Internalization and Stability of a Thymidylate Synthase Peptide

Oct 29, 2014 - Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 183, 41125 Modena, Italy. ‡. Department of Chemical an...
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Brief Article

The internalization and stability of a Thymidylate synthase peptide inhibitor in ovarian cancer cells. GIUSEPPE CANNAZZA, Addolorata Stefania Cazzato, Chiara Marraccini, Giorgia Pavesi, Silvia Pirondi, Remo Guerrini, Michela Pela, Chiara Frassineti, Stefania Ferrari, Gaetano Marverti, Glauco Ponterini, and Maria Paola Costi J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501397h • Publication Date (Web): 29 Oct 2014 Downloaded from http://pubs.acs.org on November 18, 2014

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Journal of Medicinal Chemistry

Internalization and stability of a Thymidylate synthase peptide inhibitor in ovarian cancer cells. Giuseppe Cannazza1§, Addolorata Stefania Cazzato1§, Chiara Marraccini1, Giorgia Pavesi1, Silvia Pirondi1, Remo Guerrini2, Michela Pelà2, Chiara Frassineti3, Stefania Ferrari1, Gaetano Marverti3, Glauco Ponterini1, Maria Paola Costi1*. 1

Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 183, 41125, Modena, Italy; 2 Department of Chemical and Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara 17-19, 44100 Ferrara, Italy; 3 Department of Biomedical Sciences, Metabolic and Neuroscience, Section of Pharmacology and Molecular Medicine, University of Modena and Reggio Emilia, Via Campi 287, 41125, Modena, Italy.

Abstract. Information on the cellular internalization and stability of the ovarian cancer cell growth inhibitor peptide, LSCQLYQR (LR), is vital for lead optimization. Ad-hoc-synthesized LR/fluorescent-probe conjugates were used to monitor the internalization of the peptide. Mass spectrometry was used to identify adducts resulting from the thiol reactivity of the cysteine residue in LR. A mechanistic model is proposed to explain the observed change in intracellular peptide amount over time. Structural modifications can be foreseen to improve the peptide stability. KEYWORDS: peptidic drugs, human thymidylate synthase (hTS) inhibitors, intracellular stability, liquid chromatography mass spectrometry, mechanistic in-out model.

Introduction Epithelial ovarian cancer (EOC) is a serious disease that relies on platinum drugs for first-line therapy. Drug resistance occurs rapidly in EOC; therefore, novel and specialized drugs are critically needed to overcome this problem. Recently, specific octapeptides that reduce platinum-resistant EOC cell growth by inhibiting the enzyme human thymidylate synthase (hTS) have been identified1. TS is an enzyme of the folate metabolic pathway that plays a key role in DNA synthesis by catalyzing the conversion of 2′-deoxyuridine-5′-monophosphate (dUMP) to 2′-deoxy thymidine-5′-monophosphate (dTMP). N5,N10-methylene tetrahydrofolate (MTHF) acts as a cofactor for this reaction. hTS was found to be over-expressed in many cancers, including colorectal cancer, mesothelioma and ovarian cancer. Thus, hTS is considered to be an important drug target, and several clinically relevant anti-cancer drugs, such as raltitrexed, pemetrexed and the prodrug 5-fluorouracil (5FU), exert their biological activities through TS inhibition.2 Prolonged treatment with anti-hTS compounds results in drug resistance;3-5 therefore, we aimed to discover new therapeutic agents with a novel mechanism of action.1,6,7 The octapeptide LSCQLYQR (LR, structure in Figure 1) was designed to specifically target the monomer-monomer interface of hTS and inhibit the intracellular protein.1 A concentration of 510 µM of LR inhibited approximately 50% of the growth of both cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines.1 The cellular mechanism involves the inhibition of target protein interactions, down-regulation of folate-dependent enzymes and proteasomal and ribonucleoprotein alterations.7 Similarly to the majority of peptides, LR cannot cross the cell membrane and requires a protein-specific system for transport into the cells. We obtained efficient peptide delivery using a pol-

ymer named SAINT-PhD (Synvolux Therapeutics, Groningen, NL), which has been used for protein and peptide transfection.1,8,9 SAINT-PhD is a surfactant molecule bearing a cationic pyridinium head and a lipid chain. The cationic surfaces of the vesicles formed by this surfactant have a high affinity for the negatively charged cell surface. The peptide is then released from the lipid chains of the molecule into the cytoplasm. This delivery system did not alter cell growth when used at low concentrations.1,6 The transfection efficiency is critical for peptide activity and for the determination of the effective intracellular concentration, stability and localization of the peptide. Commonly used methods for the study of the internalization, localization and quantification of small peptides inside cells are indirect and often based on fluorescence. The peptides are covalently coupled to a fluorophore and quantified fluorometrically. Confocal microscopy can be used to determine the localization of the probes inside living cells. Though convenient, these methods have drawbacks. Indirect peptide detection through a fluorescent label may be influenced by the stability of the probe-peptide conjugate once inside cells; therefore, a direct method for determining the integrity of the peptide is needed.10 In the present study, we preliminarily studied the biological effects of the LR peptide complexed with SAINT-PhD on C13* cell lines. We then monitored the SAINT-PhD-mediated internalization of the LR peptide using fluorescence confocal microscopy. Afterwards, we identified the major adduct products of the LR-peptide thiol reactivity in a buffer, in the cellular growth medium and cell lysates using LC Chip Q-TOF. Finally, we characterized the time-dependent changes of the LR peptide concentration in reducing conditions and absence of disulfide adducts, inside and outside C13* cells using an LC-MS/MS method specifically developed and validated for this purpose. 11-13

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ment was confirmed by the suppression of the second peak and recovery of the first one after addition of dithiothreitol (DTT), which reduces the disulfide bond and yields back the two thiols. The LC chip Q-TOF TIC chromatogram of LR in DMEM consisted of signals of the LR monomer and dimer and featured another peak that eluted prior to LR with an m/z ratio of 377.186, corresponding to a three-charged ion signal ([M+3H]3+ m/z 377.186) ) with a MS/MS spectra similar to that of LR. The LR and this peaks showed similar mass fragmentation spectra. Based on the molecular mass and DMEM composition, which included cysteine, we assigned the unknown peak as a disulfide-bridged LR-cysteine nonapeptide (structure in Figure 1). The assignment was confirmed by the following observations: i) the same peak with a similar MS/MS spectra was detected during the analysis of a water solution of the LR peptide and cysteine (1:3) that was incubated for 1 hour, and ii) the m/z 377.186 peak was found to decrease following incubation with 1 mM DTT. As expected, the LR-cysteine disulfide bridge was disrupted. The LC Chip Q-TOF analysis of LR added to C13* cell lysates identified the two peaks corresponding to the LR monomer and dimer signals and a new modification of LR. Based on the experimentally calculated masses and MS/MS spectra, the new peak was assigned as a conjugation product of the LR peptide with a glutathione molecule (structure in Figure 1). The assignment was confirmed by suppressing the unknown peak with the addition of DTT to the cell lysate. These results highlight the high reactivity of the -SH group of the LR cysteine versus other -SH containing compounds. Analysis of LC chip Q-TOF chromatograms of incubated LR samples indicated the absence of other possible degradation products from reactions such as oxidation, reduction, hydrolysis or de-amidation products and peptides fragments14. For the subsequent quantitative experiments described below, all cell lysates were treated with DTT to reconvert the LR-including disulfides formed in these reactions to the thiolic form of the peptide. Indeed, no evidence of S-S-linked adducts was found after this treatment. Time-dependent changes in LR peptide concentration during incubation with C13* cells. The LR peptide content was measured in the DMEM medium (extracellular concentration) and cell lysates (intracellular concentration) during incubation with C13* cells using liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-MS/MS). An analytical LC-MS/MS method was developed for the detection and quantification of the low levels of LR to overcome the problem of the complexity of cell lysate matrix. The extracellular concentration of the LR peptide was determined at different times (see the data in Table 1). LR-peptide levels normalized to the highest value obtained are displayed in Figure 4. The experiments were performed with two different initial LR concentrations in the medium, namely, 6.7 and 35 µM. The amount of SAINT-PhD was kept constant at a non-toxic level. The ratios of the µg of peptide and µL of SAINT-PhD used were 1:16 and 1:3 for the low and high initial amounts of LR, respectively. In both experiments, a pronounced LR depletion was observed in the medium. The LR levels decayed exponentially with time. The empirical rate constants, determined with a first-order kinetic analysis, were 0.44±0.03 and 0.54±0.06 h-1 for the low and high initial LR amounts, respectively. These correspond to a lifetime of approximately 2 h and a half-life of approximately 1.4 h for LR. To investigate the stability of the LR peptide, parallel experiments were performed with the LR peptide in DMEM without cells. The results showed that

The degradation kinetics was analyzed using a simple mechanistic model. The information achieved about the cellular pharmacokinetics will allow the cell-based activity optimization of the lead and will help in turning a good inhibitor into a drug lead. Results Biological effects of the LR peptide on C13* cell lines. C13* are EOC cell lines resistant to cis-platin,; therefore, they represent a good model for our purpose, i.e., showing that the LR peptide is active against a drug-resistant cell model. The toxicity of SAINT-PhD on C13* cells was checked through flow-cytometry analysis. The fluorescence of propidium iodide, which directly correlates with the nuclear DNA content, clearly indicates that no significant difference is observed on the distribution of the cell cycle phases in C13* cells treated with SAINT-PhD compared to untreated cells, after 24 and 72 hours (See ‘Effects of SAINTPhD on cell cycle progression’ and Figure SI-2 in the Supplementary Information). According to these findings, we can assess that, at the concentrations employed (20 µL/mL culture medium), the delivery system does not influence either cell proliferation or the cell cycle progression. Inhibition of cell growth by the LR peptide and 5FU (as a reference drug) for C13* cells is shown in Figure 2. While SAINT-PhD-induced cell death after 72 hours of treatment is only around 4% compared to untreated cells, a decrease in cell survival between 25 and 40% is observed after treatment with LR at 1-5 µM, comparable with the effect of 5 µM 5FU. Fluorescence confocal microscopy of the LR peptide in ovarian cancer cell lines. The intracellular localization of the LR peptide was determined using a peptide-fluorophore conjugate consisting of LR bound to LR-HL405, Figure 3. Fluorescence microscopy after a 2 hr incubation with the LR peptide-probe in the presence of the SAINT-PhD delivery system showed clear evidence of an efficient internalization of the conjugate. The treated C13* cells displayed an intense blue LR-HL405 signal in the nuclei (Figure 3, bottom row). The fluorescent signal from within the cells was very weak when the conjugate was added directly to the cell growth medium in the absence of SAINT-PhD (Figure 3, top row). We have tested other probes such as BODIPY conjugated with LR and other cell lines such as HEK293. In all cases we obtained clear internalization of the LR peptide-probe conjugate when complexed with the SAINT-PhD delivery system (Fig SI-4 in the Supplementary information). Thiol reactivity of the LR peptide in different environments. LC Chip Q-TOF operating in a wide dynamic range (details are in the Experimental Section) was employed to examine the reactivity of the LR peptide in HEPES-buffered saline (HBS), Dulbecco’s modified Eagle’s medium (DMEM) or C13* cell lysates. The extracted ion chromatogram (EIC) of the LR peptide and its MS/MS spectra prior to and after a 4-hr incubation are reported in Figure SI-5 of the Supplementary Information. In HBS, an LR peak ([M+2H]2+ m/z 505.758) with a retention time of 7.9 minutes was observed at time zero. After an incubation of 4 hours at room temperature, this peak was no more detected and was replaced by a peak that eluted about 4 minutes later (tr=12.1 minutes), corresponding to a four-charged species with an m/z ratio 505.266 ([M+4H]4+ m/z 505.266). MS/MS spectra showed that the peaks at m/z 505.758 and 505.266 yielded a similar abundance of fragments. Based on these data and the molecular weight, we assigned the second species as an LR-dimer generated by Cys3 side chain oxidation (structure in Figure1). This assign-

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Journal of Medicinal Chemistry of a parallel, first-order time-decay of LR content inside and outside the C13* cells in experiment (a) is the basis for the schematic mechanistic model shown in Figure 5. Here, we have adopted the assumption that all of the overall processes involving the peptide, i.e., internalization, expulsion from the cells and degradation outside and inside cells, are first-order with respect to LR. The corresponding pseudo-first-order rate constants, kin, kout, kdo and kdi, likely include the concentrations of other species that are assumed to be constant over time. The observed stability of the LR peptide in DMEM without cells implies the rate of degradation in the medium, represented by kdo in Figure 5, is negligible. Our kinetic observations are consistent with the assumption of fast equilibration between the peptide inside and outside of the cells, such that kin, kout ⨠ kdi, kdo (dynamic equilibrium conditions). In other words, the degradation process is much slower than the in-out exchange of the peptide. An analysis of the kinetic scheme in these conditions22 yielded decay rates for the peptide inside and outside of the cells with the same apparent first-order rate constant, λ= (kdo+kdiK)/(1+K) ~ kdiK/(1+K), with K=([LRin]Vin/([LRout]Vout)) at equilibrium. Assuming λ = 0.43±0.03 h-1 and knowing that K represents the ratio of the LR peptide masses inside and outside of the cells, i.e., ~ 1/125 = 8 x 10-3, we can estimate the apparent intracellular degradation rate constant, kdi ~ 50 h-1. This equilibration hypothesis rests on the assumption that the peptide is involved in in/out trafficking. This assumption is supported by the observation that P-glycoprotein (P-gp, MDR1), the first discovered component of the ATP binding cassette (ABC) protein family, can transport both linear and cyclic peptides.23 Evidence of involvement in peptide secretion has also emerged for other members of the ABC-transporter family, such as MRP1, which has been shown to be responsible for the secretion of the tripeptide N-acetyl-leucyl-leucylnorleucinal.24 Furthermore, it has been observed that the C13* cell membrane has a high quantity of MDR pumps. Indeed, cross-resistance to the P-gp substrate daunorubicin and a reverted sensitivity to DNA-intercalating bipyridyl–thiourea–Pt(II) complexes following pre-treatment with the well-known P-gp blocker verapamil were found to correlate with an increased intracellular drug accumulation.25 The estimated value of the intracellular degradation pseudo-rate constant, kdi ~ 50 h-1, indicates that the processes responsible for this degradation are quite efficient. In situations where the peptide cannot be supplied to the cell from the medium, this value would correspond to a half-life of only approximately 1 minute. This scenario applies after cessation of compound administration. Conclusions LC Chip Q-TOF was employed to identify the major Cys3 thiol-reactivity products of the LR peptide, i.e., the LR dimer and its conjugates with cysteine in the cell-growth medium and with GSH in C13* cell lysates. A high-sensitivity LC-MS/MS method was developed to evaluate LR peptide levels in DMEM and C13* cell lysates in presence of DTT reducing agents, during an 8 hr incubation. The intracellular concentration decreased from 37 to 3 µM, but remained approximately 15 times larger than the concentration detected in the culture medium. The depletion of the LR peptide in the presence of C13* cells followed similar kinetics inside and outside cells, with a half-life of 1.4 hours. According to a simplified kinetic model, this finding suggests the existence of a fast-equilibrium mechanism that combines in/out trafficking and degradation pathways within cells. The peptide sta-

the peptide concentration remained constant after 2 hours. To analyze the LR peptide levels within the C13* cells over time, the LR peptide content was measured in the lysates after 2, 4, 6 and 8 hours of incubation (Table 2, Figure 4). These values were converted into intracellular concentrations using a mean cellular volume of 2 x 10-12 liters15 and a total of 2 x 105 lysed cells. The observed time evolution of LR was strongly dependent upon the initial amount of LR present in the medium. We observed an exponential decay with an empirical rate constant of 0.43±0.03 h-1 with the low initial LR concentration (column (a) in Table 2 and blue empty circles in Figure 4). This decay rate constant is indistinguishable from the one measured in the cell growth medium, suggesting a roughly constant ratio between the peptide levels inside the cells and in the medium. From the results in columns (a) of Tables 1 and 2 this ratio holds between 1/150 and 1/100 in terms of masses, but it holds between 12 and 17 in terms of concentrations: the intracellular LR concentrations are about 15 times larger than the extracellular concentrations. Contrary to experiments with a lower starting concentration of LR, higher initial amounts of LR afforded an unchanged concentration between 2 and 4 hours (column (b) in Table 2, and red empty circles in Figure 4). Subsequently, the peptide concentration decreased following a sigmoidal trend. The stable concentration of LR observed at early time points with high initial amounts of the peptide may have resulted from the saturation of the SAINT-PhD delivery system due to a large excess of LR. Discussion. Fluorescence microscopy confirmed that an efficient internalization of the peptide-probe conjugate into C13* cells was facilitated by SAINT-PhD. MS experiments on cell lysate performed in the presence of the reducing agent DTT, determined that, over the 8-hour incubation, the intracellular concentration decreased from 37 to 3 µM, but remained approximately 15 times larger than the concentration detected in the culture medium. In native conditions, we found that the thiol reactivity of the LR peptide can produce a disulfide-bound complex with glutathione (GSH) inside cells. Indeed, GSH is a low-molecular-weight thiol that plays a critical antioxidant role and protects cells against the damage caused by reactive oxygen species. When in a disulfide complex with LR, GSH becomes unavailable for counteracting cellular oxidative stress. However, the estimated maximum intracellular quantity of the LR/GSH conjugate was approximately 100 µM (the extrapolated initial concentration in experiment (a), Table 2). This concentration is far lower than the cellular concentration of glutathione that range between 0.5 and 10 mM, depending on the cell type and organ.16,17 The highest levels are found in the liver (5–10 mM), followed by the kidney, spleen, small intestine, brain, pancreas, lung, heart, and muscle.18,19 In A2780 cancer cells the concentration of glutathione is reported to be around 1mM.20 Therefore, it seems unlikely that the observed cytotoxic effect of LR can be attributed to a depletion of GSH. The observed LR dimer and the LR-GSH heterodimer may be a way to accumulate LR in the disulfide oxidized form inside the cells that could be a reservoir of peptide; the LR storage as disulfide form can protect LR itself from the proteolityic degradation thus increasing its intracellular half-life21. MS experiments performed in the presence of the reducing agent DTT, determined that, over the 8-hour incubation, the intracellular concentration decreased from 37 to 3 µM, but remained approximately 15 times larger than the concentration detected in the culture medium. In presence of DTT the observation

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Journal of Medicinal Chemistry bility represents an issue for in vivo delivery and pharmacological activity. The cellular half-life observed for LR peptide, based on our kinetic model, suggests that more chemical changes should be performed for an acceptable in vivo pharmacokinetics. The cysteine thiol group is a reactive metabolic function but it is known to play an important role in LR binding to the hTS protein, as observed in the LR-hTS x-ray complex1, because it is at a canonical S-S binding distance from Cys180. However the thiol group can be a source of structural liability for the LR peptide. We will focus on careful modification of position 3 (now with Cys) of the LR peptide to improve both stability and efficacy. LR and derivatives are expected to work in combination with more aggressive chemotherapeutic agents to improve the efficacy, fight platinum-induced drug resistance and reduce the overall drug combination toxicities thus adding new opportunities to the anticancer drugs pipeline. Experimental Section. Chemicals. For chemicals see SI pag.2. Synthetic chemistry The LR peptide was synthesized and purified as previously reported.1 The conjugation of LR with probes was achieved using the classic thiol-Michael reaction. A solution of fluorescent probe (1 mg, 1 eq.) in CH3CN (250 µl) was added to a stirred solution of LR peptide (1.1 eq) in 250 µl of H2O and addition of 25 µl of NaHCO3 5% was done. The mixture was stirred in the dark under nitrogen atmosphere at ambient temperature for 15 minutes. The reaction was monitored by HPLC and MS analysis. After completion of reaction, HPLC purification conducted as described above, afforded the fluorescent conjugate in quantitative yield. For the synthesis of LR-Hilyte Fluor 405 C2 maleimide (HL405), 1 mg of HL405 (8.96*10-4 mol) and 1.3 mg of LR peptide (1.05*10-3 mol) was obtained in quantitative yield. More details and the analytical HPLC profile and mass spectra of the final compounds are available as SI pag.3 and pag.22. Effect of LR peptide and SAINT-PhD on C13 cell lines. Flow cytometry was used to evaluate the percentage of cells in each phase of the cell cycle after 24 and 72 hours of treatment with SAINT-PhD (20 µL/mL culture medium). SI pag.7. Cell growth inhibition assay. The Crystal Violet assay was performed to assess the percentage of cell survival after 72 hours of treatment with LR peptide at different concentrations. (Details are reported in SI pag.8). Sample preparation The 2008, C13* and HEK293 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) plus 20 mM L-Gln at 37°C with 5% CO2. Details of the sample preparation for mass spectrometry studies and confocal microscopy experiments are reported in the Supporting Information. (SI pag.9). Laser imaging. Confocal imaging was performed using an inverted Leica TCS SP2 confocal microscope interfaced with a blue laser diode (405 nm/25 mW) and an argon laser (488 nm/20 mW), equipped with AOBS and operating at magnifications of 40x (HCX PL APO 40x/1.25 - oil) and 63x (HCX PL APO 63x/1.40 - oil - Lambda blue correction). The images were processed using LCS Lite (Leica Microsystems, Heidelberg). SI Pag.12 Liquid chromatography quadrupole time of flight (LC– QTOF). Analyses were carried out on an Agilent 1200 series nanoflow LC system Chip with an enrichment channel of 40 nL and a 75 µm (i.d.) x 43 mm (L) separation channel. The Chip and

enrichment columns were packed with Zorbax 300 SB-C18 (5 µm) stationary phase. Details are reported in SI pag. 13-21. Liquid chromatography triple quadrupole (LC-MS/MS) These experiments were performed using an Agilent HP 1200 liquid chromatograph (Agilent technologies, Milan, Italy). A Merck Hitachi L-6200A pump (Merck KGaA, Darmstadt, Germany) was employed as the second pump. A Rheodyne 7000 valve was used to switch the mobile phase flow (Jasco Europe, Italy, Milan). Details are reported in SI pag.15-21.

Figure 1. Structure of LR and its derivatives: dimeric LR, LRcysteine adduct, LR-glutathione adduct.

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Cell Survival (% of control)

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75

50

25

0

C13* cell line

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Figure 2. Cell growth inhibition by LR peptide and 5µM 5FU after 72 h exposure in C13* cells. Black bar, control cells. Light gray bar, SAINT-PhD delivery system. Dark gray bar, 5µM 5FU. Blue bar, 1 µM dark blue bar, 5µM LR peptide. Cell survival percentages are the mean ± S.E.M. of at least three separate experiments performed in duplicate.LR peptide; light blue bar, 2.5 µM LR peptide.

Figure 5. Mechanistic model of intra- and extracellular trafficking (in-out model) of the LR peptide. kdi and kdo represent the rate constants of intracellular (in) and extracellular (out) degradation, respectively. kin and kout are the rate constants for the peptide trafficking into and out of cells. Table 1. Extracellular amount and concentration of LR peptide after 2, 4, 6 and 8 incubation hours. (a) The initial concentration was 6.7 µM (4712 ng). (b) The initial concentration was 35 µM (24850 ng).

(a)

Figure 3. Confocal imaging of ovarian cancer cells transfected with LR-HL405 after 2 hours incubation. C13* cells incubated with LR-HL405 for 2 hours without (top row) and with the Saint PhD delivery system (bottom row). Left, C13* cells incubated with LR-HL405 for 2 h at 37°C (blue signal); center, corresponding bright field; right, overlay of C13* cells stained with LRHL405 and bright field images. The probe-peptide conjugate enter the nuclei.

(b)

Amount of LR (ng)a

[LR] (µ µM)

Amount of LR (ng)

[LR] (µ µM)

2

1574.9 ± 87.6

2.22

7234.8 ±91.6

10.32

4

952.0 ± 49.5

1.36

4349.1 ±92.2

6.21

6

306.7 ± 39.1

0.08

502 ±12

0.13

8

< 175

0.04

405 ± 69

0.10

Time (h)

normalized peptide amount

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Table 2. Intracellular amount and concentration of LR peptide in C13* cells after 2, 4, 6 and 8 incubation hours. (a) The initial concentration was 6.7 µM (4712 ng). (b) The initial concentration was 35 µM (24850 ng

1.0

(a)

0.5

Amount of LR (ng)a

[LR] (µ µM)

Amount of LR (ng)a

[LR] (µ µM)

2

15.0 ± 4

37.1

9.2 ± 4

22.8

4

6.2 ± 2

15.3

9.1 ± 1

22.5

6

2.3 ± 2

5.7

6.9 ± 1

17.1

8

1.3 ± 1

3.2

1.3 ± 1

3.2

Time (h) 0.0 0

2

4 6 incubation time / h

(b)

8

Figure 4. Time-dependent changes to the extracellular and intracellular levels of the LR peptide. The amount of peptide in DMEM and extracellular medium are represented by full circles, and the intracellular amount is represented by empty circles. The initial LR content was 4712 ng (blue, lower LR: SAINT-PhD ratio), or 24850 ng (red, higher LR: SAINT-PhD ratio). Exponential curves were fitted to the extracellular data and to the intracellular ones from the lower initial LR level. The error bars for the DMEM data points are smaller than the representative circle symbols.

ASSOCIATED CONTENT Supporting Information. The supporting information includes the structures of the LR peptide and its derivatives, details on sample preparation, details on the LC Chip–QTOF and LC-MS/MS, and the LC-MS/MS method validation. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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(9) Van der Gun, B. T. F.; Maluszynska-Hoffman, M.; Kiss, A.; Arendzen, A. J.; Ruiters, M. H. J.; McLaughlin, P. M. J.; Weinhold, E.; Rots, M. G. Targeted DNA methylation by a DNA methyltransferase coupled to a triple helix forming oligonucleotide to down-regulate the epithelial cell adhesion molecule. Bioconjug. Chem. 2010, 21, 1239–1245. (10) Bechara, C.; Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013, 587 (12), 1693–1702. (11) Bronsema, K. J.; Bischoff, R.; van de Merbel, N. C. Highsensitivity LC-MS/MS quantification of peptides and proteins in complex biological samples: the impact of enzymatic digestion and internal standard selection on method performance. Anal. Chem. 2013, 85 (20), 9528–9535. (12) Rauh, M. LC–MS/MS for protein and peptide quantification in clinical chemistry. J. Chromatogr. B. 2012. 883–884, 59–67. (13) Gomez, J. A.; Chen, J.; Ngo, J.; Hajkova, D.; Yeh, I. J.; Gama, V.; Miyagi, M.; Matsuyama, S. Cell-penetrating penta-peptides (CPP5s): masurement of cell entry and protein-transduction activity. Pharm. Basel Switz. 2010, 3 (12), 3594–3613. (14) Katsila T, Siskos AP, Tamvakopoulos C. Peptide and protein drugs: the study of their metabolism and catabolism by mass spectrometry. Mass Spectrom Rev. 2012, 31(1),110-33. (15) Ligabue, A.; Marverti, G.; Liebl, U.; Myllykallio, H. Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression. PLoS ONE. 2012, 7, e47318. (16) Pastore, A.; Federici, G.; Bertini, E.; Piemonte, F. Analysis of glutathione: implication in redox and detoxification. Clin. Chim. Acta 2003, 333, 19–39. (17) Wu, G.; Fang, Y. Z.; Yang, S.; Lupton, J. R.; Turner, N. D. Glutathione metabolism and its implications for health. J. Nutr. 2004, 134, 489–492. (18) Commandeur, J. N.; Stijntjes, G. J.; Vermeulen, N. P. Enzymes and transport systems involved in the formation and disposition of glutathione S-conjugates. Role in bioactivation and detoxication mechanisms of xenobiotics. Pharmacol. Rev. 1995, 47, 271–330. (19) Aoyama, K.; Nakaki, T. Impaired glutathione synthesis in neurodegeneration. Int J Mol Sci. 2013, 14, 21021–21044. (20) Godwin, A. K.; Meister, A.; O’Dwyer, P. J.; Huang, C. S.; Hamilton, T. C.; Anderson, M. E. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc. Natl. Acad. Sci. USA 1992, 89, 3070-3074. (21) Góngora-Benítez M, Tulla-Puche J, Albericio F. Multifaceted roles of disulfide bonds. Peptides as therapeutics. Chem Rev. 2014 Jan 22;114(2):901-26. (22) Ponterini, G.; Mialocq, J. C. Formation and decay kinetics of the TICT state of 4,4’-diaminodiphenylsulphone. New J. Chem. 1989, 13, 157-164. (23) Schinkel, A. H.; Jonker, J. W. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Advanced Drug Delivery Reviews 2012, 64, 138–153. (24) De Jong, M. C.; Slootstra, J. W.; Scheffer, G. L.; Schroeijers, A. B.; Puijk, W. C.; Dinkelberg, R.; Kool, M.; Broxterman, H. J.; Meloen, R. H.; Scheper, R. J. Peptide transport by the multidrug resistance protein MRP1. Cancer Res. 2001 ,61, 2552-2557. (25) Marverti, G.; Cusumano, M.; Ligabue, A.; Di Pietro, M.,L.; Vainiglia, P.,A.; Ferrari, A.; Bergomi, M.; Moruzzi, M.,S.; Frassineti, C. Studies on the antiproliferative effects of novel DNA-intercalating bypiridylthiourea-Pt(II) complexes against cisplatin-sensitive and -resistant human ovarian cancer cells. J. Inorg. Biochem. 2008, 102, 699-712.

Corresponding Author

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* Corresponding author: Maria Paola Costi, Dipartimento di Scienze della Vita, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy. Phone: 0039-059-205-5134. Fax: 0039-059-2055131. Email: [email protected]. Author Contributions Conceived, designed, monitored the experiments: MPC, GC. GP, CF, SF; Synthesis: RG, MP; Sample preparation: CM, SP, GM; MS experiments : ASC, GC; Laser imaging: GP, SP; Mechanistic model GP. All the authors contributed in writing the manuscript and participate to the AIRCDROC project. §These Authors contribute equally to the work performance. Funding Sources This work was financially supported by the Italian Association for Cancer Research (AIRC-DROC, IG 10474 to M.P.C.). ACKNOWLEDGMENT The Authors thank CIGS (Centro Interdipartimentale Grandi Strumenti) of Unimore (www.cigs.unimore.it). ABBREVIATIONS DMEM, Dulbecco’s Modified Eagle’s Medium; DTT, dithiothreitol; HBS, HEPES buffered saline;hTS, human Thymidylate Synthase; LC, liquid chromatography; MS, Mass Spectrometry; QTOF, quadrupole time-of-flight; TS, Thymidylate Synthase. REFERENCES (1) Cardinale, D.; Guaitoli, G.; Tondi, D.; Luciani, R.; Henrich, S.; Salo-Ahen, M. H.; Ferrari, S.; Marverti, G.; Guerrieri, D.; Ligabue, A.; Frassineti, C.; Pozzi, C.; Mangani, S.; Fessas, D.; Guerrini, R.; Ponterini, G.; Wade, R.C.; Costi, M.P. Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase. Proc. Natl. Acad. Sci. 2011, 108 (34), E542–E549. (2) Costi, M. P.; Ferrari, S.; Venturelli, A.; Calò, S.; Tondi, D.; Barlocco, D. Thymidylate synthase structure, function and implication in drug discovery. Current Medicinal Chemistry, 2005, 12, 763-771. (mettiamo quella del jemdechem) (3) Chu, E.; Koeller, D. M.; Casey, J. L.; Drake, J. C.; Chabner, B. A.; Elwood, P. C.; Zinn, S.; Allegra, C. J. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. PNAS 1991, 88 (20), 8977–8981. (4) Chu, E.; Allegra, C. J. The role of thymidylate synthase as an RNA binding protein. Bio Essays News. Rev. Mol. Cell. Dev. Biol. 1996, 18(3), 191–198. (5) Chu, E.; Callender, M. A.; Farrell, M. P.; Schmitz, J. C. Thymidylate synthase inhibitors as anticancer agents: from bench to bedside. Cancer Chemother. Pharmacol. 2003, 52 (1), 80–89. (6) Pelà, M.; Saxeena, P.; Luciani, R.; Santucci, M.; Ferrari, S.; Marverti, G.; Marraccini, C.; Martello, A.; Pirondi, S.; Genovese, F.; Salvadori, S.; D’Arca, D.; Ponterini, G.; Costi, M. P.; Guerrini, R. Optimization of peptides that target human tymidylate synthase to inhibit ovarian cancer cell growth. J. Med. Chem. 2014, 57, 1355-1367. (7) Pirondi, S. Folate receptor targeting through a pteroyl-nonapeptide conjugate on ovarian cancer cell lines. PhD Degree Thesis. Doctorate school: Science and Tecnologies of health products. XXVI Cycle, AA 2011-2013, University of Modena and Reggio Emilia. (8) Hay, E.; Buczkowski, T.; Marty, C.; Da Nascimento, S.; Sonnet, P.; Marie, P. J. Peptide-based mediated disruption of N-cadherin-LRP5/6 interaction promotes Wnt signaling and bone formation. J. Bone Mineral Res. 2012, 27, 1852–1863.

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