of the mevalonate pathway, its precursors and some

Fig. 2 .

 2 Fig. 2. Synthesis of isopentenyl-triphospho(5´)adenosine (iso-pppA) from

Fig. 3 .

 3 Fig. 3. Synthesis and characterization of isopentenyl-

Fig. 4 .

 4 Fig. 4. Synthesis of ATP derivatives of compounds of the mevalonate

Fig. 5 .

 5 Fig. 5. Synthesis of the ATP derivatives of compounds of the mevalonate

Fig. 6 .

 6 Fig. 6. Inhibition by excess of substrate, of the synthesis of far-pppA and

A c c e p t e d M a n u s c r i p tFig. 7 .

 7 Fig. 7. Synthesis of isopentenyl-triphospho(5´)adenosine (iso-pppA)

Fig. 8 .Fig. 9 .

 89 Fig. 8. Synthesis of the ATP derivatives of compounds of the mevalonate

A c c e p t e d M a n u s c r i p t

Figure A c c e p t e d M a n u s c r i p

  Figure

Figure A c c e p t e d M a n u s c r i p t

  Figure

Figure

  Figure

Figure

  Figure

Figure A c c e p t e d M a n u s c r i p t

  Figure

Table 1 -

 1 -Reaction models catalyzed by ligases

Table 2 .

 2 V max , K m and K cat values of T4 RNA ligase towards substrates of the mevalonate pathway

	Substrate	V max (mU/mg)	K m (M)	K cat (s -1 )	K cat /K m (s -1 M -1 )

X, a co-substrate of a ligase; NTP, a nucleoside triphosphate; (p) n A, adenosine 5'-polyphosphate; (P) n , a polyphosphate; P-C(R1)(R2)-P, a bisphosphonate; iso-PP, isopentenyl diphosphate; mev-PP and mev-PPP, components of the mevalonate pathway with a terminal pyrophosphate or triphosphate, respectively.

Table 3 .

 3 V max , K m and K cat values of T4 DNA ligase towards substrates of the mevalonate pathway

	Substrate	V max (mU/mg)	K m (M)	K cat (s -1 )	K cat /K m (s -1 M -1 )
	Farnesyl triphosphate	36.7±1.5	0.15±0.03	34.9x10 -3	232
			x10 -3
	Isopentenyl triphosphate	76.7±3.0	0.63±0.20	72.9x10 -3	116
			x10 -3
	Geranyl triphosphate	33.6±5.7	0.31±0.07	31.9x10 -3	103
			x10 -3
	Farnesyl diphosphate	2.9±0.4	0.05±0.01	2.8x10 -3	56
			x10 -3
	Isopentenyl diphosphate	33.3±10.6	2.3±0.93 x10 -3	31.6x10 -3	13.8
	Dimethylallyl diphosphate	51.8±1.5	4.0±0.2 x10 -3	49.2x10 -3	12.3
	Geranyl diphosphate	0.85±0.03	0.21±0.02 x10 -3	0.8x10 -3	3.8

Acknowledgements

This work was supported by grants from Dirección General de Investigación Científica y Técnica (BFU 2006-04307/BMC and BFU 2008-00666/BMC). We thank Antonio Fernández and Sandra Morillo (Erasmus student from ENCPB, Paris, France) for technical assistance. J. E. F. T. and J. A. D. C. S. were Erasmus students from the Faculdade de Farmacia, Universidade do Porto, Portugal.