Reactions of PtII diamine anticancer complexes with trypanothione and octreotide

JOURNAL OF

Inorganic Biochemistry Journal of Inorganic Biochemistry 100 (2006) 1946–1954 www.elsevier.com/locate/jinorgbio

Reactions of PtII diamine anticancer complexes with trypanothione and octreotide Vivienne P. Munk, Sarah Fakih, Piedad del Socorro Murdoch, Peter J. Sadler

*

School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, UK Received 28 June 2006; received in revised form 23 August 2006; accepted 24 August 2006 Available online 5 September 2006

Abstract The products formed in reactions of the square-planar platinum(II) anticancer complexes, [Pt(en)Cl2] and [Pt(R,R-dach)Cl2] where en = ethylenediamine and dach = diaminocyclohexane, with trypanothione, a glutathione analogue found in some parasites, and octreotide, a synthetic analogue of the hormone somatostatin, have been investigated. Mononuclear and binuclear platinum adducts were formed in reactions of the cyclic disulfides in their oxidised and reduced forms, and were analysed by UV–visible spectroscopy and liquid chromatography–mass spectrometry (LC–MS). NMR and molecular modelling studies were carried out on the mononuclear adducts.  2006 Elsevier Inc. All rights reserved. Keywords: Trypanothione; Octreotide; Platinum diamine; HPLC; Anticancer; NMR

1. Introduction The anticancer activity of platinum complexes has been extensively investigated since the 1960s [1]. Cisplatin is one of the most successful drugs used in the clinic for the treatment of testicular, bladder, lung and ovarian cancers [2]. Many second and third generation platinum drugs, such as oxaliplatin, [Pt(1R,2R-dach)(oxalate)], have been used to circumvent some of the side-effects of cisplatin [3]. Despite the success of cytotoxic platinum complexes, reactions of intracellular thiols, such as glutathione (GSH), which is found in relatively high concentrations in cells (up to 10 mM) [4], are believed to deactivate the drugs by preventing them reaching their biological target, DNA [5–8]. We have investigated reactions of [Pt(en)Cl2] (en = ethylenediamine) 1, and [Pt(1R,2R-dach)Cl2] (dach = diaminocyclohexane) 2, with GSH and oxidised GSH (GSSG). These platinum complexes can cleave the disulfide bond of glutathione disulfide, giving rise to adducts of glutathione itself (even when the disulfide is the reactant), a sulfur-bridged *

Corresponding author. Tel.: +44 131 650 4729; fax: +44 131 650 6453. E-mail address: [email protected] (P.J. Sadler).

0162-0134/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2006.08.011

dimer, and a novel macrochelate with the formula [(Pt(Am))2SGH1] (where Am = amine) [9,10]. In this current study, we have investigated whether such anticancer complexes can reductively cleave disulfide bonds in cyclic peptides by isolating and characterising adducts with the biologically important disulfide bridged peptides trypanothione, [N1,N8-bis(glutathionyl)spermidine] 3, and octreotide, 4 (see Fig. 1). Trypanothione disulfide, TS2, is a glutathione analogue which was first identified and isolated from the pathogenic protozoa Trypanosoma and Leishmania by Fairlamb et al. in 1985 [11,12]. Trypanothione is unique to trypanosomatids and acts as the functional equivalent of glutathione in these organisms by playing a role in intracellular protection against oxidative stress [13–16]. Trypanosomes have been identified as the causative agents of many tropical diseases, such as African sleeping disease, Chagas disease, Nagana cattle disease, Kala-azar and Oriental sore [17]. Octreotide, also known as Sandostatin, is a synthetic analogue of somatostatin, a hormone which is widely distributed within the body and used as a treatment for endocrine, gastrointestinal and immune system disorders [18–20]. However, due to unfavourable pharmacokinetics

V.P. Munk et al. / Journal of Inorganic Biochemistry 100 (2006) 1946–1954 NH2

NH2

Cl

Pt NH2

Cl

NH2

(1) Glu(1)

Gly(1)

O

H N

H' N N H

-OOC O

O +H2N O

S S

O

H N

-OOC N H

NH3+

Glu(2)

Cl

(2) Cys(1)

NH3+

Cl Pt

Spermidine

N H''

O

Cys(2)

Gly(2)

(3) TS2

D-Phe1

Phe3

2.3. Preparation of samples

O

H N

H N

+H3N

N H

O

S O

H N

HO

Thr(ol)8

HN H N

N H

O

NH3+ O

HO

Cys7

N H

O

S

HO

˚, 250 nm, and a RP C18 column (250 · 4.6 mm, 100 A 5 lM, Hichrom). A gradient of acetonitrile (+0.1% trifluoroacetic acid) in water (+0.1% trifluoroacetic acid) was applied. Positive ion electrospray mass spectrometry (ESI-MS) was performed using a Platform II mass spectrometer (Micromass, Manchester, UK). A Corning 240 pH meter equipped with an Aldrich microcombination electrode standardised with Aldrich buffers at pH 4, 7 and 10 was used to make pH measurements. 1D and 2D 1 H NMR spectra were recorded in 5 mm NMR tubes on either Bruker DMX 500 MHz (for 1H), Bruker AVA 600 MHz or Bruker AVA 800 MHz spectrometers, using dioxane (d(1H) 3.76 ppm) as an internal standard. Molecular modelling was carried out using SYBYL (version 6.9, Tripos). The Tripos force-field was used to energy-minimise models to a constant energy.

D-Trp4

Cys2

O

1947

Lys5

Thr6 (4) OctS2

Fig. 1. Structures of (1) [Pt(en)Cl2], 1, (2) [Pt(R,R-dach)Cl2], 2, (3) trypanothione disulfide (TS2) and (4) octreotide disulfide (OctS2).

and selectivity, the usefulness of somatostatin is limited [21,22]. Octreotide has been shown to be up to 70 times more potent than somatostatin in the inhibition of growth hormone [23] and is effective in the treatment of certain cancers, namely endocrine tumours and breast and prostate cancers [24,25]. 2. Experimental 2.1. Materials [Pt(en)Cl2], 1, and [Pt(R,R-dach)Cl2], 2, were synthesised and characterised as previously described [9,10]. Trypanothione disulfide (N1,N8-bis(glutathionyl)-spermidine disulfide, TS2) was purchased from Bachem. Octreotide disulfide (OctS2) was a gift from Novartis. All other chemicals were purchased from Aldrich. 2.2. Instrumentation Electronic absorption spectra were recorded over the range 800–200 nm at 310 K on Perkin Elmer Lambda-16 or Shimadzu 250UV spectrometers, using a 1 cm path length cell. Liquid chromatography was carried out using a Waters Separation Module Alliance 2795 pump, a Waters 486 tuneable absorbance detector at 240 or

TS2 and OctS2 were readily reduced by tris(2-carboxyethyl)phosphane and purified using HPLC techniques [26–28] to yield reduced trypanothione, T(SH)2, and reduced octreotide, Oct(SH)2. The formation of the dithiols was confirmed by mass spectrometry, and Ellman’s reagent [29] was used to determine the thiol concentration of the solutions. Reactions of 2 with T(SH)2 and TS2, and of 1 with Oct(SH)2 and OctS2 were studied. In each case, the pH of the peptide solution was adjusted to 7 with NaOH. Freshly dissolved platinum complex in water was added to 0.5 mol equiv of peptide (pH 7), and the reaction mixtures were incubated at 310 K for 24 h or 7 d. The reactions were monitored by UV/vis, and the reaction products were analysed by LC–MS and NMR. Extinction coefficients were calculated based on the total concentration of platinum. 3. Results 3.1. UV–visible (UV/vis) spectroscopy The reaction of 0.5 mol equiv of oxidised trypanothione TS2 (1 mM, 300 lL, pH 7) with 1.0 mol equiv [Pt(R,Rdach)Cl2], 2, (1 mM, 600 lL, pH 7) in water at 310 K was followed over time using UV/vis spectroscopy (Fig. 2a). One intense band appeared at 240 nm (De = ca. 1950 M1 cm1) and a second weaker peak at 325 nm (De = ca. 200 M1 cm1). The intensity of these peaks increased with time, and the reaction was complete after ca. 30 h. The reaction of 0.5 mol equiv of the reduced peptide T(SH)2 (50 lM, 400 lL pH 7) with 2 (100 lM, 400 lL pH 7) was also monitored, and the resulting difference spectrum is shown in Fig. 2(c). Due to the low concentration of the reduced peptide, the concentration of reactants used in this reaction was lower than those used for the reaction of the disulfide. The intensities of bands at 228 nm (De = ca. 6600 M1 cm1) and at

1948

V.P. Munk et al. / Journal of Inorganic Biochemistry 100 (2006) 1946–1954

600 lL, pH 7) in water at 310 K was followed over time by UV/vis spectroscopy (Fig. 2b). This reaction was significantly slower than the reaction of 2 with TS2. New bands appeared at 250 nm (De = ca. 2950 M1 cm1) and 330 nm (De = ca. 320 M1 cm1) which increased in intensity with time, and the reaction appeared to reach equilibrium after ca. 140 h. Analogous reactions of reduced peptide, Oct(SH)2, (75 lM, 400 lL pH 7) with 1 (150 lM, 400 lL pH 7) were also monitored by UV–vis spectroscopy. The resulting difference spectra are shown in Fig. 2(d). The intensity of the new bands at 233 nm (De = ca. 10,000 M1 cm1) and 333 nm (De = ca. 300 M1 cm1) increased with time. The reaction appeared to reach equilibrium after ca. 25 h. 3.2. Liquid chromatography–mass spectrometry (LC–MS) TS2 was readily reduced to form T(SH)2 by reaction with tris-(carboxyethyl)phosphane. Both the oxidised and reduced peptides were characterised by mass spectrometry (Figs. 3a and 4a). The mass spectrum for TS2 showed a cluster of ions at m/z (+1) 722.2, consistent with oxidised trypanothione, ([C27N9O10S2H48]+, {TS2}+, calc. m/z (+1) 722.0), and that of T(SH)2 a cluster of ions at m/z (+1) 724.2, consistent with reduced trypanothione, ([C27N9O10S2H50]+, {T(SH)2}+, calc. m/z (+1) 724.0), Fig. 3a. Similarly, the mass spectrum of OctS2 showed a cluster of ions at m/z (+2) 510.5, consistent with oxidised octreotide, ([C49N10O10S2H68]2+, {OctS2}2+ calc. m/z (+2) 510.2) and the MS of Oct(SH)2 showed a cluster of ions at m/z (+2) 511.3, consistent with the expected mass of the reduced octreotide, ([C49N10O10S2H70]2+, {Oct(SH)2}2+, calc. m/z (+2) 511.2), Fig. 4a. The disulfides TS2 and OctS2 and dithiols T(SH)2 and Oct(SH)2 were incubated with platinum complexes [Pt(R,R-dach)Cl2], 2, and [Pt(en)Cl2], 1, respectively, for 7 d at 310 K and the reaction mixtures were analysed by LC–MS. The following products were prepared and characterised by LC–MS techniques. TðSHÞ2 þ 2 ! A þ B TS2 þ 2 ! C þ D OctðSHÞ2 þ 1 ! E þ F OctS2 þ 1 ! G þ H

Fig. 2. Difference UV–vis spectra showing the increase in absorbance with time for aqueous reactions of (a) 2 (1 mM, 600 lL) with 0.5 mol equiv of TS2, (b) 1 (1 mM, 600 lL) with 0.5 mol equiv of OctS2, (c) 2 (100 lM, 400 lL) with 0.5 mol equiv of T(SH)2 and (d) 1 (150 lM, 400 lL) with 0.5 mol equiv of Oct(SH)2. Measurements were taken at regular intervals at 310 K.

325 nm (De = ca. 1000 M1 cm1) increased with time, and the reaction appeared to have reached equilibrium after 2 h. The reaction of 0.5 mol equiv of oxidised octreotide, OctS2, (1 mM, 300 lL, pH 7) with [Pt(en)Cl2], 1, (1 mM,

The reverse-phase HPLC traces for the reaction mixture of TS2 and 2 and OctS2 and 1 (both reactions initially at pH 7, 310 K, 7 d) are shown in Fig. 5. Some unreacted platinum complex was still present in both reactions after 24 h. For the reaction of TS2 and 2, peaks for two major products containing Pt were observed, the first eluting at ca. 16 min. (peak C), and the second at 19 min (peak D), with a peak area ratio of ca. 0.3:1. Products with similar retention times were also isolated from the reaction products of T(SH)2 and 2, peaks denoted as A and B, respectively. The chromatogram for the reaction products of OctS2+1 also showed two major peaks with a peak area ratio of 0.3:1,

V.P. Munk et al. / Journal of Inorganic Biochemistry 100 (2006) 1946–1954

1949

511.30

724.2

511.79

725.1

512.23

722.2

510.48

511.04

723.2

511.54

715

720

725

730

m/z 505

510

520

515

Modelled (m/z +2) Pt2C39H75N13O10S2

671.2

Modelled (m/z +2) Pt2C53H82N14O10S2

764.3

672.7 670.2 673.2

763.8 765.3

Observed Peak A

671.0

m/z

764.5

Observed Peak E

670.8 672.4 765.6 764.2

516.7 516.2

Modelled (m/z +2) PtC33H63N11O10S2

517.7

638.2

Modelled (m/z +2) PtC51H76N12O10S2

638.8

516.6 516.1

Observed Peak B 638.5

517.3

Observed Peak F

639.0

500 550 600 650 700

670.2

m/z Modelled (m/z +2) Pt2C39H73N13O10S2

620 660 700 740 780 763. 3 762.8

669.7 671.2 671.7

670.0

763.4

Observed Peak C

Observed Peak G 762.5

670.4

761.8

Modelled (m/z +2) PtC33H61N11O10S2

515.6

Modelled (m/z +2) Pt2C53H80N14O10S2

762. 3 765. 3

669.4

515.1

764.8

m/z

637.2

516.6

Modelled (m/z +2) PtC51H74N12O10S2

636.2

515.7

Observed Peak D

514.9

637.2

516.8

Observed Peak H 636.4

500

550

600

650

700 m/z

Fig. 3. (a) Mass spectra of T(SH)2 and TS2, (b) mass spectra of the two major reaction products separated by HPLC from the reaction of 2, initially at pH 7, 310 K, 7 d with 0.5 mol equiv of T(SH)2 for 7 d (peaks A and B) and (c) with 0.5 mol equiv TS2 under similar conditions (peaks C and D). In each case, the upper spectrum is that the first peak eluted by the HPLC, and the lower spectrum that of the second peak eluted. The observed and modelled isotope splitting patterns are shown for the two products formed in each reaction i.e. (b) 1Pt:1 T(SH)2 and 2Pt:1 T(SH)2 and (c) 1Pt:1TS2 and 2Pt:1TS2.

the first eluting at ca. 7 min (peak G) and the second at ca. 8 min (peak H). Products with similar retention times were also eluted from the reaction of Oct(SH)2 with 1 (peaks denoted as E and F).

620 660 700 740 780

m/z

Fig. 4. (a) Mass spectra of Oct(SH)2 and OctS2, (b) mass spectra of the two major reaction products separated by HPLC from the reaction of 2, initially at pH 7, 310 K, 7 d with 0.5 mol equiv of Oct(SH)2 for 7 d (peaks E and F) and (c) with 0.5 mol equiv OctS2 under similar conditions (peaks G and H). In each case, the upper spectrum is that the first peak eluted by HPLC, and the lower spectrum that of the second peak eluted. The observed and modelled isotope splitting patterns are shown for the two products formed in each reaction i.e. (b) 1Pt:1Oct(SH)2 and 2Pt:1Oct(SH)2 and (c) 1Pt:OctS2 and 2Pt:1OctS2.

Mass spectra of the two separated platinated products formed in the reaction of 2 with T(SH)2 showed clusters of ions at m/z 671.0 for peak A and at m/z 516.6 for peak

1950

V.P. Munk et al. / Journal of Inorganic Biochemistry 100 (2006) 1946–1954 Table 1 Observed and calculated mass spectral peaks for reactants and HPLC isolated products from the reactions of 1 or 2 with the cyclic disulfides in their oxidised and reduced forms

2

D

Sample

A240 TS2

C

10

15

20

Retention time (min) Solvent front

1

A250

OctS2

H G

5

T(SH)2 TS2 Oct(SH)2 OctS2 1 2 T(SH)2 + 2 T(SH)2 + 2 TS2 + 2 TS2 + 2 Oct(SH)2 + 1 Oct(SH)2 + 1 OctS2 + 1 OctS2 + 1 a

10

15

20

Retention time (min) Fig. 5. HPLC trace of the reaction of (a) 0.5 mol equiv TS2 and 2 and (b) 0.5 mol equiv OctS2 and 1 (initially at pH 7) for 7 d at 310 K. Unreacted Pt complex and peptide are still present in the reaction mixture, but, two new peaks for products in each reaction are observed.

Peaka

A B C D E F G H

Observed m/z

Formula

Calculated m/z

724.2 722.2 511.3 510.5 – – 671.2 516.6 670.0 515.7 764.5 638.5 763.4 637.2

[C27N9O10S2H50]1+ [C27N9O10S2H48]1+ [C49N10O10S2H70]2+ [C49N10O10S2H68]2+ [PtC2N2H8]2+ [PtC6N2H14]2+ [Pt2C39N13O10S2H75]2+ [PtC33N11O10S2H63]2+ [Pt2C39N13O10S2H73]2+ [PtC33N11O10S2H61]2+ [Pt2C53N14O10S2H82]2+ [PtC51N12O10S2H76]2+ [Pt2C53N14O10S2H80]2+ [PtC51N12O10S2H74]2+

724.0 722.0 511.2 510.2 – – 671.2 516.7 670.2 515.6 764.3 638.2 763.3 637.2

(+1) (+1) (+2) (+2)

(+2) (+2) (+2) (+2) (+2) (+2) (+2) (+2)

(+1) (+1) (+2) (+2)

(+2) (+2) (+2) (+2) (+2) (+2) (+2) (+2)

HPLC peak.

HPLC for the reactions of OctS2 and 1 are consistent with the 2:1 adduct ([Pt2C53H80N14O10S2]2+, {(Pt(en))2 (OctS24H)}2+, calc. m/z (+2) 763.3) and 1:1 adduct ([PtC51H74N12O10S2]2+, {(Pt(en))(OctS22H)}2+, calc. m/z (+2) 637.2) Pt :OctS2 products, respectively. All observed and calculated m/z values are shown in Table 1.

3.3. NMR spectroscopy B (Fig. 3b). Isotope modelling showed that these peaks are consistent with a Pt:peptide ratio of 2:1 for peak A ([Pt2C39H75N13O10S2]2+, {(Pt(dach))2(T(SH)23H)}2+, calc. m/z (+2) 671.2), and a 1:1 ratio for peak B ([PtC33H63N11O10S2]2+, {(Pt(dach))T(SH)2–H}2+, calc. m/z (+2) 516.7). The mass spectra of the two HPLC-separated platinum-peptide products formed from reactions of 2 with oxidised TS2 (peaks C and D) are shown in Fig. 3c. Mass spectrometry of peak C, formed by reaction of oxidised TS2 and 2, gave a cluster of ions with a mass consistent with a 2:1 Pt:peptide adduct at m/z 670.0 ([Pt2C39H73 N13O10S2]2+, {(Pt(dach))2(TS23H)}2+, calc. m/z (+2) 670.2) and peak D, a cluster ions consistent with a 1:1 Pt: peptide adduct at m/z 515.7 ([PtC33H61N11O10S2]2+, {(Pt(dach)) (TS2H)}2+, calc. m/z (+2) 515.6). Mass spectra for the HPLC-separated platinated fractions formed during 7 d of incubation of 1 with Oct(SH)2 and OctS2 are shown in Fig. 4b and c, respectively. For the products formed in the reaction of Oct(SH)2 and 1, an ion cluster with m/z 764.5 was observed for peak E and m/z 638.5 for peak F (Fig. 4b). These peaks are consistent with a 2:1 Pt:Oct(SH)2 species ([Pt2C53H82N14O10S2]2+, {(Pt(en))2(Oct(SH)24H)}2+, calc. m/z (+2) 764.3) and 1:1 Pt :Oct(SH)2 ([PtC51H76N12O10S2]2+, {(Pt(en)) (Oct (SH)22H)}2+, calc. m/z (+2) 638.2). Likewise, the two ion clusters at m/z 763.4 and m/z 637.2 observed for the platinated products G and H, respectively, isolated by

NMR samples of the 1:1 Pt(R,R-dach)/TS2 and Pt(en)/ OctS2 adducts (peaks D and H, respectively) were prepared from fractions isolated by HPLC. Large scale mixtures of the platinum complex (1 mM, 8.5 mL, 8.5 lmol) and oxidised peptide (10 mM, 425 lL, 4.25 lmol, pH 7) were incubated at 310 K for 7 d. The 1:1 Pt : peptide adducts were collected by HPLC, characterised by MS, lyophilised, redissolved in 90%/10% H2O/D2O and the pH was adjusted to 7.4 (concentrations ca. 0.5 mM for Pt:TS2, and ca. 0.1 mM for Pt:OctS2). 1D 1H NMR spectra were recorded for the unplatinated and platinated peptide samples at 298 K. All the peaks were assigned, and the chemical shifts are shown in Tables 2 and 3. Changes in chemical shift (d Pt(peptide)–d (peptide), ppm) were calculated (Tables 2 and 3). For the TS2 sample, significant changes in chemical shift (Dd>0.1 ppm) are observed upon platination only for the Glu a-CH protons. For the 1:1 Pt/OctS2 sample, peak H, several protons experience chemical shift changes of 0.1–0.3 ppm upon platination of OctS2. The TOCSY spectrum of the 1:1 Pt(dach):TS2 adduct isolated by HPLC (sm = 60 ms) is shown in Fig. 6, and allowed full assignment of the proton resonances. In the 2D 1H MHz ROESY (sm = 150 ms) spectrum of this sample, through-space connectivities (ROEs) between ligand–ligand (en–en), peptide–peptide (TS2–TS2) and ligand–peptide (en–TS2) protons are observed, including

V.P. Munk et al. / Journal of Inorganic Biochemistry 100 (2006) 1946–1954 Table 2 1 H NMR chemical shifts (d) for free TS2 and the 1:1 Pt :TS2 adduct isolated by HPLC (peak D)

1951

Table 3 1 H NMR chemical shifts (d) for free OctS2 and the 1:1 Pt :OctS2 adduct isolated by HPLC (peak H)

Group

Proton

d (TS2)

d (Pt(TS2))a

Dd (ppm)b

Group

Proton

d(OctS2)

d (Pt(OctS2))a

Dd (ppm)b

Glu(1)/Glu(2)

a-CH b-CH2

3.745 2.161

3.641, 3.641 2.091, 2.216

D-Phe1

c-CH2

2.546

2.517, 2.610

NH a-CH b-CH2

c

–

3.141 3.126, 2.778 7.195 7.492 7.461 –

d

d

d

3.039, 3.031

Gly(1)/Gly(2)

NH a-CH2

8.647 3.910

8.641, 8.641 3.852, 3.926

8.736 3.240 1.641 1.575 3.045 3.045 1.880 3.340 7.836 8.091 –

NH

–

8.732 3.257 1.629 1.587 3.045 3.006 1.880 3.326 7.850 8.090 1.140, 1.573, 2.358 5.294, 5.005,

3.084, 2.941 8.191 3.971 3.096, 2.721 7.271 7.394 7.367 – 4.274 2.873, 2.721 7.141 7.567 7.214 7.282 7.517 10.200 7.922 3.833 1.589, 1.317 0.377 0.550 2.725 8.341 – 4.328 4.432 1.254 8.020

3.224, 3.049 8.443 4.035 3.141, 2.936 7.263 7.398 7.461 –

dach

NH a-CH2 b-CH2 c-CH2 d-CH2 c 0 -CH2 b 0 -CH2 a 0 -CH2 NH 0 NH00 CH

a-CH b-CH2 d-H e-H f-H c NHþc 3 a-CH b-CH2 NH a-CH b-CH2 d-H e-H f-H NH a-CH b-CH2 d-CH f2-CH g2-CH f3-CH e3-CH e1-NH NH a-CH b-CH2 c-CH2 d-CH2 e-CH2 NH þðaÞ NH3 a a-CH b-CH2 c-CH3 NH a-CH b-CH2 NH a-CH b-CH2 b 0 -CH2 c-CH3 NH CH2 NH

3.196 2.938, 2.721 7.271 7.415 7.367 –

4.690 3.037

0.104 0.070, 0.055 0.029, 0.064 – – 0.002, 0.006 0.006 0.058, 0.016 0.004 0.017 0.012 0.012 0.000 0.039 0.000 0.014 0.014 0.001 –

0.055 0.188, 0.076 0.077 0.094 – – 0.140, 0.252 0.064 0.045, 0.008 0.004 0.094 – – 0.038, 0.054 0.083 0.049 0.116 0.041 0.045 0.186 0.009 0.174, 0.109 0.115 0.053 0.248 – 0.437 0.221 0.027 0.023 – 0.140, 0.129 0.092 0.016 0.062, 0.002 0.289 – –

Cys(1)/Cys(2)

1.264, 2.038,

Cys2

Phe3

D-Trp4

Lys5 5.689, 5.646

–

a

HPLC Peak D. Dd (ppm) = [dPt(TS2)][d(TS2)]; both samples analysed at pH 7.4 and 298 K. c Signal not observed due to fast exchange. d Hidden under H2O. b

those between dach NH protons and Glu a-CH protons (Fig. 7). The pH and temperature stability of the 1:1 Pt:TS2 adduct was investigated using 1D 1H NMR. Little change in the 1D NMR spectrum was observed at different pH values (pH 2–11.5) and temperatures (278 K–308 K), except that NH proton exchange with solvent was rapid at pH > ca. 7, and the chemical shift of one NH peak was slightly temperature dependent. Subsequent incubation of the sample with an equimolar amount of 5 0 -GMP resulted in no reaction, showing that the coordination sphere of Pt in the adduct is saturated with strongly binding ligands. No ROEs between the en ligand and peptide protons were observed in the ROESY spectrum for the 1:1 Pt:OctS2 sample, perhaps because the signal-to-noise ratio was too low because the sample was dilute. 3.4. Molecular modelling A number of 1Pt:1TS2 adducts can form in the reaction of 2 with TS2, and molecular modelling was used to visualise two which are consistent with peaks observed in the mass spectrum. Energy-minimised adducts of a bifunctional adduct formed via the a-carboxylate acid O and a-amino N of Glu1 and a bifunctional adduct via the a-car-

Thr6

Cys7

Thr(ol)8

en

d

2.911, 2.778 7.195 7.650 7.263 7.398 7.476 10.245 7.736 3.824 1.415, 1.342 0.268 0.435 2.778 8.589 – 3.891 4.211 1.281 8.043

d

d

3.084, 2.941 8.624 3.872 4.036 3.744, 3.646 1.160 7.692  –

3.224, 3.049 8.753 3.964 4.052 3.682, 3.554 1.158 7.981 3.421 3.009

0.057

0.108

0.215

0.057

0.025

0.108

0.092

a

HPLC Peak H. Dd (ppm) = [dPt(OctS2)]–[d(OctS2)]; both samples analysed at pH 7.4 and 298 K. c Signal not observed due to fast exchange. d Hidden under H2O. b

boxylate acid O and a-amino N of Glu2 were built. In both cases, the {Pt(dach)}2+ group sits out of the plane of the peptide, causing no significant steric clashes between the platinum complex and the peptide. Similarly, molecular models with formulae consistent with those observed in the mass spectra were built for some of the possible 1:1 {Pt(en)}2+ and OctS2 adducts. No steric barriers were observed in any of these models, with the platinum complex sitting out of the plane of the peptide.

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V.P. Munk et al. / Journal of Inorganic Biochemistry 100 (2006) 1946–1954

4. Discussion

Fig. 6. 2D 1H 600 MHz TOCSY (sm = 60 ms) NMR spectrum for the 1Pt(dach):1TS2 adduct isolated by HPLC (peak D), from the reaction of 2 with TS2 (initially at pH 7) for 7 d at 310 K. The dach CH peaks are boxed. The peak for NHþ 4 arises from contamination during freeze drying. b-Cys protons are shown by * and the dioxane standard is shown by **.

Fig. 7. 2D 1H 600 MHz ROESY (sm = 150 ms) NMR spectrum for the 1Pt(dach):1TS2 adduct isolated by HPLC (peak D), from the reaction of 2 with TS2 (initially at pH 7) for 7 d at 310 K. The dach NH ROEs are boxed. No other significant ROEs are observed. The peak for NHþ 4 arises from contamination during freeze drying.

The interactions of cytotoxic PtII complexes with intracellular thiols have important implications for the effectiveness and delivery of this class of drug [1]. A number of reports have been published investigating the binding of PtII to thiols, and even disulfides [30–32]. Previously, we have shown that PtII [9,10] and PdII [33] complexes are able to cleave the disulfide bond of oxidised glutathione, resulting in the formation of potentially reactive species. The manner in which the platinum centre reacts with the peptide is likely to be dependent on the nature of the environment of the disulfide bond. The disulfide containing peptides trypanothione and octreotide are both cyclic, and their SS bonds are sterically less accessible than that of GSSG. Previous studies have shown that T(SH)2 is not as good a nucleophile as GSH, but it is more reactive [34]. The T(SH)2/TS2 redox couple has a potential of 0.242 V, which is more strongly reducing than GSH (0.230 V) and indicative of the increased stabilization of the oxidised (cyclic) form of trypanothione [35]. The crystal structure of TS2 in a complex with trypanothione reductase ˚ resolution, with the TS2 (TR) has been determined to 2.4 A binding to the TR active site asymmetrically [36]. The c-Glu–Cys–Gly component of TS2 interacts more with the active site of TR than the hydrophobic spermidine bridge. Sun et al. [26,37] have investigated the binding of T(SH)2 to antimony(III) and antimony(V) complexes of tartrate and gluconate. In reactions of SbV gluconate, they observed the pH-dependent reduction of SbV to SbIII, and subsequent rapid binding of SbIII to two peptide thiols. A stable complex with the stoichiometry of SbIIIT(S)2 formed. As far as we are aware, the physical properties of octreotide (pKa, Eo, etc.) have not been reported. In the X-ray crystal structure of OctS2 [38], Phe1 occupies the space around the disulfide bridge, thus protecting the sulfurs from enzymatic attack. The solution structure of octreotide, as determined by NMR, is consistent with the crystal structure [39]. In the present study, we have investigated reactions of PtII anticancer complexes with both the oxidised and reduced forms of trypanothione and octreotide. The reaction of 1, [Pt(en)Cl2] with OctS2 in aqueous solution at 310 K is significantly slower than the reaction of 2, [Pt(1R,2R-dach)Cl2], with TS2. The slower kinetics of the former reaction is likely to be due to the different Pt binding environments of the peptides, especially the presence of three hydrophobic aromatic side-chains in octreotide, although a small contribution to the difference may arise from the change in diamine (from en to dach). The extinction coefficients of the absorption bands appeared to be greater for products from the octreotide reactions. UV– vis spectra for reactions with the dithiols T(SH)2 and Oct(SH)2 showed two bands, the major band with a lower kmax (ca. 15 nm) and a higher relative extinction coefficient (by ca. 5000 M1 cm1) than those observed in reactions with the disulfides (TS2 and OctS2). The wavelengths and

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intensities of the absorption bands observed in the Pt(dach)/T(SH)2 and Pt(en)/Oct(SH)2 reactions are consistent with previous findings for the Pt(dach)/glutathione system, in which thiolate–platinum charge-transfer bands were observed at ca. 220 nm (De > 30,000 M1 cm1) [9,10,40], suggesting that the PtII complexes bind to the thiolate groups of T(SH)2 and Oct(SH)2. There appear to be few reports of UV–vis studies on Pt-(O,N) peptide binding, although there have been numerous NMR studies [40,41]. Our studies of the binding of glycine to [PtCl2(R, R-dach)] (data not shown) suggest that carboxylate and amino platinum binding would give rise to much weaker bands. Thus, absorption bands observed in the UV–vis spectra for the reactions of Pt(dach)/TS2 and Pt(en)/OctS2 may arise from transitions associated with PtII-disulfide interactions. Fazlur Rahman et al. have reported the coordination of disulfides to {Pt(dien)}2+ (dien = diethylenetriamine) to form isolatable {Pt-(RS–SR)}2+ complexes [42]. MS analysis showed that the two major products isolated by HPLC from reactions of the PtII complexes (2 mol equiv) with both the dithiols and disulfides were 1:1 and 2:1 Pt:peptide adducts. The 1:1 and 2:1 products from reactions of both reduced and oxidised peptides have similar retention times which might suggest they have similar structures. Indeed, the profiles of MS peaks are similar, except that the masses of the products from the reactions of oxidised peptides, TS2 and OctS2, are 2 mass units lower. This would imply that the increase in absorption at 240– 250 nm in these reactions is associated with a disulfide-toPtII electronic transition and that the disulfide bond is not reduced. Although such species have been isolated for alkyl disulfides [42], they do not appear to be have been characterised by electronic absorption spectroscopy. We were able to characterise the 1:1 adducts from reactions of both TS2 and OctS2 from reactions with 2 and 1, respectively, (Peaks D and H) by 1H NMR spectroscopy. In the case of TS2, the only significant chemical shift changes, compared to free TS2, are for the a-CH of the terminal Glu (Table 2). ROESY crosspeaks between the Glu a-CH and dach NH protons are also observed (Fig. 7). Examination of a model suggested that binding of {Pt(dach)}2+ to the disulfide sulfurs would bring the Glu ˚ ) to give rise to a-CH close enough to a dach NH (<4 A an ROE. For the 1:1 adduct from reaction of 1 with OctS2, the 1H NMR shift changes relative to free OctS2 were larger and distributed throughout the molecule (Table 3). Taking into consideration the UV–visible and MS data, they suggest that the binding of {Pt(en)}2+ to the disulfide bond causes structural perturbations throughout the cyclic peptide, perhaps as a consequence of the smaller ring size compared to TS2 (20 versus 24 atoms) and the presence of more hydrophobic side-chains. The conformational changes may be triggered by the displacement of the phenyl ring of residue Phe1 which occupies the space around the disulfide bond in the structure of free OctS2 [38] by {Pt(en)}2+.

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5. Abbreviations dach en GSH GSSG HPLC LC–MS Oct(SH)2 OctS2 ROESY TOCSY T(SH)2 TS2

1,2-diaminocyclohexane ethylenediamine glutathione oxidized glutathione high performance liquid chromatography liquid chromatography–mass spectrometry reduced octreotide oxidized octreotide rotating frame Overhause effect spectroscopy total correlated spectroscopy reduced trypanothione oxidized trypanothione

Acknowledgements We thank the BBSRC and Wellcome Trust (Edinburgh Protein Interaction Centre) for their support for this work, Novartis for their gift of octreotide, Mr Juraj Bella for assistance with NMR, Mr Robert Smith for assistance in LC–MS and colleagues in EC COST Action D20 for stimulating discussions. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23]

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