Trypanothione, a metabolite specific to trypanosomatid parasites, is enzymatically synthesized from spermidine and glutathione by the consecutive action of the ATP-dependent carbon-nitrogen ligases, glutathionylspermidine synthetase and trypanothione synthetase. As part of our programme aimed at developing inhibitors of these enzymes, we have synthesized a series of analogues of glutathione (gamma-L-Glu-L-Cys-Gly) and tested them as substrates or inhibitors of glutathionylspermidine synthetase. Recognition at the gamma-glutamyl moiety appears to be essential, as any modification of this part of glutathione results in a total loss of activity as a substrate. Alkylation of the thiol side chain of cysteine with methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (kcat/Km) as substrates equivalent to or better than glutathione. In contrast, the bulkier S-butyl analogue was a much poorer substrate. Substitution of L-Cys by amino acids with an alkyl side-chain is also well tolerated giving relative catalytic efficiencies of 1.1 and 1.5 for peptide analogues containing L-Val and L-Ile respectively. Other analogues, where the bulk of the alkyl chain is increased further (as in L-Leu or L-Phe) or where the glycine moiety is replaced with L-Ala, are inhibitors rather than substrates.

A series of phosphonic and phosphinic acid derivatives of glutathione were synthesized as potential inhibitors of glutathionylspermidine synthetase, an essential enzyme in the biosynthesis of trypanothione in trypanosomatids. The compounds showed moderate activity.

Intracellular levels of glutathione and glutathionylspermidine conjugates have been measured throughout the growth phases of Escherichia coli. Glutathionylspermidine was present in mid-log-phase cells, and under stationary and anaerobic growth conditions accounted for 80% of the total glutathione content. N1,N8-bis(glutathionyl)spermidine (trypanothione) was undetectable under all growth conditions. The catalytic constant kcat/Km of recombinant E. coli glutathione reductase for glutathionylspermidine disulphide was approx. 11,000-fold lower than that for glutathione disulphide. The much higher catalytic constant for the mixed disulphide of glutathione and glutathionylspermidine (11% that of GSSG), suggests a possible explanation for the low turnover of trypanothione disulphide by E. coli glutathione reductase, given the apparent lack of a specific glutathionylspermidine disulphide reductase in E. coli.

Trypanothione reductase, central to the redox defense systems of parasitic trypanosomes and leishmanias, is sufficiently different in its substrate-specificity from mammalian glutathione reductase to represent an attractive target for chemotherapeutic intervention. Previous studies of the physiological substrates trypanothione (N1,N8-bis(glutathionyl)spermidine) and N1-glutathionylspermidine disulphide established that the spermidine moiety of these substrates can be replaced by the 3-dimethyl-propylamide group (N1-glutathionyl-N3-dimethyl-propylamide). With this modification, the specificity for the gamma-glutamyl moiety of the substrate was examined. Kinetic analysis of a series of substrate analogues indicated that neither the alpha-carboxylate or alpha-amino functions of the L-gamma-glutamyl group is essential for recognition, since this group could be replaced by uncharged benzyloxycarbonyl or t-butyloxycarbonyl groups with relative catalytic efficiencies (kcat/Km) of 58 and 11%, respectively, of N1-glutathionyl-N3-dimethylpropylaminedisulphide. Other substitutions are less well tolerated (e.g., beta-L-aspartyl or aminobutyryl) or not at all (e.g., glutaryl). These findings are discussed in relation to the structural model of TR from Trypanosoma congolense. The successful structural replacements achieved have potential application for drug delivery.

The enzyme trypanothione reductase has been identified as a prime target for the rational design of inhibitors which may have clinical use in the treatment of tropical diseases caused by the genera Trypanosoma and Leishmania.To aid the design or identification of new inhibitors of this enzyme we have elucidated the structural detail of a trypanothione reductase complexed with one of the naturally occurring substrates, N1-glutathionylspermidine disulphide, by single-crystal X-ray diffraction methods at 0.28-nm resolution. The model for the Crithidia fasciculata enzyme-substrate complex has an R-factor of 14.8% and root-mean-square deviations of 0.0015 nm and 3.3-degrees on bond lengths and angles respectively. Hydrogen bonding and van der Waals interactions between the enzyme and substrate are dominated by the amino acid side chains. The substrate binds in a rigid active site such that one glutathione moiety is in a V-shape, the other in an extended conformation. One spermidine moiety binds closely to a hydrophobic patch in the active site formed by a tryptophan and a methionine. Distances between the methionine Sdelta and the terminal N of this spermidine suggest that a hydrogen bond may supplement the hydrophobic interactions in this part of the active site.

Two enzymes involved in the biosynthesis of the trypanosomatid-specific dithiol trypanothione - glutathionylspermidine (Gsp) synthetase and trypanothione (TSH) synthetase - have been identified and purified individually from Crithidia fasciculata. The Gsp synthetase has been purified 93-fold and the TSH synthetase 52-fold to apparent homogeneity from a single DEAE fraction that contained both activities. This constitutes the first indication that the enzymatic conversion of two glutathione molecules and one spermidine to the N1, N8-bis(glutathionyl)spermidine (TSH) occurs in two discrete enzymatic steps. Gsp synthetase, which has a k(cat) of 600/min, shows no detectable TSH synthetase activity, whereas TSH synthetase does not make any detectable Gsp and has a k(cat) of 75/min. The 90-kDa Gsp synthetase and 82-kDa TSH synthetase are separable on phenyl Superose and remain separated on gel filtration columns in high salt (0.8 M NaCl). Active complexes can be formed under low to moderate salt conditions 0.0-0.15 M NaCl), consistent with a functional complex in vivo.

D,L-dihYdrolipoamide and D,L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10(9) and 4.51 x 10(9) M-1, respectively. These complexes possess 6-membered cyclic dithioarsenite rings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500-fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione. L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate. The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 +/- 4.3 and 9.7 +/- 2.9 pmol (10(8) cells)-1, respectively). The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 +/- 1.4 and 47.8 +/- 3.7 mU mg-1, respectively). However, the K(m) for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains. Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane. These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs.

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (> 200-fold resistance to melarsen oxide and > 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.

Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.

Trypanosomatids are mainly responsible for heterogeneous parasitic diseases: Leishmaniasis, Sleeping sickness, and Chagas disease and control of these diseases implicates serious challenges due to the emergence of drug resistance. Redox-active biomolecules are the endogenous substances in organisms, which play important role in the regulation of redox homeostasis. The redox-active substances like glutathione, trypanothione, cysteine, cysteine persulfides, etc., and other inorganic intermediates (hydrogen peroxide, nitric oxide) are very useful as defence mechanism. In the present review, the suitability of trypanothione and other essential thiol molecules of trypanosomatids as drug targets are described in Leishmania and Trypanosoma. We have explored the role of tryparedoxin, tryparedoxin peroxidase, ascorbate peroxidase, superoxide dismutase, and glutaredoxins in the anti-oxidant mechanism and drug resistance. Up-regulation of some proteins in trypanothione metabolism helps the parasites in survival against drug pressure (sodium stibogluconate, Amphotericin B, etc.) and oxidative stress. These molecules accept electrons from the reduced trypanothione and donate their electrons to other proteins, and these proteins reduce toxic molecules, neutralize reactive oxygen, or nitrogen species; and help parasites to cope with oxidative stress. Thus, a better understanding of the role of these molecules in drug resistance and redox homeostasis will help to target metabolic pathway proteins to combat Leishmaniasis and trypanosomiases.

We have obtained well-ordered single crystals of the flavoenzyme trypanothione reductase from Crithidia fasciculata. The crystals are tetragonal rods with unit cell dimensions a = 128·6 Å, c = 92·5 Å. The diffraction pattern corresponds to a primitive lattice, Laue class 4/m. Diffraction to better than 2·4 Å has been recorded at the Daresbury Synchrotron. The accurate elucidation of the three-dimensional structure of this enzyme is required to support the rational design of compounds active against a variety of tropical diseases caused by trypanosomal parasites.

Book publishing in anglophone middle Africa must be interpreted against a background of illiteracy, an emphasis on achievement reading, and other social and infrastructural elements. International market forces acting through metropolitan publishers determine general and nonfiction publishing, whereas the major market governing African creative writing is found in African schools and universities. The actions of British publishing multinationals only diverge slightly from the pattern of multinational action in less developed countries. Educational publishing is dominated by these multinationals who, through localization and because of the barriers to African educational publishing, have generally retained their position. State publishing, retarded by problems, has not yet proved very successful. Only limited government action has been taken to adjust the balance in favor of indigenous publishing because a symbiotic relationship exists between the educational system, multinational publishers, bureaucrats and transnationalism.