Synthesis and Characterization of Pt(IV)-Peptide Conjugates
Cisplatin and other platinum(II) drugs are generally administered to patients intravenously, whereas platinum(IV) agents can be taken orally because of their greater stability in the gastro-intestinal (G. I.) tract (31
). One such compound, cis,trans,cis
-amine(cyclohexylamine)diacetatodichloroplatinum(IV), or satraplatin, is currently in a Phase III clinical trial for hormone-refractory prostate cancer (37
). Whereas the half-life of satraplatin in tissue culture medium and human plasma is long (22 h and 5.3 h, respectively), the Pt(IV) compound undergoes rapid biotransformation in blood to a Pt(II) species, with a half-life of only 6.3 minutes (38
). The Pt(II) metabolite of satraplatin, cis
-amine(cyclohexylamine)dichloroplatinum(II), forms DNA cross-links that are structurally very similar to those of cisplatin (39
A targeted Pt(IV) complex requires a sufficiently long half-life to reach the tumor environment. Because the average cardiac output in an adult resting human is 5 liters per minute, and the average total circulating volume in an adult male is 5 liters, it takes approximately 1 minute for the blood to circulate once through the body. Based on the satraplatin half-life in blood, we estimate that an intravenous administration of a targeted Pt(IV) compound should have sufficient time to reach the tumor site before its conversion to Pt(II).
By exploiting integrin- and APN-specific peptide recognition as the guiding strategy, we have designed new Pt(IV) anticancer drug candidates for targeting angiogenic tumor endothelial cells and tumor cells expressing such integrins. The RGD and NGR containing tri- and pentapeptide sequences in these molecules are tethered as axial ligands to the Pt(IV) center. Following selective binding to the tumor cell surfaces, these Pt(IV) species have the potential to enter the cells where they will be readily reduced by glutathione or other intracellular reductants to afford cisplatin (40
). If successful, the level of toxicity of these complexes towards normal cells should be greatly diminished.
), was employed as the precursor in the synthesis of all Pt-peptide conjugates. Compound 1
, prepared as reported elsewhere (24
), is suitable for additional derivatization at its axial positions by appropriate coupling with the terminal carboxylate groups of the succinato moieties. A similar strategy was employed successfully in our laboratory to obtain a series of estrogen-tethered platinum(IV) compounds, which are capable of inducing the over-expression of HMGB1 (high mobility group binding protein 1) in MCF-7 cells and thus sensitizing them to the conjugates (24
Three different tripeptide sequences, RGD, NGR, and AGR, and the disulfide-bridged pentapeptide, (CRGDC)c, were prepared by solid-phase synthesis. The –SH groups of cysteine residues in linear CRGDC peptide were oxidized in air to form intrapeptide S–S bridged (CRGDC)c. The peptides were purified by reverse-phase HPLC on a C18 column and characterized by LC-MS, MALDI, and ESI-MS. The cyclopentapeptide, (RGDfK)c, was prepared by following the literature procedure (32
Five monofunctionalized PtIV
-peptide conjugates, denoted as Pt-RGD-mono (2a
), Pt-NGR-mono (3a
), Pt-AGR-mono (4a
), Pt-(CRGDC)c-mono (6
), and Pt-(RGDfK)c-mono (7a
), and four difunctionalized analogues designated Pt-RGD-bis (2b
), Pt-NGR-bis (3b
), Pt-AGR-bis (4b
), and Pt-(RGDfK)c-bis (7b
), were synthesized (). These complexes form upon treatment of 1
with peptide molecules in aqueous solution in the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as activator and coupling reagents. In the case of (RGDfK)c, the ε-amino group of the lysine side chain was activated with a base, DIPEA, before it was allowed to react with 1
. Except for the (CRGDC)c peptide, these reactions afforded both mono- and diconjugated Pt-peptide species, which were separated and purified by reverse-phase HPLC. An HPLC chromatogram for the purification of linear RGD conjugated Pt(IV) complexes, 2a
, is shown in Figure S1 (Supporting Information)
. For the (CRGDC)c peptide, no difunctionalized complex was detected in the reaction mixture. In addition to the aforementioned complexes, a single amino acid residue, glycinamide, was tethered to the succinato group(s) in a similar fashion to obtain mono and difunctionalized Pt(IV) conjugates, 5a
. These complexes were characterized by LC-MS and high-resolution ESI-MS. The experimental mass-spectral data are in excellent agreement with the calculated values and display the proper isotopic mass distribution patterns (see Figure S2, Supporting Information
, for representative mass spectra). Lower yields were obtained for the platinum-peptide conjugates by comparison to the glycinamide-platinum conjugates. The decreased yield is most likely the consequence of steric effects generated by the longer amino acid side chains of tri- and pentapeptides. In contrast, glycinamide is a single amino acid containing hydrogen as a side chain; therefore, the coupling reaction is facilitated.
Synthesis and Labeling of Complexes
Concentration-Response Curves of the Pt(IV)-Peptide Conjugates on Proliferating Cells
In vitro activity studies of the monofunctionalized (2a
) and difunctionalized (2b
) platinum(IV) complexes, as well as cisplatin and 1
, were performed on proliferating endothelial and tumor cells that express APN and/or the integrins αv
, discussed above. Cisplatin was used as positive control; compounds 4a
, and 5b
, and complex 1
were investigated as negative controls. We used primary cultures of endothelial cells, including bovine capillary endothelial (BCE) cells, human microvascular endothelial cells (HMVEC), and human umbilical vein endothelial cells (HUVEC). The tumor cell lines glioblastoma (U87), pancreatic carcinoma (ASPC1), uterine sarcoma (MES-SA and MES-SA/Dx-5), and cervical (HeLa) were also tested based on their expression of at least one of the two αv
), which we determined by fluorescence-activated cell sorting (Figure S4
). The expression of aminopeptidase-N (CD13) was also confirmed in BCE and HMVEC cells. As shown in and supplementary figure S4
, only endothelial cells and the tumor lines U87 and ASPC1 expressed αv
; all cells expressed αv
Expression Level of αvβ3 and αvβ5 Integrins in Different Cell Lines
Cell growth was evaluated in sub-confluent, serum- and growth factor-stimulated endothelial cells or serum-stimulated tumor cells in the presence or absence of Pt compounds. BCE cells were stimulated with basic fibroblast growth factor (bFGF); HMVEC and HUVEC were stimulated with endothelial growth media containing bFGF, insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). The activities of the platinum complexes were determined after 72 h by comparing the number of viable cells as a function of platinum concentration in treated versus untreated cells. Short pre-incubation times (2 h or 6 h) of cisplatin and 1 with BCE and HMVEC cells afforded limited decrease in cell viability, indicating the rates of internalization of platinum complexes to be relatively slow. The activities of these complexes were independent of serum concentration, tested at 5 and 10% (data not shown).
Concentration-response curves of the platinum-peptide complexes in all the cells were compared to those with cisplatin and 1. To ensure unbiased data, two additional independent double-blinded experiments with all the complexes were performed in BCE cells, with reproducible results.
Inhibition of BCE and HMVEC Proliferation by Pt(IV) Conjugates
The IC50 values of these complexes, defined as the concentration at which 50% of the cells are viable, were determined for BCE and HMVEC and are summarized in .
IC50 (μM) Values of Platinum Complexes for BCE and HMVEC Cells
The concentration-response curves of the monoconjugated and diconjugated platinum(IV) complexes, together with that of cisplatin and 1, toward the BCE cells are plotted in , respectively. Among all these complexes, cisplatin was most efficient at inhibiting cell growth (IC50 ≈ 1.1 μM). All Pt(IV) compounds containing conjugated targeting moieties were significantly more inhibitory than 1, with difunctionalized and monofunctionalized compounds being equally effective. Targeted complexes 2a, 2b, containing the linear RGD tripeptide, and 7a, 7b, containing the (RGDfK)c pentapeptide, were the most active (IC50 ≈ 2.1 - 3.4 μM), followed by 3a and 3b consisting of NGR tripeptide. In contrast, all platinum(IV) conjugates containing non-targeting peptides or amino acid residues were equal to or less active than 1. The monofunctionalized compounds 4a and 5a are comparable in activity to 1 (IC50 ≈ 18.5 μM), whereas their difunctionalized counterparts are less active (4b, 5b) (IC50 ≈ 29 μM).
Figure 1 Concentration-response curves of the platinum complexes on BCE cells. (A) Monofunctionalized platinum(IV) complexes along with cisplatin, and 1. (B) Difunctionalized platinum(IV) complexes along with cisplatin, and 1. (C) Pt-RGD complexes and the RGD-containing (more ...)
A similar inhibitory trend was observed when the complexes were tested on HMVEC (). The Pt(IV)-RGD conjugates, mainly 6, 7a and 7b, were most potent at inhibiting proliferation (IC50 ≈ 2.7-3.4 μM) and comparable to cisplatin (IC50 ≈ 1.2 μM), followed by 2a, 2b and the Pt-NGR conjugates, 3a and 3b (IC50 ≈ 4.5-5.9 μM). Non-targeting platinum(IV) complexes, 1, 4a, 4b, 5a and 5b were approximately an order of magnitude less potent (IC50 ≈ 33–55 μM) (see ).
Figure 2 Concentration-response curves of platinum complexes on HMVEC cells. (A) Monofunctionalized platinum(IV) complexes along with cisplatin and 1. (B) Difunctionalized platinum(IV) complexes along with cisplatin, and 1. The cells were treated for 72 h with (more ...)
The enhanced activity of RGD-containing Pt(IV) compounds was not caused by the targeting peptide moiety, as shown in . None of the free peptides, RGD, (CRGDC)c, or (RGDfK)c inhibited BCE growth, even at very high concentrations. In addition, non-conjugated 1:1 and 2:1 mixtures of free-RGD and 1 did not improve the concentration-response of 1 (IC50 ≈ 36 μM) (data not shown). These results are consistent with the inhibitory properties of RGD peptides in vitro. RGD-peptides interfere with cellular adhesion and migration by antagonizing the interaction between integrins and the extracellular matrix, but do not affect cellular proliferation when administered to sub-confluent pre-adherent cells, as is the case in our study.
Competition Studies of 2 with Free RGD-Containing Peptides in BCE
To determine whether the increased inhibitory response of RGD-containing platinum compounds could be blocked with an excess of RGD-containing peptides, we performed competition studies in BCE cells using 2a as the RGD-platinum compound, and RGD, (RGDfK)c and (CRGDC)c as the free peptides. As shown in , the more constrained cyclic pentapeptides (CRGDC)c (purple) and (RGDfK)c (light green) could partially decrease the inhibitory effect of 2a in BCE cells treated with 2 μM of the Pt-RGD compound, and this effect was statistically significant. In contrast, the RGD tripeptide had no effect, even at mM concentrations (light blue). By themselves, none of the free peptides affected BCE proliferation ((CRGDC) in gray, (RGDfK)c in dark green, and RGD in dark blue), consistent with our previous findings (). The fact that the integrin-specific αvβ3 and αvβ5 ligand (RGDfK)c could reduce the inhibitory effect of the mono-functionalized RGD-Pt(IV) compound suggests that these integrins are playing a role in the recognition of RGD-containing Pt(IV) compounds.
Competition of 2a with RGD peptides in BCE cells
Concentration-Response curves in HMVEC-d lacking αv, β1, and β3 integrins
Although the RGD tripeptide can bind to eight of the 24 known integrin heterodimers of the integrin family, only three of these, α5
, and αv
have been reported to be expressed in endothelial cells (reviewed in (48
)). Integrin levels α5
are increased, mobilized, or induced during angiogenesis, respectively, and they can promote apoptosis of angiogenic endothelial cells in vitro and in vivo (reviewed in (48
)). To determine whether some or all of these integrins are responsible for the increased sensitivity of cells toward RGD-containing platinum compounds, we performed RNAi interference studies on HMVEC-d cells. HMVEC-d cells were transfected with β1
or control RNAi, and loss of the integrins was confirmed by Western analysis at the end of the experiment (120 hours post-transfection). Complete knockdown of the integrins was obtained for β1
but not for αv
, for which a significant decrease in protein expression was achieved (). An increase in β3
protein expression was observed in cells transfected with β1
, respectively, suggesting compensation by other integrin pairs. This was not the case of β3
-transfected cells, for which a decrease in αv
integrin was also observed.
Integrin beta-3 partially mediates Pt(IV)-RGD inhibition of proliferation in HMVEC-d cells
Cells were plated 24 hours after RNAi transfection, treated with either cisplatin or 2a, and counted 3 days after treatment. We found poor cell growth in HMVEC-d transfected with β3 RNAi. The cell numbers in β3 knockdown cells 72 h post-treatment were nearly identical to the number of plated cells, demonstrating the critical role of β3 integrin in endothelial cell proliferation (See ). In contrast, HMVEC-d transfected with control RNAi, αv, and β1 integrins proliferated and had comparable cell numbers at the end of the experiment.
Despite differences in cell growth between the integrin knockdown HMVEC cells, cisplatin treatment afforded identical concentration-response curves (). In contrast, a shift in IC-50 was obtained with 2a (), particularly with β3 knockdown (5 to 13 μM), demonstrating that this integrin is involved in 2a-mediated inhibition of cell growth (See also ). Integrin β5 could be compensating for the remaining targeting in the β3 knockdown cells. Although, αv RNAi transfections were intended to remove the contribution from αvβ3 and αvβ5 integrins, it was not possible to knock down αv entirely (), possibly due to the importance of integrin αvβ3 in endothelial cell proliferation. The low levels of αv found in αv-transfected HMVEC-d may be sufficient to mediate an inhibitory effect by RGD-Pt(IV). Given the differences in β1 expression in β1 and αv-transfected cells () and their similarities in IC50 values (), β1 is most likely not involved in the inhibition of cell proliferation by 2a.
Inhibition by Cisplatin and RGD-Targeted Pt(IV) Compounds in Endothelial and Non-Endothelial Cells
Because the (RGDfK)c pentapeptide specifically binds to αv
, and our results with RGD- and (RGDfK)c-Pt(IV) conjugates are comparable, recognition of the Pt(IV) complex by the cells most likely occurs, at least in part, through either the αv
integrin. This conclusion is supported by our RNAi knockdown studies. Incorporation of NGR-containing Pt(IV) conjugates 3a
may also be mediated in part by integrin αv
, because this integrin binds NGR peptides, albeit with lower affinity than their RGD analogues (19
). APN does not appear to be solely responsible for recognition of 3a
given that the IC50
values were comparable in BCE and HMVEC despite differences in their APN/CD13 expression levels ().
Inhibition of Cell Proliferation by RGD-Containing Pt(IV) Conjugates in αvβ3 Positive and αvβ3 Negative Cells
To determine whether αv
could mediate delivery of the RGD-containing complexes in the absence of αv
, we compared the inhibition of Pt(IV) conjugates in endothelial cells or tumor lines that were positive (HUVEC, U87) and negative (MES-SA, MES-SA/DX-5, HeLa) for αv
. The concentration-response curves of the Pt-RGD conjugates were comparable on HUVEC, U87, ASPC1, MES-SA, and HeLa cell lines. The IC50
values of these complexes and cisplatin are summarized in (See Figure S3, Supporting Information
, for U87). Because each cell type had a different sensitivity to cisplatin, the IC50
ratio between 2a
/cisplatin and 7a
/cisplatin was used to compare the RGD and (RGDfK)c conjugates (see and ). The best inhibitory effect was obtained in BCE, U87, ASPC1, and HeLa; because the last two cell lines are negative for αv
, our results suggest that αv
is equally efficient at delivering Pt(IV) into the cell.
Targeting Efficiency: Linear vs. Cyclic Peptides
According to literature reports, the cyclic RGD and NGR peptides containing cysteine residues that form disulfide bonds mark angiogenic endothelial cells with more efficiency compared to their linear counterparts (20
). It is believed that the presence of disulfide linkage in the cyclic peptides provides the RGD and NGR motifs a constrained conformational arrangement resulting in stronger interactions with the integrins expressed on the cell surfaces. In some cases, the cyclic peptides containing the RGD and NGR motifs were 10- to 200-fold more effective over the corresponding linear peptides in recognizing the receptors (21
). In the present study we examined both linear RGD and cyclic CRGDC peptides to serve as the marker to transport the attached platinum complexes to the tumor cells. We also included the cyclic pentapeptide, (RGDfK)c, which is a potent inhibitor of αv
The differential recognition of linear vs cyclic RGD peptides to integrins was indirectly confirmed in our competition studies, because both cyclic pentapeptides, (CRGDC)c and (RGDfK)c, could partially compete the inhibitory effect of 2a
in BCE cells, whereas the linear tripeptide RGD was ineffective. Although we have not yet determined the binding affinities of the linear RGD-containing Pt(IV) compounds and the cyclic RGD containing Pt(IV) complexes, our results indicate that the linear RGD tri-peptide containing Pt(IV) complexes are equally effective in inhibiting cell proliferation and cell killing, Only for about half of the cells tested did we find that the mono and diconjugated (RGDfK)c compounds were slightly superior to the mono and bis-conjugated RGD compounds (HMVEC, U87, MES-SA). As supported by the values listed in , the IC50
ratios of the monoconjugated RGD containing Pt(IV) complexes, 2a
, with respect to cisplatin are quite comparable for a given cell type. We have also demonstrated that at least some of the anti-proliferative effect of 2a
is mediated by the αv
integrins, as (1
) the αv
integrin-specific ligand, (RGDfK)c, and (CRGDC)c could partially compete off 2a
in BCE cells, and (2
) Loss of β3 in HMVEC decreased the IC50
but not of cisplatin. It is possible that upon conjugation to the succinate group in Pt(IV), the RGD linear tripeptide and the cyclic RGD pentapeptide targeting moieties become comparable at recognizing αv
integrins. The Pt(IV) center could sterically hinder binding of the cyclic pentapeptides, decreasing their enhanced binding, or the Pt(IV) center may impose a more rigid conformation to the RGD linear targeting moiety enhancing its binding potential to the αv
integrins. A high degree of targeting ability with linear RGD tripeptide attached to glycosylated porphyrins was also observed in photodynamic cancer therapy against K562 leukemia cell lines (50
Cell Internalization of Pt-Peptide Conjugates
The platinum complexes can enter the cells selectively, through internalization by a surface marker such as αv
integrin, or non-selectively, by diffusion across the cell membrane or via other transporters or carriers. Cisplatin, a small neutral molecule, enters cells by passive diffusion (4
) and, to a degree not yet quantified, other transporters (51
). Differences in the size and overall charge on the complex will affect passive diffusion of the complexes through the cell membrane, with smaller, planar, and neutral complexes being more readily incorporated than large, non-planar, and charged ones. Some of our platinum-peptide complexes are neutral at pH 7 in aqueous solution, whereas a few of the complexes are positively charged and several of them are negative (). All of the Pt(IV) complexes have an octahedral geometry which, together with their variable overall charges, makes it unlikely that they would be internalized by passive diffusion. Except for cisplatin, the non-targeted Pt-complexes should therefore enter cells by selective uptake. The toxicity profiles of the negatively charged mono-glycinamide-tethered complex (5a
), the neutral mono-AGR (4a
) and bis-glycinamide complexes (5b
) and the positively charged bis-AGR complexes (4b
), are quite similar to that of 1
, which is smaller and negatively charged in aqueous solution. Although the monoconjugated RGD complexes are negatively charged, and the diconjugated complexes are neutral in water, they have very similar inhibitory effect and are significantly more potent than the non-targeted Pt(IV) complexes. No significant differences in anti-proliferative effect were observed between the targeted mono and di-conjugated complexes, suggesting that only one ligand is sufficient for cell surface recognition. These results indicate that the cellular uptake of the targeted platinum(IV) complexes is most likely mediated by internalization by surface markers, αv
integrins, which would account for their greater potency compared to the non-targeted analogues.
From the perspective of evaluating compounds for further study in animal models and, eventually, human cancer treatment, it would have been valuable to test them against cells that were αvβ5 negative. The cell lines that we tested were all positive for this integrin (), however. Nevertheless, the significantly improved effectiveness of the targeted RGD, NGR, and (CRGDC)c conjugates compared to the non-targeted AGR, Gly, and disuccinate precursor clearly establishes the value of targeting receptors involved in tumor vasculature as a strategy for improving the efficacy and selectivity of platinum-based drugs.