Introduction

The trypanosomatid parasite Leishmania causes a wide range of human diseases including kala azar and other less severe forms of leishmaniasis. Millions of people have clinical symptoms, and 400,000 new cases are reported each year [1]. Two pentavalent antimonial drugs, sodium stibogluconate (Pentostam) and meglumine antimonite (Glucantime), have been widely used for the treatment of human visceral leishmaniasis for several decades [2, 3, 4, 5]. Despite their extensive use, the mechanism of antileishmanial action, including its side effects, still remains unclear. It has been suggested that SbV is a prodrug and is intracellularly reduced to trivalent form (SbIII), the active form of the drugs at or near the site of action. The antileishmanial activity of SbV has been found to be dependent on its reduction to SbIII inside parasites [6]. The greater susceptibility of SbV to intracellular amastigotes than promastigotes suggests that the reductive activation of the drug occurs within the intracellular amastigotes. Arsenate (AsV), the analogue of antimonial, is known to be reduced to arsenite (AsIII) by arsenate reductase (e.g., ArsC and Acr2p) in yeasts, bacteria and even mammalian systems, and a mechanism for catalysis has been proposed [7, 8, 9, 10, 11, 12, 13]. Surprisingly, SbV cannot be reduced by this enzyme [14], and no analogous reductive enzyme in Leishmania has been identified.

Trypanothione [T(SH)2], a conjugation between the tripeptide glutathione and the polyamine spermidine [i.e., N 1,N 8-bis(glutathionyl)spermidine], is the most important and abundant (>80%) low molecular mass thiol within the Leishmania species [15, 16, 17]. In combination with trypanothione reductase (TR), T(SH)2 provides an intracellular reducing environment in parasites, in contrast to the glutathione found in mammalian systems, and is crucial for the survival of the parasites [18, 19]. T(SH)2 is also involved in the detoxification of metal ions, and an ATP-coupled AsIII/SbIII-T(S)2 pump (PGPA protein) has been described which extrudes the metal-(TS2) complex from parasites [17, 20]. Overproduction of trypanothione leads to resistance to antimonials and arsenicals [21]. Trypanothione metabolism has therefore long been regarded as an attractive target in antileishmanial and antitrypanosomal drug design [22], and one of the major targets for antimonial drugs is probably T(SH)2.

Low molecular mass thiols such as glutathione have already been shown to reduce antimonial and arsenate; however, this process is too slow to be biologically significant [10, 23, 24, 25, 26]. In this paper, we report an unexpectedly rapid reduction of SbV to SbIII by trypanothione and then characterize the complexation of SbIII to trypanothione by electrospray ionization mass spectrometry (ESI-MS) and NMR spectroscopy. The SbIII(TS2) complex is thermodynamically stable but kinetically labile, and forms a ternary complex upon addition of monothiols such as cysteine and glutathione. The interaction between antimony and T(SH)2 and other thiols (e.g., GSH and enzymes) may play a role in the pharmacology of antimony.

Materials and methods

Materials

Antimony trichloride (Aldrich), N-acetyl-l-cysteine (Sigma), oxidized trypanothione (Bachem), potassium antimony tartrate (Aldrich), reduced glutathione (GSH) (Sigma), tris(2-carboxyethyl)phosphine (TCEP) (Aldrich), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma), bromopyrogallol (BPR) (Sigma), dithiothreitol (DTT) (Sigma), NaNO3 (99.99%, Aldrich), NaOD, DCl (Aldrich) and D2O (Sigma), and 13C-labeled cysteine (98%, Cambridge Isotope Laboratories) were used as received. Sodium stibogluconate (batch GW387208A) was provided by GlaxoWellcome (Stevenage, UK).

Sample preparation

In all experiments, the concentration of reduced trypanothione was determined by titration with DTNB [27]. ESI-MS experiments were carried out in a solution of SbCl3 and T(SH)2. Other ligands such as tartrate and citrate used in the experiments were prepared from tartaric acid and citric acid using 1 M ammonia for pH adjustment. All the samples were adjusted to pH≈7 by ammonia.

NMR experiments were carried out in D2O or H2O/D2O (90/10) in the presence of 10 mM phosphate buffer. Solutions containing 5 mM of antimony complexes and various ratios of T(SH)2 were used in the experiments. Adjustment of pH* was made with NaOD and the solutions were then exposed to nitrogen (99.9%) for at least 10 min to minimize the oxidation of T(SH)2. All solutions used for the study of formation constants contained 5 mM SbIII(tart), 5 mM T(SH)2 and 0.1 M NaNO3 in D2O. Measurements of pH* were made with a Corning 440 pH meter equipped with a micro combination electrode (Aldrich), calibrated with standard buffer solutions. The meter readings for D2O solutions were recorded as pH* values without correction for isotope effects.

Electronic absorption spectroscopy studies were carried out in a solution containing 1 mM SbV(gluconate) and 2 mM T(SH)2 in the presence of 10 mM phosphate buffer at different pH values. In the kinetics studies, the analytical solutions were kept under a nitrogen atmosphere to minimize the oxidation of T(SH)2.

Electronic absorption spectroscopy

Determination of the SbIII content was carried out on a Varian Cary 50 UV-vis spectrometer, as described previously [23]. A solution containing 700 μL of 15 μM bromopyrogallol (BPR) was used for SbIII detection. Aliquots of the sample solution (5–20 μL) were added to the BPR cuvette. SbIII tartrate was used as a standard. The absorbance of BPR at 560 nm decreased proportionally to the amount of SbIII in the medium, and a calibration curve was generated for calculating the SbIII concentration. We checked that neither SbV nor T(SH)2 (and GSH) in the analyte solution interfered with the colorimetric test. For kinetics studies of SbV reduction, aliquots of the sample were added to the BPR-containing cuvette to monitor the SbIII formation profile. The rate of reduction (k) was calculated from a non-linear square fit of the data by assuming a second-order reaction between SbV and T(SH)2.

NMR experiments

All 1H NMR experiments were carried out on Bruker DRX500 and AV600 spectrometers at 278–298 K. Typical acquisition conditions for 1H NMR were 45° pulse length (5 μs), 2.0 s relaxation delay (4 s for determination of formation constants), 16k data points and 16–32 transients. Spectra were processed using an exponential function with a line-broadening coefficient of 0.3 Hz. Standard TOCSY, NOESY and ROESY pulse sequences were used with a spectrum width of 10 ppm and a relaxation delay of 2–4 s. Several mixing times were chosen in the range of 20–75 ms (2D TOCSY), 200–400 ms (2D NOESY) and 150 ms (2D ROESY) respectively, with 2k data points in the t 2 dimension and 256 increments in the t 1 dimension. The spectra were zero-filled to a 2k×1k matrix. The standard NOESY pulse sequence was used to perform the 2D exchange spectroscopy (EXSY). A 1D heteronuclear single-quantum coherence spectrum (HSQC or 13C edited 1H NMR spectrum) was recorded using the first increment of the 2D HSQC sequence. In all experiments, the solvent resonance (residual water) was suppressed via presaturation during the relaxation delay, and the resonance of the water signal for experiments performed in H2O/D2O (90/10) was suppressed by the WATERGATE pulsed-field-gradient sequence [28].

Electrospray ionization mass spectrometry

Positive ion electrospray mass spectrometry was performed on an LCQ spectrometer (Finnigan). The sample was infused at 3 µL min−1 and the ions produced in an atmospheric pressure ionization (API)/ESI ion source. The source temperature was 453–473 K and the drying gas flow rate was 0.9 L min−1. A potential of 3.5 kV was applied to the probe tip, and a cone voltage of −10 V over 200–2000 Da was used. The quadrupole was scanned at 100 amu s−1. The mass accuracy of all measurements was within 0.5 m/z units. The acquisition and data processes were performed on a Microsoft Windows NT operating system.

Preparative HPLC separations of trypanothione

Reduced trypanothione was separated by a cation-exchange column (a preparative MN-nucleosil 100-10 SA column, pore size 100 Å, particle size 10 μm) equipped with a Waters 510 HPLC pump and a Waters 410 differential refractometer as the detector. Isocratic elution (8 mL min–1) of 1 M KNO3 was applied as a solvent system. After initial separation by the cation-exchange column, the fraction containing trypanothione was freeze-dried and redissolved in a minimum amount of the mobile phase as described below and then desalted by a GL Science Intertsil PREP ODS reverse-phase column (pore size 100 Å, particle size 10 μm). Isocratic elution (8 mL min–1) with 10 mM NaClO4 and 0.1% v/v of 85% H3PO4 was applied.

Results

HPLC separation of trypanothione

The reduced form of trypanothione [T(SH)2] was obtained via reduction of its oxidized form [T(S-S)] using tris(2-carboxyethyl)phosphine (TCEP) [29], and was further purified by HPLC. Since trypanothione has a very strong ion-pair interaction with the stationary phase (sulfonate group) via the spermidine amine group, a higher ionic strength mobile phase (1 M KNO3) was used to elute trypanothione from the column. Reduced [T(S-S)] and oxidized trypanothione [T(SH)2] were eluted at 19 and 20 min, respectively, while TCEP was eluted at 10 min (Fig. 1). After the fraction containing trypanothione was collected, it was freeze-dried and desalted as described (Materials and methods), to produce an appropriate ionic strength for NMR experiments.

Fig. 1.
figure 1

HPLC profile of the separation of reduced trypanothione [T(SH)2], oxidized trypanothione [T(S-S)], (NAC)SbIII(TS2) and tris(2-carboxyethyl) phosphine (TCEP). Insert: structure of T(SH)2

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Reduction of SbV complex by reduced trypanothione

UV-vis spectroscopy was used to investigate the reduction of antimony(V) by trypanothione with bromopyrogallol (BPR) as indicator. When T(SH)2 was incubated with SbV (as sodium stibogluconate) in the molar ratio of 2:1 at pH 5.0 and 310 K, a gradual decrease in absorption at 560 nm was observed, indicative of the reduction of SbV to SbIII [30]. These changes can be converted to the increase in the concentration of SbIII (Fig. 2). The concentration of SbIII increased exponentially and the rate of reduction of SbV (or formation of SbIII) was calculated at 6.82 M−1 min−1 with t 1/2=73±5 min. A slower reduction rate was observed when the sample was incubated at the higher pH of 6.4 and 7.4 (4.42 and 1.53 M−1 min−1, respectively), showing that the rate of reduction was pH-dependent and favored under acidic pH. The rate of formation of SbIII was much slower (1.66 M−1 min−1) at the lower temperature of 298 K (pH 5.0), with t 1/2=300±5 min. Little SbIII was detected when SbV was incubated in the absence of reduced trypanothione. This result indicated that T(SH)2 plays a significant role in the reduction of SbV to SbIII. For comparison, dithiothreitol (DTT), E°1/2=−0.330 V [vs. −0.242 V for T(SH)2], also a dithiol [17], was tested for its ability to reduce SbV to SbIII. A very slow reduction process for DTT was observed under identical conditions, in contrast to the effects achieved with T(SH)2 (Fig. 2).

Fig. 2.
figure 2

Kinetics of the reduction of SbV in aqueous solution at pH 5.0, 6.4 and 7.4 and 310 K by T(SH)2 or DTT. The insert is the rate of reduction by T(SH)2 at 310 K and different pH values; simulation of the reaction rate is described in Materials and methods. The solution was maintained in a nitrogen atmosphere

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Formation of a binary and novel ternary complex

Following the reduction of SbV to SbIII, a complex of SbIII with T(SH)2 may form. Owing to the presence of potassium in the SbIII tartrate complex, which greatly suppresses the ionization of the ions and results in dramatic loss of ESI-MS sensitivity, antimony trichloride (SbCl3) was used in our study. In the ESI-MS spectrum of T(SH)2 at pH≈7, there was a cluster of ions at m/z 724.9, corresponding to C27H50N9O10S2 (calcd 724.3). A mixture of SbCl3 and T(SH)2 (1:1 molar ratio) at pH 7.4 gave a prominent cluster of ions related to the natural isotopic distribution of Sb (121Sb:123Sb=57:43), with predominant peaks [M+] at m/z 842.3 and 844.3 (intensity ratio 5.5:4.5), which can be assigned to SbIII(TS2) (1, C27H48N9O10S2Sb, calcd 843.2 and 845.2). The minor peaks at m/z 843.6, 845.6, 846.6 and 847.6 were due to the isotopic distribution of 13C, and the distribution pattern of Sb and C agreed well with the simulations (Fig. 3A, insert). A minor cluster of ions at m/z 1567.2 and 1569.3 corresponded to SbIII(TS2)2 complexes (C54H97N18O20S4Sb, calcd 1566.5 and 1568.5). There were no further changes after addition of total 1.5 and 2 mol equiv of T(SH)2 to the mixture, except an increase in the intensity of the peak at m/z 724.9, arising from T(SH)2.

Fig. 3.
figure 3

ESI-MS spectra of A SbIII(TS2) (1), B after addition of N-acetyl-l-cysteine and C glutathione. Note the new clusters formed at the larger m/z, indicative of formation of a ternary complex

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Interestingly, the addition of one mol equiv of N-acetyl-l-cysteine (NAC) to 1 at the same pH value resulted in a new major cluster of ions at m/z 1005.3 and 1007.1 with an intensity ratio of about 5:5 in the ESI-MS spectrum, corresponding to a new complex (NAC)SbIII(TS2) (C32H56N10O13S3Sb, calcd 1005.4 and 1007.4 with intensity ratio 57:43) (Fig. 3B). No other major changes were observed after the further addition of NAC. Similarly, the addition of one mol equiv of the tripeptide glutathione (GSH) to 1 led to a major cluster of ions at m/z 1149.3 and 1151.2 with an intensity ratio 4.5:5.5, respectively. The cluster can be assigned to a ternary complex (GS)SbIII(TS2) (C37H64N12O16S3Sb, calcd 1149.5 and 1151.5 with intensity ratio 57:43) due to GSH binding to 1 (Fig. 3C). Again, the further addition of GSH did not produce any significant changes in the spectrum.

In order to investigate whether the formation of the ternary complex depends on the binary complex, we first incubated SbCl3 with NAC at pH 7.4 for 1 h at room temperature to obtain an SbIII(NAC)3 complex which gave a prominent cluster [M+] at m/z 607.5 and 609.1 in the ESI-MS spectrum. After addition of one mol equiv of T(SH)2 to the reaction mixture, a predominant cluster of ions [M+] at m/z 1005.3 and 1007.1 was observed with an intensity ratio of about 5:5, corresponding to the ternary complex (NAC)SbIII(TS2). The intensity of the peaks at m/z 607.5 and 609.1 decreased dramatically. Only a very weak cluster at m/z 842.6 and 844.3 was noticed (data not shown). Similarly, after the addition of T(SH)2 to the SbIII(GS)3 solution, a prominent cluster [M+] at m/z 1149.3 and 1151.2 appeared at the expense of the initial major cluster [M+] at m/z 1039.3 and 1042.1. These two groups of clusters corresponded to (GS)SbIII(TS2) (calcd 1149.5 and 1151.5) and SbIII(GS)3 (calcd 1039.1 and 1041.1), respectively.

Other non-thiolate ligands such as tartrate or citrate were also added to SbIII(TS2) solution. Little change was observed on addition of one mole equivalent of either tartrate or citrate (pH 7.4), except for the original signal at m/z 842.3 and 844.3 corresponding to 1.

Thermodynamics and kinetics study of the binary and ternary complex by 1H NMR

SbIII binding to T(SH)2

Trypanothione is a polydentate ligand including four carboxylate groups, two thiol groups and several amino groups. 1H NMR spectroscopy was used to identify the target sites of SbIII binding. Only one set of resonances from like residues was observed, despite the asymmetry of the sperimidine moiety. This indicates an approximate symmetry of the two halves of T(SH)2, which is similar to its T(S-S) oxidized form [31]. In aqueous solution containing a T(SH)2 and SbIII tartrate mixture (1:1 mol ratio) at 298 K and pH* 7.4, the major changes in the spectrum were the disappearance of the resonances of the α and β protons of Cys of T(SH)2, and the appearance of relatively broad resonances for these protons at a relatively lower field, as well as a new singlet which appeared at 4.33 ppm (Fig. 4).

Fig. 4.
figure 4

1H NMR spectra of SbIII(TS2) and (13C-Cys)SbIII(TS2) complexes. Left: 1H NMR spectra of reduced-trypanothione [T(SH)2] (bottom), after addition of 0.5 (middle) and 1 mol equiv of SbIII to T(SH)2 (top). Right: 1H NMR spectrum of free 13C-labeled cysteine (13C-Cys) (bottom), SbIII(13C-Cys)3 (middle) and the ternary complex (13C-Cys)SbIII(TS2) (top). The spectra were acquired at pH* 7.4 and 298 K

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The 1H NMR spectra of free T(SH)2 and T(SH)2 in the presence of 0.5 and 1.0 mol equiv of SbIII(tart) at pH* 7.4 are shown in Fig. 4. Reaction of T(SH)2 with 0.5 mol equiv of SbIII at pH* 7.4 led to the appearance of new T(SH)2 peaks at 3.22 and 4.58 ppm, while the Cys βCH2 and αCH of free T(SH)2 at 2.98 and 4.51 ppm disappeared. A 2D TOCSY spectrum confirmed that the peaks at 2.98 and 4.51 ppm were coupled each other, as were the resonances at 3.22 and 4.58 ppm (data not shown). These can therefore be assigned to the βCH2 and αCH protons of the Cys residues of the free T(SH)2 and SbIII-bound trypanothione, respectively.

The further addition of 0.5 mol equiv of SbIII (total 1.0 mol equiv) to the T(SH)2 solution produced further shifts in the resonances at 3.22 to 3.36 ppm (βCH2) and the resonances at 4.58 to 4.64 ppm (αCH). Simultaneously, the intensity of the resonances of free tartrate at 4.33 ppm increased by 40%. No other evident changes in the proton signals of trypanothione were observed. The addition of another mol equiv of SbIII resulted in little changes in the resonances except for a new resonance at 4.72 ppm, which can be assigned to the bound form of tartrate. This suggests the formation of a stable SbIII(TS2) complex, which matches the ESI-MS data. The large changes in peaks for the βCH2 and αCH of the Cys residues of T(SH)2 (Δδ=0.38 and 0.13 ppm) suggest that thiolate groups of Cys are the binding sites for SbIII.

Characterization of an SbIII ternary complex by 13C-labeled cysteine

Because the 1H NMR resonances of cysteine (and GSH) overlap with cysteine residues from trypanothione, it is difficult to distinguish whether the resonances come from cysteine or a cysteine residue from GSH or T(SH)2. We therefore employed 13C-labeled cysteine in this study. As the α and β protons of the cysteine were coupled with the labeled 13C, a splitting (1JCH=145 Hz) for both protons was observed, which allowed us to readily distinguish the resonance of 13C-cysteine from GSH and trypanothione.

Figure 4 shows the 1H NMR spectra of the free 13C-labeled cysteine and 13C-labeled cysteine (13C-Cys) in the presence of 3 mol equiv of SbIII tartrate or 1 mol equiv of SbIII(TS2) (1) at pH* 7.4. Addition of 1.0 mol equiv of 13C-labeled cysteine to 1 at pH* 7.4 led to resonances at 3.15 and 3.43 ppm due to 13C and 1H coupling (1JCH=142 Hz), as well as another set of broad peaks at 3.98 and 4.28 ppm (1JCH=145 Hz). A 13C-edited 1H spectrum shows only two resonances at 3.08 and 2.99 ppm (βCH2), and 3.97 ppm (αCH, data not shown), which confirmed that these splittings resulted from the coupling between the carbon of 13CH2 of Cys with its attached protons. Resonances of the β and α protons of free 13C-Cys at the same pH value appeared at 2.90 and 3.18 ppm, and 3.75 and 4.05 ppm, respectively. None of the other parts of the resonance of bound trypanothione was affected by the addition of 13C-Cys. Details of the assignments are listed in Table 1. The large low-field shifts of the resonances for the α and β protons (Δδ=0.23 and 0.25 ppm) of 13C-Cys suggested that the thiolate sulfur of cysteine coordinated to the SbIII center.

Table 1. Chemical shifts of 13C-labeled cysteine
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Both NOESY and ROESY experiments of the ternary complex (as 13C-labeled cysteine) with different mixing time were performed. However, few NOE or ROE cross-peaks were observed between protons of trypanothione and those of 13C-Cys, probably due to the flexible structure of the complex in the solution.

The exchange behavior of thiolate groups between free and bound forms of T(SH)2 in 1 was investigated by 2D 1H exchange spectroscopy (EXSY). A 2D EXSY spectrum (mixing time 400 ms) of SbIII(TS2) at pH* 4 showed cross-peaks at 3.39/2.98 ppm and 4.64/4.51 ppm, which are assignable to Cys β and α protons exchange between free and bound forms of T(SH)2 on the 1H NMR timescale (Fig. 5A and Fig. S1). Similarly, another set of cross-peaks at 4.70/4.33 ppm was observed, due to the exchange between free and bound forms of tartrate (Fig. S1).

Fig. 5A, B.
figure 5

Exchange of trypanothione between bound and free forms, and exchange of 13C-labeled cysteine between its bound and free forms in the ternary complex. A 2D EXSY spectrum (mixing time 500 ms) of SbIII in the presence of T(SH)2 at pH 4.0, showing the cross-peaks for exchange between bound and free T(SH)2 at 3.36/2.98 ppm. B 13C-edited 1H NMR spectrum of a solution containing 5 mM SbIII in the presence of T(SH)2 and 13C-labeled cysteine at a mole ratio of SbIII:T(SH)2:13C-labeled cysteine=1:1:2 at pH 4.1–7.4 at 298 and 310 K, showing the broadening and coalescence of free and bound 13C-labeled cysteine at 3.97/4.12 ppm (αCH) and 3.09, 3.10/3.42, 3.61 ppm (βCH2) as the pH increases

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The exchange behavior of cysteine between free and bound forms in the ternary complex was investigated by the 13C-edited 1H NMR technique (1D-HSQC). A solution containing SbIII, T(SH)2 and 13C-Cys with the ratio 1:1: 2 was used to study the exchange behavior at various pH values. At pH 4.08 and 298 K, separate resonances of free and bound forms of αCH (3.97/4.12 ppm) and βCH2 (3.09, 3.10/3.42, 3.61 ppm) of 13C-Cys were observed (Fig. 5B). As the pH was increased to 6.49, both the free and bound resonances broadened and gradually coalesced. At pH 7.40 and 310 K, only an average resonance was observed, indicative of a rapid exchange between free and bound forms of 13C-labeled Cys in the ternary complex. The rate of exchange was estimated at 5–10 s−1 at pH* 4.1–4.7, 298 K and >500 s−1 at pH* 7.40 and 310 K, based on previous calculations [32]. No detailed lineshape analysis was attempted due to the presence of a pH-dependent equilibrium of the ternary complex.

pH dependence of SbIII(TS2)

To investigate the pH* stability of 1, 1H NMR spectra of T(SH)2 and SbIII tartrate at a 1:1 mol ratio were recorded over the pH range 2–9 at 298 K. At pH* 2.4, both free and bound forms of Cys βCH2 at 2.98 and 3.36 ppm, and αCH at 4.51 and 4.68 ppm, were observed, respectively (Fig. S2), as were the free and bound forms of tartrate at 4.62 and 4.70 ppm. Both βCH2 and αCH resonances of free Cys decreased in intensity with the increase in pH* value, while an increase in intensity of the bound form was observed. Simultaneously a new resonance appeared at 4.62 ppm and later shifted to 4.33 at pH*>4.2. At pH* 7.4, the free Cys βCH2 and αCH resonances at 2.98 and 4.51 ppm completely disappeared, and the intensity of the resonance at 4.33 ppm reached its maximum. The chemical shifts of peaks at 4.68 and 3.36 ppm were almost independent of pH* up to 8.6, while the chemical shifts of Glu and Gly residues of [SbIII(TS2)] were almost the same as those in free T(SH)2, suggesting that SbIII binds to T(SH)2 only at the thiolate sulfur of Cys when forming an [SbIII(TS2)] complex.

Determination of the stability constant for [SbIII(TS2)]

A reversible reaction was observed between SbIII tartrate and T(SH)2. At low pH* values, SbIII binds strongly to tartrate, whereas at higher pH* values, T(SH)2 displaces tartrate gradually. The stability constants can therefore be calculated, based on the integration of free and bound βCH2 of Cys (Table 2), with the following procedures. We consider Sb(tart) as a monomer, although it is a dimer in the solid state [33]. An equilibration reaction occurs in aqueous solution between the monomer and dimer, and at higher pH values, and an ionic strength of 0.1 M, most of this compound will be present as monomers. Firstly, the pH*-dependent stability constant (K) is determined at different pH* values by integration of the appropriate 1H NMR peaks:

Table 2. Stability constants (K) for reaction between antimony tartrate and 1 mol equiv of T(SH)2 at different pH* values, conditional stability constants for \({{\rm{Sb}}{\left( {{\rm{TS}}_{2} } \right)}\;{\left( {{K}'_{{{\rm{Sb}}{\left( {{\rm{TS}}_{2} } \right)}}} } \right)}}\) and derived stability constants \({{\left( {K_{{{\rm{Sb}}{\left( {{\rm{TS}}_{2} } \right)}}} } \right)}}\) at 298 K
Full size table
$$ \left[ {{\rm{Sb}}\left( {{\rm{tart}}} \right)^ - } \right] + {\rm{TS}}_{2\left( {\rm{t}} \right)} \to \left[ {{\rm{Sb}}\left( {{\rm{TS}}_{\rm{2}} } \right)} \right] + {\rm{tart}}^{4 - } \;\;\;\;\;\;\;K = {\textstyle{{\left[ {{\rm{Sb}}\left( {{\rm{TS}}_{\rm{2}} } \right)} \right]\left[ {{\rm{tart}}^{4 - } } \right]} \over {\left[ {{\rm{Sb}}\left( {{\rm{tart}}} \right)^ - } \right]\left[ {{\rm{TS}}_{\rm{2}} } \right]_{\left( {\rm{t}} \right)} }}} $$
(1)

where TS2(t) is the total free trypanothione, i.e. [TS2](t)=[TS2 2−]+[T(SH)2]. The stability constant for [Sb(tart)−], K tart, is known (logK tart=14.8 at 293 K, I=0.1 M) [34]:

$$ {\rm{Sb}}^{{\rm{III}}} + {\rm{tart}}^{4 - } \to \left[ {{\rm{Sb}}\left( {{\rm{tart}}} \right)^ - } \right]\;\;\;\;\;\;\;K_{{\rm{tart}}} = {\textstyle{{\left[ {{\rm{Sb}}\left( {{\rm{tart}}} \right)^ - } \right]} \over {\left[ {{\rm{Sb}}^{{\rm{3 + }}} } \right]\left[ {{\rm{tart}}^{4 - } } \right]}}} $$
(2)

Hence the pH*-dependent stability constant of [Sb(TS2)] (K′TS2) can be obtained by means of the relationship K′TS2=K×K tart:

$$ {\rm{Sb}}^{{\rm{III}}} + {\rm{TS}}_2^{2 - } \to \left[ {{\rm{Sb}}\left( {{\rm{TS}}_{\rm{2}} } \right)^{\rm{ + }} } \right]\;\;\;\;\;\;\;K'_{TS_2 } = {{\left[ {{\rm{Sb}}\left( {{\rm{TS}}_{\rm{2}} } \right)^ + } \right]} \over {\left[ {{\rm{Sb}}^{{\rm{III}}} } \right]\left[ {{\rm{TS}}_2 ^{2 - } } \right]}} $$
(3)

The pH-independent stability constant (K TS2) can be obtained by assuming the pK a value of T(SH)2 is 7.40 [35], with an average value of logK TS2 of 23.6±0.4 in 0.1 M NaNO3 (Table 2).

In order to compare the SbIII binding affinity of T(SH)2 with that of arsenic, the pM value was calculated and is listed in Table 3 together with the pM values of metal-glutathione complexes, where pM=−log[M], and [M] is the concentration of unchelated unhydrolyzed metal ion at equilibrium in a pH 7.4 solution of 1 μM metal and 10 μM ligand.

Table 3. pM values of selected metal-GSH complexes and the binary complex of SbIII(TS2)
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Discussion

The chemistry of antimony is well known at the molecular level. However, its biological chemistry and molecular pharmacology are poorly understood even though it has been used medicinally for several decades, as are its potential carcinogenic and genotoxic mechanisms [36]. No X-ray crystal structure of antimony and arsenic with biomolecules was available until recently [37]. In contrast to its analogue arsenic, the uptake process of SbV into cells and the mechanism of reduction or activation of antimonial drugs still remain unclear. T(SH)2 is the most important low molecular mass thiol (>80%) inside parasites (e.g., ~5 mM in Leishmania donovani) [17], and plays a crucial role in maintenance of cellular redox homeostasis. Elevated cellular T(SH)2 has been reported and found to be related to antimonial resistance in Leishmania [21]. The lack of information on the SbIII-trypanothione complex prompted us to investigate the complexation of T(SH)2 to antimonial.

The reduction of arsenate (AsV) to arsenite (AsIII) in bacteria, yeasts and mammals is catalyzed by the arsenate reductase and has been well documented [7, 8, 9, 10]. In contrast, little is known about the chemical fate of antimonials in trypanosomatids and only a small percentage of SbV in human bodies was reduced to SbIII after 12 h [36]. Surprisingly, we found that SbV can be reduced to SbIII by T(SH)2 at 310 K and pH 6.4 (k=4.42 M−1 min−1, t 1/2=113±5 min), in contrast to its analogue glutathione (days). Interestingly, other reducing dithiols such as DTT were found to reduce SbV to SbIII at a comparable rate to GSH, although a better reduction rate (E°1/2=−0.330 V) had been expected. This indicates that trypanothione is unique for the reduction (activation) of SbV. We should bear in mind that the actual concentration of T(SH)2 inside parasites may remain constantly high because of the presence of a NADPH-dependent TR reducing cycle, and hence a higher reduction rate would be expected in vivo. The reduction process is both pH- and temperature-dependent and favors at an elevated temperature and an acidic pH. Although the intracellular cytosolic pH of both promastigotes and amastigotes is only slightly acidic (pH=6.5–6.8) [38], promastigotes are known to grow at 298 K while amastigotes are cultured at 310 K [39]. Therefore reduction of antimonials probably occurs at amastigotes (intracellular). These data are in good agreement with two recent reports on the reduction of SbV by low molecular thiols [40] and on intracellular antimonial (SbV) reduction in Leishmania donovani, based on hydride generation-inductively coupled plasma-mass spectrometry (HG-ICP-MS) [6], which showed that the intracellular reduction of SbV is a kinetically controlled process. Up to 30% of the SbV was reduced to SbIII in amastigotes (310 K) and little reduction of SbV to SbIII was observed in promastigotes (299 K) after 12 h. This suggests that trypanothione might play an important role in antimonial activation. However, we cannot exclude the possibility of the presence of an enzyme that catalyzes SbV reduction. For example, although AsV is reduced by GSH and other low molecular thiols, this process is too slow and cells use arsenate reductase instead [23, 24]. The reduced SbIII may subsequently bind to trypanothione.

Our 1H (and 13C) NMR and ESI-MS data show that SbIII forms a stable complex with T(SH)2 with the stoichiometry SbIIIT(S)2, and that the trypanothione is bound by deprotonated thiolate sulfurs. This is obvious from a comparison of the SbIII-induced changes of shifts in 1H NMR spectrum with those of free T(SH)2, which are ~0.38 and 0.13 ppm for the Cys βCH2 and αCH, respectively, whereas the changes of shifts for other residues of T(SH)2 are not evident. Therefore, the interaction of antimonial (SbV) with T(SH)2 involves two steps: reduction and complexation, and each step requires one mol equiv of T(SH)2 and hence the overall reaction can be represented as (H+ is omitted in the equation):

$$ {\rm{Sb}}^{\rm{V}} + 2{\rm{T}}\left( {{\rm{SH}}} \right)_2 \to {\rm{Sb}}^{{\rm{III}}} \left( {{\rm{TS}}_{\rm{2}} } \right) + {\rm{T}}\left( {{\rm{S - S}}} \right) $$
(4)

We suggested earlier, in a brief report, that a water molecule probably binds to the SbIII center, in addition to two thiolate sulfurs from T(SH)2 [41]. Therefore, complex 1 can be represented as (H2O)SbIII(TS2), in contrast to its glutathione complex, SbIII(GS)3 [32]. This is probably because the sterically hindered T(SH)2 prevents the third sulfur from an adjacent T(SH)2 from binding to an SbIII center. Both ESI-MS and NMR data indicate that 1 is a monomer with two sulfurs from T(SH)2 and probably an oxygen from a water molecule. This is consistent with the recent crystal structure of an AsIII/SbIII-translocating ArsA ATPase [37]. In this SbIII-protein complex, a novel SbIII cluster was observed and SbIII interacts with ArsA as a soft metal. Each SbIII coordinates to three donate atoms, two of them from protein residues (e.g., Cys, His or Ser), and the third from a non-protein ligand (probably Cl−). The Sb–S bond distances are between 2.58 and 2.62 Å, slightly longer than those in SbIII(SC6H2Pri 3-2,4,6)3 [42]. Both complex 1 and SbIII-ArsA ATPase could be regarded as an intermediate, and coordination of the non-protein (peptide) ligand may allow it to be readily replaced by other thiols (vide infra).

Complexation of T(SH)2 to the arsenical drug melarsen oxide has been characterized and shown to bind at thiolate sulfurs only [43]. The much larger pM value of 1 compared with the melarsen oxide-trypanothione complex clearly indicates that binding of T(SH)2 to SbIII is much stronger than to AsIII. Similarly, the affinity of SbIII for T(SH)2 is about 100 times higher than that for GSH, probably due to the chelate effect and slightly lower pK a value of the thiolate group (~7.4) [35]. This conclusion is also supported by the displacement of either GSH or cysteine by T(SH)2 from SbIII(GS)3 and SbIII(Cys)3 complexes, but not the reverse. The strong binding of SbIII to thiolate sulfur of T(SH)2 suggests that proteins and enzymes are the biological target for SbIII in parasites (and cells).

Surprisingly, the addition of monothiols such as cysteine and GSH results in the formation of a ternary complex. The ternary complex is independent of the order of the binary complex, i.e. either from 1 or SbIII(GS)3 and SbIII(NAC)3. Our 1H NMR data demonstrated that SbIII in 1 binds to the thiolate group of the 13C-Cys, forming a (13C-Cys)SbIII(TS2) ternary complex. The formation of the ternary complex indicates that a water molecule initially bound to the SbIII center of the SbIII(TS2) can readily be displaced by a thiolate group. A ternary complex between proteins (and enzymes) and 1 is thus possible and may account for the biological function of antimony.

In spite of its high thermodynamic stability, complex 1 is kinetically labile toward T(SH)2 exchange. The rate of exchange of T(SH)2 on SbIII is pH dependent, from slow exchange at lower pH (ca. 4) to fast exchange at biological pH. Moreover, exchange between the free and bound 13C-Cys in the ternary complex was also observed (Fig. 5). The presence of minor Sb(TS2)2 species in the ESI-MS spectrum indicates that it may be an exchange intermediate. T(SH)2 may therefore play an additional role in controlling the free concentration of SbIII in parasites and could perhaps be used as a chaperone to deliver SbIII via thiolates exchange to target proteins and enzymes intracellularly (e.g., trypanothione reductase). Such an exchange of metals (e.g., AsIII and CuI) between thiolate sulfurs has been noted previously for low molecular mass ligands and proteins [44, 45, 46].

We therefore propose a five-step model for the action of antimonials (Fig. 6). The first step involves the uptake of antimonials (SbV) into parasites. Our initial results have demonstrated that only SbV binds to the iron transport protein lactoferrin rather than SbIII, and the uptake of SbV is probably protein mediated (Yan and Sun, unpublished data). The second step is the reduction/activation of SbV to SbIII by trypanothione. However, direct interaction of SbV with nucleosides at acidic conditions (pH≈5) may also be possible, as suggested recently [47]. The third step involves complexation of SbIII to trypanothione to form LSbIIIT(TS2) (L=H2O, Cl− or other thiol ligands). This is followed by the transfer of SbIII via thiolate exchange and finally targeting proteins or enzymes. Finally, the resistance of parasites to antimonials is due to pumping of the complex LSbIII(TS2) out of cells. An intracellular metal-thiol (AsIII/SbIII-glutathione/trypanothione) transporter ATPase (PGPA) has been identified in Leishmania [20]. Further biological experiments will be needed to confirm the above hypothesis.

Fig. 6.
figure 6

A proposed mechanism for antimonial in Leishmania. The first step is the protein-mediated uptake of SbV into parasites. The second step is the intracellular reduction of SbV to SbIII by T(SH)2 (or an "enzyme"), and the oxidized trypanothione [T(S-S)] is reduced by trypanothione reductase (TR). The third step involves binding of SbIII to T(SH)2 and is followed by targeting the enzyme via thiolate exchange. Simultaneously, the SbIII(TS2) complex can be pumped out of cells via an ATP-coupled transporter [20]

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