Mechanistic Insight into the Inhibition of Matrix Metalloproteinases by

J. Med. Chem. 2009, 52, 7847–7855 7847 DOI: 10.1021/jm900845t

Mechanistic Insight into the Inhibition of Matrix Metalloproteinases by Platinum Substrates† Fabio Arnesano,‡ Angela Boccarelli,§ Daniele Cornacchia,‡ Fiorentin Nushi,‡ Rossella Sasanelli,§ Mauro Coluccia,*,§ and Giovanni Natile*,‡ ‡

Dipartimento Farmaco-Chimico, University of Bari “A. Moro”, Via Edoardo Orabona 4, 70125 Bari, Italy, and §Dipartimento di Scienze Biomediche e Oncologia Umana, University of Bari “A. Moro”, Piazza Giulio Cesare 11, 70124 Bari, Italy Received June 10, 2009

Platinum compounds are among the most used DNA-damaging anticancer drugs, however they can also be tailored to target biological substrates different from DNA, for instance enzymes involved in cancer progression. We recently reported that some platinum complexes with three labile ligands inhibit matrix metalloproteinase activity in a selective way. We have now extended the investigation to a series of platinum complexes having three chlorido or one chlorido and a dimethylmalonato leaving ligands. All compounds are strong inhibitors of MMP-3 by a noncompetitive mechanism, while platinum drugs in clinical use are not. Structural investigations reveal that the platinum substrate only loses two labile ligands, which are replaced by an imidazole nitrogen of His224 and a hydroxyl group, while it retains one chlorido ligand. A chlorido and a hydroxyl group are also present in the zinc complex inhibitor of carboxypeptidase A, whose active site has strong analogies with that of MMP-3.

† This work is dedicated to the countless members of the Medicinal Chemistry Division that in 100 years of intensive research have deeply contributed to the advancement of life sciences and to the well-being of humanity. *For M.C.: phone, (þ39) 080 5478412; fax, (þ39) 080 5478524; E-mail, [email protected]. For G.N.: phone, (þ39) 080 5442774; fax, (þ39) 080 5442230; E-mail: [email protected]. a Abbreviations: AHA, acetohydroxamic acid; AMP, diethyl(aminomethyl)phosphonate; CD, circular dichroism; dien, diethylene triamine; dmm, dimethylmalonate; DMSO, dimethyl sulfoxide; DNTB, 5,50 -dithiobis(2-nitrobenzoic acid); ESI-MS, electrospray mass spectrometry; FFT, fast Fourier transform; IC50, median inhibition concentration; His, histidine; HSQC, heteronuclear single quantum coherence; INEPT, insensitive nuclei enhanced by polarization transfer; MMP, matrix metalloproteinase; NMR, nuclear magnetic resonance; NNGH, N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid; NOESY, nuclear Overhauser effect spectroscopy; OD, optical density; RF, resistance factor; SD, standard deviation; SE, standard error; SMP, diethyl[(methylsulfinyl)methyl]phosphonate; TCA, trichloroacetic acid; TOCSY, total correlation spectroscopy; TPPI, time proportional phase increment.

different from those of cisplatin and its analogues in clinical use and have encouraged the strategy of designing platinum complexes with different modes of DNA binding. The pharmacodynamic properties of platinum complexes can also be modified in a markedly different way by targeting biological substrates distinct from DNA. Very recently, platinum complexes able to inhibit matrix metalloproteinases (MMPsa) through a noncompetitive mechanism have been reported.16 [PtCl2(SMP)] and [Pt(dmm)(SMP)], characterized by bearing the bisphosphonate-analogue ligand diethyl[(methylsulfinyl)methyl]phosphonate (SMP), are slight inhibitors of MMP-2 but markedly inhibit MMP-3, MMP-9, and MMP-12. Platinum complexes able to inhibit enzymes involved in cancer progression are likely to have a wide therapeutic potential provided that the inhibition is selective and the mechanistic basis of their inhibitory activity is known.17,18 Studies of the interaction of platinum complexes with proteins indicate that histidine, methionine, and cysteines represent preferential binding sites.19-25 The comparative analysis of MMP-2, MMP-3, MMP-9, and MMP-12 sequences16 has shown that potential binding sites are quite far from the substrate pocket in the case of MMP2; in contrast, MMP-3, MMP-9, and MMP-12 contain binding sites (MMP3, His224; MMP9, Met404; MMP12, Met87) in regions of primary importance for binding of substrates.26-28 Therefore, the presence of candidate anchoring sites in regions critical for the enzyme activity could be the reason for a selective MMP inhibition by [PtCl2(SMP)] and [Pt(dmm)(SMP)] complexes. Interestingly, the platinum drugs in clinical use (cisplatin, carboplatin,16 and oxaliplatin (this work)) are not able to inhibit MMP enzymes, thus suggesting that peculiar structural features of platinum complexes are required for MMPinhibition activity. We hypothesized that a platinum complex with MMP-inhibition activity should be able (i) to interact with anchoring sites in regions critical for the enzymatic

r 2009 American Chemical Society

Published on Web 09/16/2009

Introduction Cisplatin, carboplatin, and the recently introduced oxaliplatin are among the most used DNA-damaging anticancer drugs, as shown also by the numerous clinical trials involving platinum-containing regimens.1 The widening of the spectrum of activity, along with the ability of overcoming resistance, is a major challenge of platinum-based anticancer therapy, and the progressive elucidation of both the chemical properties and the mode of action of cisplatin has driven the design and preclinical development of new derivatives.2-5 Since the introduction of platinum-based drugs, several active compounds that violate the classical geometry-based structure-activity relationships have been reported, including platinum complexes with trans geometry,6,7 platinum(IV) complexes (targeted to specific cellular functions or switchable),8-11 polynuclear platinum complexes,12 and cationic complexes.13-15 Importantly, most of these “rule-breaking” compounds are characterized by DNA interaction properties

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Figure 1. Structures of [PtCl2(SMP)], K[PtCl3NH3], K[PtCl3(DMSO)], K[PtCl3(AMP)], K[PtCl(dmm)(AMP)], cisplatin, carboplatin, oxaliplatin, transplatin, trans-[PtCl2(NH3){Z-HNdC(OCH3)CH3}], [PtCl(dien)]Cl.

activity and (ii) to determine additional binding events able to inhibit the catalytic functionality. Platinum-SMP complexes were found to easily detach the phosphonate group; in this way, the platinum atom can offer three coordination sites for interaction with the protein (the site of the phosphonate in addition to those of the leaving ligands).29 This is not possible for bifunctional complexes such as cisplatin or carboplatin, which occurred to be inactive. To verify the hypothesis that the presence of three potential coordination sites represents a necessary condition for a platinum complex to be endowed with MMP-inhibitory activity, we have extended the investigation to other platinum complexes characterized by having three chlorido or one chlorido and a dimethylmalonato as leaving groups, and ammonia (NH3), dimethyl sulfoxide (DMSO), or diethyl(aminomethyl)phosphonate (AMP) as nonleaving ligand (Figure 1). In parallel, the MMP-inhibitory activity of another bifunctional platinum complex with cis geometry (oxaliplatin), of two bifunctional complexes with trans geometry (transplatin and trans-[PtCl2(NH3){Z-HNdC(OCH3)CH3}]30,31), and of monofunctional [PtCl(dien)]Cl were investigated. All compounds were subjected to tests for MMP inhibition. Moreover, we have investigated the interaction between MMP-3 and [PtCl2(SMP)] by ESI-MS, CD, and NMR spectroscopy. Results Inhibition of Matrix Metalloproteinase Activity. The inhibitory effects of K[PtCl3(NH3)], K[PtCl3(DMSO)],

K[PtCl3(AMP)], and K[PtCl(dmm)(AMP)] have been investigated upon MMP-3 and compared to those of cisplatin, carboplatin, oxaliplatin, transplatin, trans-[PtCl2(NH3){Z-HNdC(OCH3)CH3}], and [PtCl(dien)]Cl. MMP-3 is involved in cancer progression32 and is characterized by the presence of a potential anchoring site (His224) at the edge of the substrate pocket. MMP-3 was chosen for this comparative investigation being strongly inhibited by [PtCl2(SMP)] and [Pt(dmm)(SMP)] complexes (IC50 = 5.3 and 4.4 μM, respectively).16 Recombinant catalytic domain was used for inhibition studies, and the proteolytic activity was evaluated through spectrophotometric measurements of a thiopeptolide substrate. Compound’s concentrations able to inhibit the enzymatic activity by 50% (IC50) after 1 h of interaction between compound and enzyme are reported in Figure 2. The most active compound was K[PtCl3(DMSO)] (IC50 = 1.7 ( 0.5 μM) whose inhibitory activity is even greater than those of the [PtCl2(SMP)] and [Pt(dmm)(SMP)] complexes previously investigated.16 K[PtCl3(NH3)] and K[PtCl3(AMP)] showed similar inhibitory activities (IC50 =25.0 ( 3.6 and 26.0 ( 3.0 μM, respectively), while K[PtCl(dmm)(AMP)] was slightly less effective (IC50 of 41.2 ( 9.7 μM). In contrast, oxaliplatin, transplatin, trans[PtCl2(NH3){Z-HNdC(OCH3)CH3}], and [PtCl(dien)]Cl displayed no inhibitory activity up to a 300 μM concentration (not shown), thus behaving as cisplatin and carboplatin previously investigated. To investigate the mechanism of MMP-3 activity inhibition by platinum compounds, the enzymatic activity was

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Table 1. Inhibitory Effect of K[PtCl3(NH3)], K[PtCl3(DMSO)], K[PtCl3(AMP)], K[PtCl(dmm)(AMP)], Cisplatin, and Carboblatin upon A2780 and A2780cisR in Vitro Cell Growth (IC50, μM)a IC50 (μM)a

RFb

complex

A2780

A2780cisR

A2780cisR/A2780

K[PtCl3(NH3)] K[PtCl3(DMSO)] K[PtCl3(AMP)] K[PtCl(dmm)(AMP)] cisplatin carboplatin

2.9 ( 1.0 87.1 ( 15.3 11.6 ( 4.2 0.60 ( 0.02 0.20 ( 0.01 1.3 ( 0.2

34.1 ( 9.6 167.5 ( 7.2 32.8 ( 8.7 5.9 ( 2.6 3.2 ( 0.5 23.0 ( 2.1

11.7 1.9 3 9.8 16 17.6

a IC50 (mean value ( SD calculated over at least three experiments) represents compound’s concentration able to inhibit cell growth by 50% after 96 h treatment. b RF (resistance factor, IC50 A2780cisR/IC50 A2780).

Figure 2. Inhibitory effect of K[PtCl3(NH3)], K[PtCl3(DMSO)], K[PtCl3(AMP)], and K[PtCl(dmm)(AMP)] upon MMP-3 proteolytic activity. IC50, mean value ( SD, represents the compound’s concentration (μM) that inhibits the enzymatic activity by 50% after 1 h of interaction between complex and enzyme.

Figure 3. MMP-3 inhibition by K[PtCl3(NH3)], K[PtCl3(DMSO)], K[PtCl3(AMP)], and K[PtCl(dmm)(AMP)]. The graph shows the rates of proteolysis by MMP-3 enzyme, as a function of substrate concentration, in the absence (control, 0) and in the presence of K[PtCl3(NH3)] (4), K[PtCl3(DMSO)] (3), K[PtCl3(AMP)] (2), or K[PtCl(dmm)(AMP)] (1) IC50 concentrations (25.0, 1.7, 26.0, and 41.2 μM, respectively). The rates of proteolysis (v, pmol/min) were calculated from linear plots of (OD reaction volume)/(ε l) against time (min), where ε is the extinction coefficient of 2-nitro-5thiobenzoic acid (13600 M-1 cm-1) and l is the path length of light through the sample in cm. Points are mean values ( SE over three experiments; no SE bars are seen because they are smaller than the size of the symbols. Kinetic parameters (vmax and KM, ( SE) were determined by fitting the data to the Michaelis-Menten equation using nonlinear regression analysis (GraphPad Prism, San Diego, CA).

measured as a function of thiopeptolide concentration in the presence of IC50 concentration of K[PtCl3(NH3)], K[PtCl3(DMSO)], K[PtCl3(AMP)], or K[PtCl(dmm)(AMP)]. The plots of the measured rates of proteolysis (v, pmol/min) against substrate concentrations are shown in Figure 3.

The rate of substrate conversion as a function of substrate concentration follows the Michaelis-Menten equation [v= vmax[S]/([S] þ KM)], where [S] is the substrate concentration, vmax is the maximum rate of conversion, and KM is the Michaelis constant (the substrate concentration at which the rate of conversion is half of vmax). The vmax and KM values were determined by fitting the data directly to the Michaelis-Menten equation using nonlinear regression (GraphPad Prism Software). In the presence of IC50 concentrations of K[PtCl3(NH3)], K[PtCl3(DMSO)], K[PtCl3(AMP)], or K[PtCl(dmm)(AMP)], vmax values decreased with respect to control (108.5 ( 6.9, 91.4 ( 4.7, 86.5 ( 6.9, 97.8 ( 7.9, and 190.0 ( 6.8 pmol/min for the four complexes and the control, respectively), whereas KM values were not significantly modified (141.6 ( 32.5, 112.8 ( 22.1, 108.4 ( 33.8, 184.1 ( 51.1 and 123.2 ( 16.5 μM, respectively). Therefore the results suggest a noncompetitive inhibition mechanism. The enzyme inhibitory activity of [PtCl3(DMSO)], K[PtCl3(NH3)], K[PtCl3(AMP)], and K[PtCl(dmm)(AMP)] prompted us to check if the same compounds could have tumor cell growth inhibitory effect. The growth inhibitory activity of the set of four anionic compounds was tested toward cisplatin-sensitive A2780 and cisplatin-resistant A2780cisR ovarian cancer cells (Table 1). All compounds tested displayed some growth inhibitory effect toward A2780 cells, which, however, was lower than that of cisplatin but in three out of the four complexes was of the same order of that of carboplatin. We must admit that the DMSO compound, which is the most active as MMP inhibitor, is the less active as tumor cell growth inhibitor. However, a direct parallelism between the two biological effects should not be drawn because cytotoxicity requires the drug to enter the cell while MMPs are secreted in the extracellular space and, as such, cell entry may not be required to exert anti-MMP activity. From this point of view, the poor cytotoxicity of the DMSO compound highlights its selective activity as MMP inhibitor. It is rather noteworthy the observation that some platinum complexes with three labile ligands can have cell growth inhibition potency comparable to that of carboplatin. It has already been reported in the literature that platinum compounds with only one leaving chloride can also be endowed with antitumor activity when the nonleaving ligands have particular features,13,15,33-35 thus breaking the generally accepted rule that two leaving ligands are needed in order to have antitumor activity. We have now found that platinum compounds with three leaving ligands can also exhibit


Figure 4. (A) ESI-MS spectra of the catalytic domain of MMP-3 (residues 88-248) before (in blue) and 48 h after (in red) mixing with 2 mol equiv of [PtCl2(SMP)]. Peaks corresponding to two different multiply charged states (14þ and 15þ) are shown, each state comprises two signals of nearly equal intensity corresponding to apoMMP-3 and to the enzyme with one bound Ca(II) ion, respectively. (B) Far-UV CD spectra of the catalytic domain of MMP-3 (residues 88-248) before (in blue) and 48 h after (in red) mixing with 2 mol equiv of [PtCl2(SMP)].

cytotoxicity, therefore it appears that the same rule can be questioned also from the opposite side. The fact that for some compounds the resistance factors (RF=IC50 resistant cells/IC50 sensitive cells) was very low is a clear indication that the mechanism of action of these compounds can be different from that of cisplatin. Characterization of the Adduct between MMP-3 and [PtCl2(SMP)]. The interaction between MMP-3 and [PtCl2(SMP)], the prototype of MMP inhibitors, has been investigated by ESI-MS, CD, and NMR spectroscopy. Because of the complexity of the enzyme and the presence in the native protein of several, quite labile, metal ions, mass spectrometry measurements were not straightforward due to the progressive loss of the Zn(II) and Ca(II) ions. ESI-MS spectra easy to be interpreted were only obtained under acidic conditions where most, if not all, Zn(II) and Ca(II) ions of the native protein are lost (Figure 4A), therefore, the reaction between the enzyme and the complex was carried out at 25 °C and pH 6 (as described in the Experimental Section) and 1% acetic acid was added just before injection. The ESI-MS spectrum of MMP-3, in the absence of Pt(II) complex, shows a series of multiply charged states, each state comprises two signals of nearly equal intensity corresponding to apoMMP-3 and to the enzyme with one bound Ca(II) ion, respectively. The loss of two Zn(II) and all but one Ca(II) ions can be ascribed to the low pH of the infused sample, necessary to attain a good volatilization. The ESIMS spectra of the reaction mixture show a time-dependent increase of new sets of signals indicating the formation of

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an adduct between MMP-3 and the Pt(II) complex. The mass difference between corresponding charged states indicates binding of a {PtCl(OH)(SMP)} moiety to MMP-3 (Figure 4A reports the multiply charged states 14þ and 15þ, but a similar pattern was observed also for the other charged states. We do not exclude that other platinumprotein adducts could be formed in small yield.). The fitting analysis of the CD spectrum of MMP-3, recorded in the absence of Pt(II) complex, gives a secondary structure content consistent with the folded MMP catalytic domain, which consists of three R-helices and a twisted fivestranded β-sheet. Addition of [PtCl2(SMP)] to MMP-3 produces a change in the CD profile corresponding to an increase of random coil regions at the expense of R-helical content (Figure 4B). 1 H, 15N-Edited HSQC spectra were used to map the regions of MMP-3 affected by addition of [PtCl2(SMP)]. Protein selfdigestion is usually prevented by addition of acetohydroxamic acid (AHA). However, this inhibitor requires high concentration (up to 1 M), and we worried that it could coordinate to platinum in preference to the protein. Moreover, such a big concentration of a NMR active substance could negatively affect the quality of the spectrum. For this reason, we replaced AHA with N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH), which has much greater affinity for the enzyme (KD of 13 10-8 M as compared to 17 10-3 M for AHA), thus requiring a much lower concentration. The use of NNGH for NMR and ESI-MS experiments does not compromise comparison with the results obtained from inhibition studies (ineffective low concentration of AHA) because, apart from the low concentration and poor platinum-coordination ability of NNGH, the inhibition by platinum complexes has been found to be of noncompetitive type, thus indicating that platinum coordination to MMP-3 does not interfere with substrate binding to the catalytic site. We first collected the NMR spectra of the native protein with NNGH, and afterward we added the platinum complex. Upon interaction with the Pt(II) complex, some NMR signals of 15N-labeled MMP-3 experience a decrease in intensity. Concomitantly, new peaks appear at different chemical shift values, indicating a slow exchange process on the NMR time scale between the free protein and the adduct. After assignment of the protein 1H/15N cross-peaks, the change in intensity of the peaks of the native protein and the peak shifts upon platinum binding were plotted as a function of the protein sequence (Figure 5A,B). The NMR spectral changes are localized in two well-defined regions of the protein close to His96 (residues 95-99 of strand β1, 130-133 of strand β2, and 173-174 of a loop) and His224 (residues 188-189, 192-193, and 195-198 at the N-terminal end of helix R2 and residues 223-226 and 231-232 of the specificity loop which partially defines the S10 pocket) (Figure 5C). Both histidines can act as Pt(II)anchoring residues, however binding of the complex to His224, located in the specificity loop, is expected to deeply affect the enzyme activity. Binding to this site would not alter the substrate affinity of MMP-3, consistent with a noncompetitive inhibition mechanism of the Pt(II) complex, nevertheless it may cause a conformational change at the N-terminal end of helix R2 so affecting the proximally located catalytic Zn(II) site. Indeed, the loop region 209-211, which encompasses the catalytic Zn(II)-binding residue His211, is also significantly affected by addition of [PtCl2(SMP)].

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Figure 5. (A) I/I0 profile obtained as the ratio of cross-peak intensities in 1H, 15N HSQC spectra of the catalytic domain of 15N-labeled MMP-3 in the presence and in the absence of 1 equiv of [PtCl2(SMP)]. The red line corresponds to the mean I/I0 value minus the standard deviation. (B) Weighted average chemical shift differences Δδavg(HN) of cross-peaks in 1H, 15N HSQC spectra of MMP-3 catalytic domain in the presence and in the absence of 1 equiv of [PtCl2(SMP)]. (C) Mapping on the crystal structure of the catalytic domain of MMP-3 (PDB ID 1SLN) of NMR spectral changes (in red) observed upon addition of 1 equiv of [PtCl2(SMP)]. Zn(II) ions are shown as gray spheres and Ca(II) ions as green spheres. The histidine side chains are shown as blue sticks, and the specificity loop and the helix R2 are indicated by arrows. (D) van der Waals contacts between residues of the specificity loop and of the N-terminal end of helix R2, which undergo NMR spectral changes upon addition of 1 equiv of [PtCl2(SMP)]. The side chains of these residues are shown as cyan sticks.

Discussion One outcome of this investigation was that all platinum compounds having three labile ligands also exhibit MMP-3 inhibitory activity, while compounds with only one or two labile ligand(s) are totally inactive. The three labile ligands, we thought, are likely to be lost in the reaction with the enzyme, however the picture resulting from our characterization of the site and mode of interaction of the active platinum species [PtCl2(SMP)] with MMP-3 is quite different. The reaction of MMP-3 catalytic domain with the platinum complex [PtCl2(SMP)] leads to protein adducts of the type MMP-3-{PtCl(OH)(SMP)} (ESI-MS), in which the platinum has lost only one chlorido ligand (the coordination position being taken up by a protein donor) and has added one hydroxyl group which, presumably, has taken the place of the phosphonic functionality (the SMP ligand being now semidetached). Although the conformation of the protein under acidic conditions is likely to be different from that under physiological conditions which is loaded with Zn(II) and Ca(II) ions, we are confident that the platinum fragment found in the ESI-MS spectrum corresponds to that present in the adduct of the inhibitor with the

folded protein. Such an assumption is supported by the fact that the acid has been added just before running the ESIMS experiment, and any subsequent rearrangement would have caused a loss rather then an addition of platinum donor groups. Moreover, platinum coordination compounds are usually rather inert. From the CD spectra recorded at increasing concentration of platinum substrate (Figure 4B), it is evident that only small changes in conformation do take place in accord with platinum interacting only with residues on the surface of the protein and not causing dramatic structural changes. The NMR results indicate that both histidines exposed to the solvent (His96 and His224) can act as Pt(II)-anchoring sites. With reference to the His224 anchoring site located in the specificity loop, the NMR data also show that, apart from residues close to the histidine, only residues at the N-terminal end of helix R2 and in the vicinity of the Zn(II)-binding His211 undergo spectral changes. Therefore we can assume that, relevant to the MMP-3 inhibition, is the binding to His224 of the metal fragment {PtCl(OH)(SMP)} found in ESI-MS. This binding would produce a conformational change in the S10 substrate-binding pocket close to His224 and a rearrangement of a number of contacts occurring


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Figure 6. Modeling of the adduct between MMP-3 and {PtCl(OH)(SMP)}. Two possible arrangements of the coordinating ligands around the Pt(II) ion (i.e., Cl trans to OH and SMP trans to His224, or viceversa) are shown in (A) and (B), respectively. Color code of atom spheres: zinc, gray; platinum, blue; sulfur, yellow; chlorine, green; oxygen, red. The histidine side chains are shown as blue sticks and the SMP ligand is shown in cyan. The position of His224 side chain in the crystal structure of the catalytic domain of MMP-3 is shown in light blue.

between the specificity loop and the N-terminal end of helix R2 (Figure 5D). We have attempted to model the interaction between the His224-linked {PtCl(OH)(SMP)} fragment and the protein active site. In His224-{PtCl(OH)(SMP)}, the OH and His ligands were assumed to be in cis positions because, very likely, they have entered the two most reactive sites of the starting [PtCl2(SMP)] substrate (i.e., the site trans to the translabilizing S-donor and that initially occupied by the very labile phosphonate group). Having fixed the His and OH positions, there were two possible arrangements for the Cl and SMP ligands (i.e., Cl trans to OH and SMP trans to His224, or vice versa). We have considered both possibilities and modeled the interaction with the active site by assuming the existence of a bridging hydroxyl group with the catalytic Zn(II) ion. The latter assumption was suggested by the strong analogy we noted between the {PtCl(OH)(SMP)} fragment and the complex {ZnCl(OH)} reported to efficiently inhibit the carboxypeptidase A.36,37 The catalytic Zn(II)-binding site of this latter metalloprotease comprises a glutamate, two histidines, and a water molecule and is quite similar to that of MMP-3. The Zn(OH)Cl complex was shown to create a binuclear site with the catalytic Zn(II) ion of carboxypeptidase A by displacing the coordinated water molecule.36 Analogously, the Pt(II) fragment could form a binuclear system with the catalytic Zn(II) ion of MMP-3 through a bridging hydroxyl group. Such an interaction would also be supported by the observed changes in the loop region 209-211, which encompasses the catalytic Zn(II)-binding residue His211. Both calculated structural models show that the MMP-3-linked {PtCl(OH)(SMP)} fragment can be accommodated at the lower rim of the active site defined by the specificity loop without affecting the rest of the protein structure. The specificity loop on one side and helix R2 on the other side form the S10 substrate binding pocket. No direct interaction is established between the platinum fragment and helix R2, however the specificity loop undergoes a conformational change to allow binding of His224 to the Pt(II) ion (Figure 6A,B). The movement of the specificity loop (residues 223-226) is transferred, as a secondary effect, to the N-terminal end of helix R2 (residues 192-193 and 195-198) through a series of contacts between these two regions (Figure 5D). The two ethoxy groups of the SMP ligand point toward the solvent. In the case of SMP trans to His224, the

free phosphonate group is sufficiently close to the catalytic Zn(II) ion and may eventually bind to it, while the chlorido ligand can form a hydrogen bond with an imidazole ring proton of His224 (Figure 5C). In the case of the chlorido ligand trans to His224, a direct hydrogen bond can be established between the chloride and an imidazole ring proton of Zn(II)-coordinating His211 (Figure 6B). Conclusions This investigation has clearly shown that platinum(II) substrates with three labile ligands can selectively inhibit matrix metalloproteinases. Structural investigations of the interaction between one of these inhibitors, [PtCl2(SMP)], and the catalytic domain of MMP-3 has revealed that the platinum substrate has lost only two labile ligands (which have been replaced by a protein histidine and a hydroxyl group) while it has retained one chlorido ligand. The latter unexpected result could be more than just a coincidence and could have even greater relevance in the natural system due to the localization of the MMP’s on the cell surface and the rather high chloride concentration in the extracellular medium. This view is also supported by the presence of a chlorido and a hydroxyl group in the zinc complex, which inhibits carboxypeptidase A (an enzyme which has strong analogies with the MMP-3 active site) and the possibility for this chlorido ligand to form hydrogen bonds with key histidine residues, as suggested by our structural models. This synergistic anion could play a fundamental role in the neutralization of the charged metal cores. Apart from the crucial role of His224 (located in the specificity loop of MMP-3), which anchors the platinum substrate in a key position for the subsequent interaction with the protein active site, a central role is also played by the hydroxyl group, which was postulated to bridge the platinum moiety with the catalytic zinc ion. Bridging hydroxo groups are a common feature in platinum chemistry and have been investigated also in connection with the solution fate of antitumoral cisplatin.38,39 We believe that the present results can represent a valuable starting point for the further exploitation of platinum substrates as MMP inhibitors. Experimental Section Starting Materials. [PtCl2(SMP)],29,40 K[PtCl3(AMP)],41 K[PtCl3(NH3)],42 K[PtCl3(DMSO)],43 and [PtCl(dien)]Cl44

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were synthesized as previously reported. The K[PtCl(dmm)(AMP)] complex was synthesized as follows. A solution of K[PtCl3(N-AMP)] (110 mg, 0.21 mmol) in water (10 mL) was treated with potassium dimethylmalonate containing three water molecules of crystallization (56.5 mg, 0.21 mmol) and kept under stirring for 6 h at room temperature. Subsequently, a solution of Ag2SO4 (67.7 mg, 0.21 mmol) in 20 mL of water was added dropwise and the reaction mixture was stirred in the dark for 24 h. The final solution was filtered and concentrated to onethird of its initial volume, treated with ethanol and diethyl ether, and cooled to 4 °C to afford hygroscopic yellow-brown crystals of the desired compound (38 mg, 32% yield). The K[PtCl(O,Odmm)(N-AMP)] complex was characterized by NMR in D2O (δ, ppm. 1H: 3.08 NH2CH2P; 4.29 POCH2CH3; 1.39 POCH2CH3; 1.27 CH3CCOO. 31P:=24.4, ppm, 2JH,P=15 Hz. 195Pt: -1453, 3 JP,Pt = 120 Hz) and by ESI-MS ({[PtCl(dmm)(AMP)]}-, C10H20NClO7PPt 526.7). Cisplatin and carboplatin were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). Culture media and reagents were from Euroclone (Paignton, UK). The unlabeled and 15N-enriched MMP-3 catalytic domain (residues 88-248) were purchased from Protera srl (Sesto Fiorentino, Italy). N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH) was purchased from Biomol (Plymouth Meeting, PA). All compounds, comprising those already reported in the literature were characterized by elemental analysis, IR, ESI-MS, and NMR spectroscopy. Their purity was always above 98%. The purity of protein samples was tested by HPLC and found to be well above 95%. Matrix Metalloproteinase Activity Inhibition and Kinetic Assays. MMP-3 activity inhibition and kinetic assays were performed by using the colorimetric method Biomol QuantiZyme, following the manifacturer’s protocol with minor modifications. All experiments were performed by using the recombinant human MMP-3 catalytic domain (residues 83-255) purchased from Biomol. Briefly, the proteolytic activity was measured by using a thiopeptolide substrate (Ac-PLG-[2-mercapto-4methyl-pentanoyl]-LG-OC2H5), whose peptidic bond, which is cleaved by the MMP, has been replaced by a thioester bond.45,46 MMP-mediated thioester hydrolysis produces a sulfhydryl group, which reacts with 5,50 -dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB) to form 2-nitro-5-thiobenzoic acid, whose concentration can be evaluated by measuring the absorbance at 412 nm. In 96-well microplates, recombinant human MMP-3 catalytic domain was incubated (1 h at 37 °C, if not otherwise indicated) with different concentrations of compounds under investigation in MES (50 mM), CaCl2 (10 mM), Brij-35 (0.05%), and DTNB (1 mM) at pH 6.0. After addition of the thiopeptolide substrate to the incubation mixture, the increase of absorbance was recorded at 1 min time intervals for 60 min. Data were plotted as OD versus time for each sample to obtain the reaction rate in OD/min. The percentage of residual activity for each compound concentration was calculated by the formula: % remaining activity=(rate in the presence of inhibition/control rate) 100, and the compound concentration able to inhibit the enzymatic activity by 50% (IC50) was calculated from semilogarithmic dose-response plots. The obtained results exclude the possibility that the reduction of enzymatic activity stems from interaction of the platinum complexes with the thiopeptolide and not with the enzyme. Generally, the thiopeptolide concentration was much greater than that of the platinum complex, therefore a mere reaction of the platinum complex with the thiopeptolide would have resulted in a reduction of the proteolytic activity much smaller than that observed. Furthermore, this contribution, even if present, would be negligibly small because it would be non-MMP-specific while we observe an inhibition activity which is different for different MMPs. The kinetic parameters vmax and KM were evaluated by using thiopeptolide substrate concentrations ranging from 31.25 to 2000 μM, either in the absence or in the presence of IC50


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concentrations of platinum complexes. Substrate conversion rates (v, pmol/min) were calculated from reaction rates (OD/ min) by using the equation: v = (reaction rate reaction volume)/ε l, where ε is the extinction coefficient of 2-nitro5-thiobenzoic acid (13600 M-1 cm-1), and l is the path length of light through the sample in cm. KM and vmax were calculated by using the software GraphPad Prism for nonlinear regression analysis (GraphPad Software, Inc., USA). Tumor Cell Lines and Cell Growth Inhibition Assay. A pair of ovarian cancer cell lines, A2780 (parent line from untreated patient) and A2780cisR (derived cisplatin-resistant subline), kindly supplied by the late Dr. L. Kelland (The Institute of Cancer Research, Surrey, UK), was used.47,48 A2780 and A2780cisR were maintained at 37 °C in a 5% CO2 humidified air in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, penicillin (100 U/mL), and streptomycin (0.1 mg/mL). The growth inhibitory effect of platinum complexes was evaluated by using the sulforhodamine B assay (46). Briefly, cells were seeded into 96-well microtiter plates (1000 cells/well, in 100 μL culture medium). After seeding, plates were incubated at 37 °C for 24 h prior to addition of the compounds. After this time, several samples were fixed in situ with cold trichloroacetic acid (TCA) to represent a measurement of the cell population at the time of compound addition. The compounds under investigation were freshly dissolved in culture medium and stepwise diluted to the desired final concentrations. After the addition of different compound concentrations to quadruplicate wells, the plates were further incubated at 37 °C for 96 h. Then, cells were fixed in situ by the gentle addition of cold 50% (w/v) TCA (50 μL, final concentration 10%) and incubated for 1 h at 4 °C. The supernatant was discarded, and the plates were washed 4 times with tap water and air-dried. Sulforhodamine B solution (100 μL, 0.4% w/v) in 1% acetic acid was added to each well, and plates were incubated for 30 min at room temperature. After staining, unbound dye was removed by washing 5 times with 1% acetic acid and the plates were airdried. Bound stain was then solubilized with trizma base (10 mM), and the absorbance was read on an automatic plate reader at 515 nm. The compound concentration able to inhibit cell growth by 50% (IC50 ( SD) was then calculated from semilogarithmic dose-response plots. Electrospray Mass Spectrometry. ESI-MS was performed with an electrospray interface and an ion trap mass spectrometer (1100 series LC/MSD trap system Agilent, Palo Alto, CA). The reaction between MMP-3 catalytic domain complexed with the inhibitor NNGH (20 μM) and [PtCl2(SMP)] complex (40 μM) was conducted at 25 °C in 5 mM ammonium acetate buffer (pH 6) containing 40 μM Zn(OAc)2 and 60 μM CaCl2. Aliquots of the reaction mixture were removed at different times after mixing and infused via a KD Scientific syringe pump at a rate of 10 μL/min. Acetic acid (1% v/v) was added before injection in order to obtain a good volatilization. Ionization was achieved in the positive ion mode by application of þ4 kV at the entrance of the capillary; the pressure of the nebulizer gas was 15 psi. The drying gas was heated to 350 °C at a flow of 5 L/min. Full-scan mass spectra were recorded in the mass/charge (m/z) range of 50-2200. Circular Dichroism. CD spectra were recorded at different time intervals on 20 μM protein solutions (in 5 mM ammonium acetate buffer containing 40 μM Zn(OAc)2 and 60 μM CaCl2) using a Jasco J-810 spectropolarimeter (Jasco Inc., Easton, MD), a quartz cuvette with a 1 mm optical path, a wavelength interval of 185-250 nm, and a 0.1 nm data pitch. The complex [PtCl2(SMP)] was added at a final concentration of 25 μM. Each spectrum corresponds to an average of 10 scans and was baseline corrected and then smoothed by applying adjacent averaging or an FFT filter. The ellipticity is reported as mean residue molar ellipticity (deg cm2 dmol-1) according to [θ]=100 3 [θ]obs/(CLN), where [θ]obs is the observed ellipticity in degrees, C is the molar concentration of the peptide, L is the optical path length


(in cm), and N is the number of amino acid residues in the protein (N=161). NMR Spectroscopy. NMR experiments were performed on 0.5 mM 15N-enriched MMP-3 catalytic domain complexed with the inhibitor NNGH at 25 °C. The interaction with 1 equiv of [PtCl2(SMP)] complex was monitored by 2D 1H,15N-edited HSQC spectra. The Pt(II) complex was dissolved in water 1 h before mixing with MMP-3. The spectra were collected on a Bruker Avance 600 spectrometer using a triple-resonance probe equipped with pulsed field gradients along the z axis. HSQC spectra were acquired using a gradient-enhanced sequence in which coherence selection and water suppression are achieved via gradient pulses. Twelve transients were acquired over an F2 (1H) spectral width of 13 ppm into 1024 complex data points for each of 128 t1 increments in TPPI mode with an F1 (15N) spectral width of 40 ppm centered at 118 ppm. The sequence was optimized with an INEPT delay 1/(4JNH) of 2.78 ms. A recycle delay of 1.0 s was used. Decoupling during the acquisition time was achieved using a GARP scheme. Data zero-filled in F1 were subjected to apodization using a squared cosine bell function in both dimensions prior to Fourier transformation and phase correction. Data were processed using the standard Bruker software (TOPSPIN) and analyzed through the programs CARA (The Computer Aided Resonance Assignment Tutorial, R. Keller, 2004, Cantina Verlag), developed at ETH-Zurich, and SPARKY 3 (T. D. Goddard and D. G. Kneller, University of California, San Francisco). Protein cross-peaks affected upon interaction with the Pt(II) complex were identified by comparing their intensities (I) with those of the same cross-peaks (I0) in the data set of sample lacking [PtCl2(SMP)]. The I/I0 ratios were plotted as a function of the protein sequence to obtain intensity profiles. Resonance assignment of MMP-3 complexed with NNGH was carried out by using available chemical shifts data49 with the aid of 2D TOCSY and NOESY and 3D 15 N-edited NOESY spectra. Chemical shift changes upon platination were reported as weighted average chemical shift differences Δδavg(HN) to account for differences in spectral widths between 1H and 15N resonances. Δδavg(HN) were calculated as previously described50 (i.e., Δδavg(HN) = [(ΔδH2 þ (ΔδN/5)2)/2]1/2, where ΔδH and ΔδN are chemical shift differences for 1H and 15N, respectively) and plotted as a function of the protein sequence. Modeling. A structural model of the adduct between MMP-3 and Pt-SMP was obtained with the minimization protocol of the program PSEUDODYANA,51 starting from the available X-ray structure of the catalytic domain of MMP-3 (1SLN).52 The Pt-SMP complex was introduced in the PSEUDODYANA library as a nonstandard residue using the available X-ray coordinates of the K[PtCl3(SMP-S)] complex having monodentate SMP and detached phosphonate group29 and defining nine rotatable dihedral angles for the SMP ligand. Structure calculations were performed by linking the platinum ion to an imidazole nitrogen of His224 and constraining the metal-ligand bond length in the range 1.9-2.1 A˚. In addition, a hydroxyl and a chloride ion were linked to platinum, at 2.0 and 2.3 A˚ respectively, and the platinum geometry was imposed to be square planar. Model analysis and display were performed with the program MOLMOL.53

Acknowledgment. We thank Prof. Ivano Bertini and the CERM Research Infrastructure of the University of Florence for providing access to the NMR facilities. We also thank Dr. Francesca Cantini and Dr. Simone Scintilla for assistance in the recording of NMR and CD spectra, respectively. We are grateful to the University of Bari, the Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), the Italian Ministero dell’Universit a e della Ricerca (PRIN 2005 032730), and the European Commission (COST Action D39) for support.

Arnesano et al.

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