Mithramycin: a very strong metal chelating agent

59

Biochimica et Biophysica Acta, 1158 (1993) 59-64 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-4165/93/$06.00

BBAGEN 23839

Mithramycin: a very strong metal chelating agent Cynthia Demicheli and Arlette Garnier-Suillerot Laboratoire de Chimie Bioinorganique, LPCB (URA CNRS 198) Universit~Paris Nord, Bobigny (France) (Received 11 January 1993)

Key words: Mithramycin; Chelating agent; Circular dichroism; Metal chelator

The interaction of mithramycin (MTR) with Ca2+,Cd 2+, Tb 3+, Gd 3+, Li +, Na ÷ and K ÷ ions has been studied by circular dichroism and absorption spectroscopy. Mithramycin binds strongly to Ca 2+, Cd 2+, Tb 3+ and Gd 3+ forming a 1:4 Ca 2+ :MTR entities with a left-handed screw conformation. The concentration of Ca 2+ present in water currently used being about 10/zM, this leads to the conclusion that, in most of the experiments reported in the literature, about 40/zM mithramycin were actually bound to Ca 2÷. Mithramycin also binds to Na +, forming entities with left-handed screw conformation, but not to K ÷ and Li ÷. None of these cations were able to promote the mithramycin-DNA interaction.

Introduction

Mithramycin (MTR; scheme 1) is an antibiotic that belongs to the aureolic acid group and is closely related to chromomycin h 3 [1]. Mithramycin has antitumor properties and also a calcium-lowering effect that is independent of its tumoricidal activity [2]. The hypocalcemic action is an undesirable side effect in the therapy of normocalcemic cancer patients but a desirable effect in treating humoral hypercalcemia of maligoN

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Correspondence to: A. Garnier-Sufllerot, Laboratoire de Chimie Bioinorganique, Universit~ Paris Nord, 74 rue Marcel Caehin, 93012 Bobigny, France; Fax (33) (1) 48 38 77 77. Abbreviations used: MTR, mithramycin; C-'D, circular dichroism; EGTA, ethyleneglyeol-b/s-(tg-aminoethyl ether) N,N,N',N'-tetraacetic acid,

nancy [3-5]. Mithramycin appears to inhibit bone resorption [2,6]. Mithramycin inhibits I)NA-dependent R N A synthesis by forming complexes with DNA in the presence of Mg2÷[7-11]. It has also been shown that Zn 2÷, not only can replace Mg 2÷ but is even more efficient to promote the binding of mithramycin to DNA and to polynucleotides [10,11] Recently, the lack of interaction between mithramycin and Ca 2÷ has been reported [12]. However, in the course of our studies on the type of metal ions able to promote the interaction of mithramycin with DNA [11], polynucleotides [13] and membrane, we found that, in most cases, mithramycin in aqueous solution was actually bound to Ca :+ present in the solution. This prompted us to carefully study the interaction not only with calcium but also with other cations such as Li +, K +, Na ÷, Cd 2+, Tb 3+ and Gd 3+ and to determine whether these cations were able to promote the interaction of mithramycin with DNA. We report here that mithramycin strongly binds Ca 2+, Cd 2÷, T b 3+ and Gd 3+ forming 1:4, M n+ : M T R entities with a left-handed screw conformation. In addition, mithramycin is also able to bind Na ÷ but not K ÷ and Li ÷. Contrarily to what is observed with Zn 2+ and Mg 2+ these cations were not able to promote the interaction of mithramycin with DNA. Materials and Methods

Mithramycin was a gift from Pfizer, USA and used without further purification. Concentrations were determined spectroscopically using an absorption coeffi-

60 cient of 10000 M - l cm -1 at 400 nm. CaCI2, NaCI, KC1 LiCI (Normatom grade) were purchased from Prolabo, TbCI 3 and GdCI 3 were from Aldrich. Ethyleneglycolb/s-(/3-aminoethyl ether) N,N,N',N'-tetraacetic acid ( E G T A ) was from Sigma, Chemical Co., St. Louis, MO, USA. Aqueous solutions of these cations were prepared and their concentrations determined using E D T A titration [14]. The water used for the solutions was first distilled and then passed through a Milli-Q reagent water system (Millipore Co). It was either used like that or further purified by treatment with chelex 100, chelating ion exchange resin (Bio-Rad Co.). High-molecular-mass calf thymus D N A was purchased from Sigma and dissolved in Hepes buffer at p H 7 for 3 h under vigorous stirring. A nucleotide absorption coefficient of 6600 M-~ • cm-1 was used to calculate D N A concentrations from absorbance measurements at 260 nm. Absorption spectra were recorded on a Cary 219 spectrophotometer and circular dichroism (CD) on a Jobin Yvon dichrograph Model Mark V. Results are expressed in terms of • (molar absorption coefficient) and Ae = e L - - E R (molar CD coefficient). The values of e and Ae are expressed in terms of [MTR], the molar concentration of mithramycin. Results and Discussion The absorption spectrum of mithramycin exhibits strong bands at about 400, 300, 280 and 230 nm. The band at 280 nm can be assigned to 1,4 ~ IB b transition [15]. In most cases, the CD spectrum exhibits, in this region, two Cotton effects of opposite signs. The summation of the amplitude of the two Cotton effects gives a A value: A = zae 1 - A E 2 where AE1 and AE 2 a r e the amplitudes of the Cotton effects at the longer and shorter wavelengths respectively [16]. The presence of the couplet type signal reveals the existence of a molecular association and the A value defines its chirality. Usually, the CD spectrum of mithramycin at pH > 8 exhibits a signal of the couplet type characteristic of a molecular association in the left-handed screw conformation [11,13]. The mithramycin molecule being once negatively charged at this pH, it was rather surprising to observe such an association. For this reason, we suspected the presence of cations bound to mithramycin and, to test this hypothesis we studied the interaction of mithramycin with the chelating agent EGTA.

Mithramycin in the presence of EGTA At low concentration (i.e., [MTR] < 10/xM) a solution of mithramycin in Milli-Q treated water, in the presence of 0.1 M KC1, 0.05 M Hepes buffer at p H 8.1, exhibited a CD spectrum of the couplet type (A =

'E o

6.o 30

O.C

-

×

30

30o

3~o Wavelength

46o

4~0

(rim)

Fig. 1. Circular dichroism spectra of mithramycin in chetex 100treated water. [MTR] 2 /zM ( ); 250 /zM ( - 0 " ); T = 25°C; pH = 8.5

- 106) in the 280 nm wavelength region. The addition of increasing amount of E G T A yielded a decrease of the signal and the appearance of a positive band at 275 nm (Ae = + 10), characteristic of a monomeric species. Such behavior strongly suggested the presence of cations promoting mithramycin molecular association and this was further attested by the following experiments. In the previous experiment, the water used was first distilled and then passed through a Milli-Q reagent water system. To further improve the water purity, it was treated by chelex 100 resin. Using this water, the CD spectra of mithramycin solutions of different concentrations were recorded. The spectra thus obtained were concentration dependent. As it can be seen in Fig. 1, at low concentration (2/zM), the CD spectrum in the 250-330 nm region exhibits a signal of the couplet type (A = - 1 0 6 ) , whereas at high concentration (250 /zM), only one positive band (AE = + 10) is present. These data strongly suggest that low concentrations of cations able to promote the mithramycin interaction are still present in chelex 100-treated water.

Interaction of mithramycin with Ca 2+ The ability of chelex 100 resin to remove ions from solution depends on the metal ion nature. Its affinity for Ca 2÷ being one of the lowest, the presence of small amount of Ca 2÷, in the chelex 100-treated water, could be expected. To check this point, increasing concentrations of Ca 2+ ions were added to a 250/zM mithramycin solution. As can be seen in Fig. 2, at this concentration and in the absence of Ca 2+, the CD spectrum in the UV region exhibited one positive band only, characteristic of mithramycin in the monomeric statel The addition of increasing amounts of Ca 2+ ions to this solution gave rise to the appearance of a CD signal of the couplet type (A = - 1 0 6 ) , corresponding to the spec-

61 calcium-mithramycin and calcium-EGTA complexes and the total calcium concentration was then.

trum observed at low mithramycin concentration (Fig. 1). We have plotted Ae at 275 nm as a function of the molar ratio of Ca 2÷ to mithramycin (Fig. 2 inset). The CD signal increased as the molar ratio was varied from 0 to 0.25 and then plateaued for higher values. We can infer: (i) that a 1 : 4 Ca 2÷ :MTR complex, [Ca (MTR)4] 2- is formed, (ii) that the spectrum observed at low mithramycin concentration in chelex 100-treated water is due to the presence of Ca 2÷ ( ~ 2/~M). In the absence of Ca 2+, the visible absorption spectrum exhibits one symmetric band at 410 nm which shifts to 400 nm and becomes asymmetric in the presence of Ca 2+ (Fig. 2). In order to have an estimation of the stability constant of this complex, EGTA was stepwise added to a solution containing 100 mM mithramycin and 25 t~M Ca 2÷, at pH 8.1, in the presence of 0.1 M KCI, at 25°C. In these conditions, in the absence of EGTA, the [Ca(MTR)4] 2- was present. The addition of 600 /~M EGTA was required to dissociate 50% of the complex.

[Ca 2+ ]T = [Ca E G T A ] 2 - + [Ca(MTR)4] 2-

On the other hand, at pH 8.1 most of the mithramycin molecules were once negatively charged and the concentration of free drug in the neutral form could be neglected as compared to that of free drug in the deprotonated form. It follows that the total mithramycin concentration was [MTR]T = [ M T R - ] + 4[Ca(MTR)4- ]2-

The free and complexed mithramycin concentrations, [MTR-] and [MTR]c, respectively, were calculated using CD data. The two other terms were easily calculated: [Ca EGTA] 2-

The following equilibria were taken into consideration Ca 2÷ + 4 M T R - ,

[ M T R ] c / 4 and [ E G T A 4- ]

= [ E G T A ] T - [Ca EGTA] 2-

, [Ca(MTR)4] 2-

At pH 8.1, the stability constant K of the [Ca(EGTA)] 2- complex being equal to 3.2.10 8 [17], we can estimate that the stability constant /3 for the equilibrium 4 M T R - + C a 2+ ~ > [Ca(MTR)4] 2- is equal to 1.2.10 21 .

/3 = [ C a ( M T R ) 4 ] 2 - / [ C a 2+ ]" [ M T R - ]4 Ca 2+ + E G T A 4 - ,

= [Ca]T--

, [Ca E G T A ] 2-

K = [Ca E G T A ] Z - / [ C a 2+ ]" [ E G T A 4- ] [Ca E G T A ] 2-" [ M T R - ] 4 / [ E G T A 4 - ]. [Ca(MTR)4] 2-

Interaction o f mithramycin with the monovalent cations Li + Na + and K +

Under our experimental conditions, the free calcium concentration was negligible as compared to those of

As most of the experiments were performed either in the presence of KCI or NaCI, we have checked

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250

350

400 Wavelength

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Fig. 2. Circular dichroism (left) and absorption (right) spectra of mithramycin in chelex 100-treated water in the presence of various amounts of Ca 2~-. [MTR] ffi 250 ~ M ; T ffi 25°C; pH ffi 8.1; [Ca 2+ I / [ M T R I ffi 0 ( -a- ); 0.07 (--~); 0.15 ( ~ ); 0.25 ( -o- ); 0.5 ( ). Inset: Ae at 275 nm has been plotted as a function of the molar ratio of Ca 2+ to mithramycin.

62

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Fig. 3. Circular dichroism spectra of mithramycin, in chelex 100treated water, in the presence of increasing amount of Na +. [MTR] =450 mM, T=25°C; p H = 8 . 1 ; [ N a + ] / [ M T R ] = 0 ( ); 150 ( -i- ); 400 ( -e- ); 800 ( ~ ). Inset: Ae at 275 nm has been plotted as a function of the molar ratio of Na + to mithramycin.

whether these cations were able to promote the mithramycin molecules association. The addition of KCI, up to to 0.1 M, to a chelex 100-treated aqueous solution of 450 /~M M T R at p H 8.1 did not give rise to modification of the CD signal which exhibited the positive band characteristic of the monomeric form, in the 250-330 nm region. However, when similar experiments were performed with NaCI instead of KCI, one observed the appearance of a CD signal characteristic of an association in the left-handed screw conformation (A = - 5 7 ) (Fig. 3). AE at 275 nm has been plotted as a function of the molar ratio of Na ÷ to mithramycin (Fig. 3, inset) and a plateau was reached for [ N a + ] / [ M T R ] ~ 300 The main difference between Na ÷ and K + lies at the level of their ionic radii, the ionic radius of Na ÷ being 0.095 A and that of K + 1.33 A. To determine if the ability of a cation to promote the mithramycin molecular association was related to its ionic radius value, we have studied the interaction of mithramycin with Li ÷. As in the case of K ÷, we did not observe any modification of the spectral pattern of mithramycin when Li ÷ was added. These data strongly suggest that the ionic radius value is of key importance for cation binding to mithramycin. Interaction o f mithramycin with Tb 3 + and Gd 3 + ions

It is well known that many tripositive lanthanide (Ln 3+) make excellent probes for Ca 2÷ and that (i) in forming complexes, both Ca 2÷ and L n 3 + prefer charged or uncharged oxygen donors to nitrogen donors (ii) Ca 2÷ and Ln 3+ exhibit similar and variable coordina-

tion numbers and lack strong directionality in binding donor groups [18]. Therefore, we have studied the interaction of mithramycin with two elements of the lanthanide group: Tb 3+ and G d 3+. As at p H > 6.5, hydroxo complex formation of Ln 3 ÷ begins to occurs, our experiments were performed at pH 6 in chelex 100-treated water. Tb 3+ (or G d 3+) ions were stepwise added to a 250/xM mithramycin buffer solution (0.1 M KC1 and 0.05 M acetate buffer). In the UV region the CD spectrum evolved from that characteristic of the monomeric species with one positive band at 275 nm to that of a couplet type (A = - 5 5 ) . A shift of the visible absorption band to longer wavelength was also observed. Ae at 282 nm has been plotted as a function of the molar ratio of T b 3 ÷ / M T R ( G d 3 ÷ / M T R ) (data not shown) and as in the case of the C a 2 ÷ / M T R system we observed an increase of the CD signal which plateaued for values higher than 0.25. We inferred that a 1:4 Tb 3÷ : M T R (Gd 3÷ : M T R ) complex was formed. The spectroscopic data are reported in Table I. In order to compare the stability of the Ca 2÷ : M T R and Tb 3+ : M T R complexes, Tb 3÷ was stepwise added to a 2 ~ M mithramycin solution in the presence of 20 /~M Ca 2+. Despite the presence of this large excess of calcium, a ratio of 1 Tb 3+ per 4 M T R was sufficient to displace the Ca 2÷ ions from its coordination site (Fig. 4). Interaction o f mithramycin with C d 2 + ions As C d 2+ has the same ionic charge and the same

ionic radius as Ca 2÷ there was strong probability that Cd 2+ could bind to mithramycin. Under experimental conditions strictly analogous to that used to follow the C a 2 ÷ / M T R interaction, we observed the formation of a 1:4, Cd 2÷ : M T R complex (Table I). However, the addition of a 40 fold excess of Cd 2÷ ions to 2 /.~M mithramycin in the presence of 20 /zM Ca 2÷ did not give rise to a displacement of Ca 2 ÷ from its coordination site to mithramycin.

TABLE I

Circular dichroism and absorption spectroscopic data of mithramycin and its metal ions complexes. CD MTR[Ca(MTR)4] 2[Cd(MTR) 412[Tb(MTR) 4][Na(MTR)n](n- 1)-

Absorption

~i2(Ae 2)

Al(Ae 1)

A

A(E)

275(10) 275( + 62) 276(+ 50) 282( + 32) 275(+ 35)

295(297(299(291(-

- 106 - 88 - 55 - 57

410(6100) 400(6000) 400(6200) 422(8500) 410(6500)

44) 38) 23) 22)

Data related to the CD signal in the 250-350 nm range and the absorption in the visible range have been reported only. A, e and Ae are in M - 1. c m - 1.

63

The need of Mg 2÷, which can be replaced by Zn 2+, to promote the mithramycin-DNA interaction is now well-documented [7-11]. To check if Ca 2+, Cd 2÷, Tb 3÷, Gd 3÷, Na ÷ cations were able to promote the binding of mithramycin to DNA, the following experiments were performed: DNA was stepwise added to a solution containing the M "+ :MTR complex ([MTR] --- 100 /.tM, [M"+]=25 ~M, [KCI] = 0.1 M pH=8.1). No modification of the CD spectra of the complexes was observed when the molar ratio of DNA (base pairs) to mithramycin was increased up to 50 indicating the lack of mithramycin-DNA interaction.

the case of studies performed at low mithramycin concentrations. This explains why, recently, no spectral modification of a 50/zM solution of mithramycin was observed through Ca 2+ addition [12]. From a biological point of view, it is clear that mithramycin will be transported in the biological fluids complexed to Ca 2÷ and Na + cations. On the other hand, the mechanisms of action of mithramycin as a hypocalcemic agent has never been elucidated and the observation that mithramycin has so high affinity for Ca 2÷ should open new routes for further investigations. At this point, it is interesting to remind the data which have been obtained with Mg 2÷ and Zn 2+. Depending on the molar ratio of Mg 2÷, or Zn 2+, to mithramycin, two different complexes are observed: at low molar ratios a complex (I) with a left-handed screw conformation is obtained whereas, at high molar ratios, a complex (II) with a right-handed-screw conformation is formed [19]. At a 1 : 2 metal to ligand stoichiometry, these two complexes are able to interact with macromolecular systems such as DNA and polynucleotides and in that case the right-handed complex is involved [11,13] or with small unilamellar vesicles and the lefthanded complex is then involved. The interaction of the cations tested in the present study with mithramycin is different of those of Mg 2+ and Zn2+: they are able to form one type of complex only (1:4, M "+ :MTR)which has a left-handed screw conformation. The common characteristic of these cations is the ionic radius which has a value close to 1A, the cationic charge being not a determinant factor for the coordination. In addition, these cations are not able to promote the binding of mithramycin with DNA.

Discussion

Acknowledgement

Despite the fact that mithramycin has been for a long time recognized as a metal chelating agent and that its.affinity for Mg 2÷ [7-11, 19] and Zn 2÷ [10,11] have been reported by several authors, up to now its strong affinity for Ca 2+ and to a lesser extent for Na +, which are abundant in biological fluids, have been ignored. Our data clearly demonstrate that mithramycin is a very strong Ca 2+ chelating agent. This point is very important when low mithramycin concentrations are used (1-10/~M) because, under these conditions, even in highly pure water (chelex 100-treated) ~ 1-2 /zM Ca 2÷ is still present and is able to complex ~ 4-8/~M mithramycin. In Milli-Q treated water, which is the most commonly used, the Ca 2÷ concentration is higher ( ~ 1 0 /zM) and up to ~ 4 0 /~M MTR should be complexed to Ca 2÷. This means that most of the data reported in the literature do not refer to free mithramycin but to the [Ca(MTR)4] 2- complex, specially in

This work was supported with grants from Universite Paris Nord, CNRS and Institut Curie. C.D. is grateful to CAPES (Brazil) for a fellowship.

4C

2.C

x

O.C

-2C

240

260

280 300 320 Wavelength (rim)

340

Fig. 4. Circular dichroism spectra of mithramycin in the presence of inreasing amount of Tb 3+. [MTR] = 2/zM, [Ca 2+ ] = 20 ~M, [KCI] = 0.1 M, pH=8.1, T=25°C; [Tb3+]/[MTR]=0 ( ), 0.07 ( O ), 0.14 (-e-), 0.25 ( -~- ), 0.33 ( -~- ), 0.5 and 1 ( ).

Interaction of mithramycin with DNA in the presence of the different cations

o

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