Interaction of antimonyIII compounds with thiolate molecules

SbIII is classified as a borderline metal ion and has a high affinity towards nitrogen- and thiol-containing ligands.85,86 The anti-leishmanial mechanisms of SbIII drugs are probably related to their interaction with those thiol-containing polypeptides, proteins and enzymes. One of the possible targets specific to the parasitic protozoa is the trypanothione reductase (TR), the analogoue of glutathione reductase (GR) in mammalian systems. Although TR shares structural and mechanistic similarity with its analogy GR, differences in the disul-fide binding site between TR and GR draw the interest for selective inhibition.87 Since trypanothione is responsible for the survival of the protozoa

Figure 23.3 The structures of glutathione, GSH (7-L-Glu-L-Cys-Gly) (a) and trypa-nothione, T(SH)2 (b)

Figure 23.3 The structures of glutathione, GSH (7-L-Glu-L-Cys-Gly) (a) and trypa-nothione, T(SH)2 (b)

inside the macrophage of the host, alternation of the metabolism of trypano-thione by the inhibition of TR resulted in a disruption of the reducing environment of the parasite, which is essential for the survival of parasites.

The active site of TR consists of a disulfide-binding site. In the crystal structure of TR from Trypanosoma cruzi, the two components of the GSH in trypanothione bind to the active site of TR through 25 amino acid residues.88 The mutually exclusive substrate specificity between the trypanothione-TR system and its human analogy GSSG-GR system is revealed which is probably related to the structure differences in the orientation of the domains as well as the steric and electrostatic factors of the two enzymes.

Reversible inhibition of TR by trivalent antimonials has been observed in a time-dependent manner with a pseudo-first-order rate related to antimony concentration and the inhibition occurs within the disulfide binding site, provided that the redox-active disulfide binding site is first reduced by NADPH (reduced nicotinamide adenine dinucleotide phosphate).87 This, in turn, suggested that the thiol is likely to react with the antimonial first. The interaction of the free antimonial with thiol generates a transient monothio-antimony adduct followed by a rapid intramolecular rearrangement and binding of the charge-transfer thiol from the enzyme to form a stable antimony-thiolate complex.87

Like As111 and Bi111, Sb111 has been demonstrated to be able to form a stable complex with the tripeptide GSH with a stoichiometry Sb(GS)3. The rate of uptake of the complex into red blood cells and complexation with intracellular GSH is rapid, a matter of minutes.89 Strong binding of Sb111 to trypanothione (T(SH)2) has been chemically characterized by both electrospray ionization mass spectrometry (ESI-MS) and NMR spectroscopy (pM = 24.5 and 22.1 for Sb(TS2) and Sb(GS)3, respectively, where pM = -log[M], and [M] is the concentration of unchelated unhydrolyzed metal ion at equilibrium in a pH 7.4 solution of 1 mM metal and 10 mM ligand). In contrast to the GSH complex, each Sb111 coordinates to only one T(SH)2 with two sulfurs provided by Cys residues and probably an oxygen from a water molecule (Figure 23.4).90 T(SH)2 has been known to be the most abundant low molecular mass thiol-containing ligand (>80%) in Leishmania parasites.91,92 Together with TR, it is crucial for the growth and survival of the parasites.93,94 The resistance to antimonials in Leish-mania is related to the overproduction of T(SH)2.95 For this reason, trypano-thione metabolism in parasites is considered as a valid target for drug design.

Recently, the crystal structure of SbIII-bound ArsA ATPase complex has shown that there is an SbIII cluster, and that SbIII acts as a soft metal and coordinates to three donating atoms, with two of them from the protein residues and one from a non-protein ligand (Cl~) (Figure 23.5).96 The coordination of AsIII to the protein at the metal cluster is essentially identical.97 Among the coordinated ligands, two are from residues of domains A1 and A2 and a third is from a non-protein ligand chloride (Cl~), and the binding of SbIn/AsnI brings together the two domains of the enzyme, triggering the ATP-dependent activation. Both SbIII(TS2) and SbIII-bound ArsA ATPase complexes can probably be regarded as intermediates, and the bound water or chloride (Cl~) can readily be replaced by a nitrogen (e.g. histidine) or thiolate sulfur (e.g. cysteine) from another molecule (e.g. protein or enzyme). Indeed,

Figure 23.4 Structures of Sb(T(S)2) complex together with the oxidized form of trypanothione (adapted with permission from Ref. 90 copyright Wiley-VCH) (see Plate 4)

Figure 23.4 Structures of Sb(T(S)2) complex together with the oxidized form of trypanothione (adapted with permission from Ref. 90 copyright Wiley-VCH) (see Plate 4)

Sbm cluster fEhS

Sbm cluster fEhS

C172

Figure 23.5 Overall structure of the ArsA ATPase (a) and coordination of Sb111 at the metal binding site (b) (coordinates from PDBID: 1F48). Residues His148, Cys113 and Cys172 are from domain A1 while Ser420, Cys422 and His453 are from domain A2. The purple balls represent the bound chlorides (Cl~) (see Plate 5)

C172

H453

Figure 23.5 Overall structure of the ArsA ATPase (a) and coordination of Sb111 at the metal binding site (b) (coordinates from PDBID: 1F48). Residues His148, Cys113 and Cys172 are from domain A1 while Ser420, Cys422 and His453 are from domain A2. The purple balls represent the bound chlorides (Cl~) (see Plate 5)

a ternary complex can readily form between SbIII-trypanothione and other monothiol ligands such as GSH and cysteine.83 Although they are thermo-dynamically stable, both the SbIII(TS2) and the ternary complexes are kinet-ically labile, and the free and bound forms of either trypanothione or cysteins/ GSH exchange on the *H NMR time scale. This exchange is also pH-dependent, and is relatively slow at low pH (~4), but considerably faster at pH 7. Such a facile exchange may be important for the transport and delivery of SbIn in vivo.

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