Sandra L. Oza

Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

Phenotypic analysis of trypanothione synthetase knockdown in the African trypanosome

Trypanothione plays a pivotal role in defence against chemical and oxidant stress, thiol redox homoeostasis, ribonucleotide metabolism and drug resistance in parasitic kinetoplastids. In Trypanosoma brucei, trypanothione is synthesized from glutathione and spermidine by a single enzyme, TryS (trypanothione synthetase), with glutathionylspermidine as an intermediate. To examine the physiological roles of trypanothione, tetracycline-inducible RNA interference was used to reduce expression of TRYS. Following induction, TryS protein was reduced >10-fold and growth rate was reduced 2-fold, with concurrent 5-10-fold decreases in glutathionylspermidine and trypanothione and an up to 14-fold increase in free glutathione content. Polyamine levels were not significantly different from non-induced controls, and neither was the intracellular thiol redox potential, indicating that these factors are not responsible for the growth defect. Compensatory changes in other pathway enzymes were associated with prolonged suppression of TryS: an increase in trypanothione reductase and gamma-glutamylcysteine synthetase, and a transient decrease in ornithine decarboxylase. Depleted trypanothione levels were associated with increases in sensitivity to arsenical, antimonial and nitro drugs, implicating trypanothione metabolism in their mode of action. Escape mutants arose after 2 weeks of induction, with all parameters, including growth, returning to normal. Selective inhibitors of TryS are required to fully validate this novel drug target.

Leishmania trypanothione synthetase-amidase structure reveals a basis for regulation of conflicting synthetic and hydrolytic activities

The bifunctional trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, trypanothione, glutathionylspermidine, and spermidine in Leishmania.

Chemical Validation of Trypanothione Synthetase A POTENTIAL DRUG TARGET FOR HUMAN TRYPANOSOMIASIS

In the search for new therapeutics for the treatment of human African trypanosomiasis, many potential drug targets in Trypanosoma brucei have been validated by genetic means, but very few have been chemically validated. Trypanothione synthetase (TryS; EC 6.3.1.9; spermidine/glutathionylspermidine:glutathione ligase (ADP-forming)) is one such target. To identify novel inhibitors of T. brucei TryS, we developed an in vitro enzyme assay, which was amenable to high throughput screening. The subsequent screen of a diverse compound library resulted in the identification of three novel series of TryS inhibitors. Further chemical exploration resulted in leads with nanomolar potency, which displayed mixed, uncompetitive, and allosteric-type inhibition with respect to spermidine, ATP, and glutathione, respectively. Representatives of all three series inhibited growth of bloodstream T. brucei in vitro. Exposure to one of our lead compounds (DDD86243; 2 × EC50 for 72 h) decreased intracellular trypanothione levels to <10% of wild type. In addition, there was a corresponding 5-fold increase in the precursor metabolite, glutathione, providing strong evidence that DDD86243 was acting on target to inhibit TryS. This was confirmed with wild-type, TryS single knock-out, and TryS-overexpressing cell lines showing expected changes in potency to DDD86243. Taken together, these data provide initial chemical validation of TryS as a drug target in T. brucei.

Properties of trypanothione synthetase from Trypanosoma brucei.

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.

Dissecting the essentiality of the bifunctional trypanothione synthetase-amidase in Trypanosoma brucei using chemical and genetic methods

The bifunctional trypanothione synthetase‐amidase (TRYS) comprises two structurally distinct catalytic domains for synthesis and hydrolysis of trypanothione (N1,N8‐bis(glutathionyl)spermidine). This unique dithiol plays a pivotal role in thiol‐redox homeostasis and in defence against chemical and oxidative stress in trypanosomatids. A tetracycline‐dependent conditional double knockout of TRYS (cDKO) was generated in bloodstream Trypanosoma brucei. Culture of cDKO parasites without tetracycline induction resulted in loss of trypanothione and accumulation of glutathione, followed by growth inhibition and cell lysis after 6 days. In the absence of inducer, cDKO cells were unable to infect mice, confirming that this enzyme is essential for virulence in vivo as well as in vitro. To establish whether both enzymatic functions were essential, an amidase‐dead mutant cDKO line was generated. In the presence of inducer, this line showed decreased growth in vitro and decreased virulence in vivo, indicating that the amidase function is not absolutely required for viability. The druggability of TRYS was assessed using a potent small molecule inhibitor developed in our laboratory. Growth inhibition correlated in rank order cDKO, single KO, wild‐type and overexpressing lines and produced the predicted biochemical phenotype. The synthetase function of TRYS is thus unequivocally validated as a drug target by both chemical and genetic methods.

Comparative structural, kinetic and inhibitor studies of Trypanosoma brucei trypanothione reductase with T. cruzi

As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3 Å and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6 Å over 482 Cα atoms). Kinetically, the Km for trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The Km for NADPH for the T. brucei enzyme was found to be 0.77 μM using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S = Km values for trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thioridazine and citalopram. The relative IC50 values for the two enzymes were found to vary by no more than 3-fold. Thus trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.

Characterization of recombinant glutathionylspermidine synthetase/amidase from Crithidia fasciculata.

Trypanothione [N1,N8-bis(glutathionyl)spermidine] is a unique metabolite found only in trypanosomatids, where it subsumes many of the functions of GSH in other organisms. In Crithidia fasciculata, two distinct ATP-dependent ligases, glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), are involved in the synthesis of trypanothione from GSH and spermidine. Both enzymes have been cloned previously, but expression in Escherichia coli produced insoluble and inactive protein. Here we report on the successful expression of soluble (His)6-tagged C. fasciculata GspS in E. coli. Following purification using nickel-chelating affinity chromatography, the tag sequence was removed and the enzyme purified to homogeneity by anion-exchange chromatography. The kinetic parameters of the recombinant enzyme have been determined using a coupled enzyme assay and also by HPLC analysis of end-product formation. Under optimal conditions (0.1 M K+-Hepes, pH 7.3) GspS has synthetase activity with apparent K(m) values for GSH, spermidine and MgATP of 242, 59 and 114 microM respectively, and a k(cat) of 15.5 s(-1). Glutathionylspermidine is formed as end product and the enzyme lacks TryS activity. Like E. coli GspS, the recombinant enzyme also possesses amidase activity (EC 3.5.1.78), hydrolysing glutathionylspermidine to GSH and spermidine with a k(cat) of 0.38 s(-1) and a K(m) of 500 microM. GspS can also hydrolyse trypanothione at about 1.5% of the rate with glutathionylspermidine. A single amino acid mutation (Cys-79-->Ala) is shown to ablate the amidase activity without affecting the synthetase activity.

Glutathione-like tripeptides as inhibitors of glutathionylspermidine synthetase. Part 1: Substitution of the glycine carboxylic acid group.

Glutathionylspermidine synthetase/amidase (GspS) is an essential enzyme in the biosynthesis and turnover of trypanothione and represents an attractive target for the design of selective anti-parasitic drugs. We synthesised a series of analogues of glutathione (L-gamma-Glu-L-Leu-Gly-X) where the glycine carboxylic acid group (X) has been substituted for other acidic groups such as tetrazole, hydroxamic acid, acylsulphonamide and boronic acid. The boronic acid appears the most promising lead compound (IC(50) of 17.2 microM).

Glutathione-like tripeptides as inhibitors of glutathionylspermidine synthetase. Part 2: substitution of the glycine part.

Glutathionylspermidine synthetase (GspS) is an essential enzyme in the biosynthesis of trypanothione and is an attractive target for the design of selective anti-parasitic drugs. We synthesised a series of analogues of glutathione (L-gamma-Glu-L-Leu-X) where the glycine moiety has been substituted for other amino acids. These peptides were evaluated as substrates and inhibitors of GspS. Compounds with basic side chains such as diaminopropionic acid were found to be good inhibitors (K(i): 7.2 microM). Substitution of the glycine part abolished the GspS substrate properties of the tripeptide.