Stephen Patterson

Nitro drugs for the treatment of trypanosomatid diseases: past, present, and future prospects

Highlights • Two nitro drugs are currently used in the treatment of trypanosomatid diseases.• Several new nitroaromatics are being developed against the trypanosomatid diseases.• Many nitro drugs and drug candidates act as prodrugs which require bioactivation.• Nitroaromatics can have disparate mechanisms of action in trypanosomatid parasites.

The Anti-Trypanosome Drug Fexinidazole Shows Potential for Treating Visceral Leishmaniasis

Fexinidazole, a drug in clinical testing for African sleeping sickness, shows potential as an oral treatment for another neglected tropical disease. A New Job for an Old Drug Fever, fatigue, weight loss, and swelling of the spleen and liver are all symptoms of visceral leishmaniasis—a tropical disease that is also known as kala-azar or black fever. Caused by the protozoan parasite Leishmania donovani, which is transmitted to people through the bite of a sand fly, the disease is almost always fatal if untreated. Although several drugs exist, they are costly and not always safe, effective, or easy to administer. To address the need for better drugs, Wyllie et al. investigated the possibility of using fexinidazole to treat visceral leishmaniasis. This antiparasitic compound, developed decades ago, is now undergoing early clinical trials as an oral therapy for African sleeping sickness, a disease that is caused by a related protozoan parasite called Trypanosoma brucei. Fexinidazole’s mode of action is thought to involve a trypanosome nitroreductase; the finding that a closely related enzyme is encoded by the leishmania genome inspired Wyllie et al. to pursue fexinidazole as a therapy for visceral leishmaniasis. They found that the compound and two of its metabolites (which rapidly form in vivo) showed activity against both developmental stages of L. donovani in vitro. The metabolites were cytotoxic, killing all the parasites within 30 hours. For unclear reasons, only the metabolites were active against L. donovani grown in macrophages (the cells in which the parasite reproduces during infection). In a mouse model of visceral leishmaniasis, a daily oral dose of fexinidazole for 5 days almost completely suppressed infection—an activity that is comparable to that of drugs currently in clinical use against this deadly tropical disease. Visceral leishmaniasis kills more people than any other parasitic disease except malaria. The clinical trials of fexinidazole for African sleeping sickness have already shown that the drug is extremely safe. The discovery that it may also be a viable oral treatment for visceral leishmaniasis bodes well for those afflicted with this disease. Safer and more effective oral drugs are required to treat visceral leishmaniasis, a parasitic disease that kills 50,000 to 60,000 people each year in parts of Asia, Africa, and Latin America. Here, we report that fexinidazole, a drug currently in phase 1 clinical trials for treating African trypanosomiasis, shows promise for treating visceral leishmaniasis. This 2-substituted 5-nitroimidazole drug is rapidly oxidized in vivo in mice, dogs, and humans to sulfoxide and sulfone metabolites. Both metabolites of fexinidazole were active against Leishmania donovani amastigotes grown in macrophages, whereas the parent compound was inactive. Pharmacokinetic studies with fexinidazole (200 mg/kg) showed that fexinidazole sulfone achieves blood concentrations in mice above the EC99 (effective concentration inhibiting growth by 99%) value for at least 24 hours after a single oral dose. A once-daily regimen for 5 days at this dose resulted in a 98.4% suppression of infection in a mouse model of visceral leishmaniasis, equivalent to that seen with the drugs miltefosine and Pentostam, which are currently used clinically to treat this tropical disease. In African trypanosomes, the mode of action of nitro drugs involves reductive activation via a NADH (reduced form of nicotinamide adenine dinucleotide)–dependent bacterial-like nitroreductase. Overexpression of the leishmanial homolog of this nitroreductase in L. donovani increased sensitivity to fexinidazole by 19-fold, indicating that a similar mechanism is involved in both parasites. These findings illustrate the potential of fexinidazole as an oral drug therapy for treating visceral leishmaniasis.

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.

Dihydroquinazolines as a Novel Class of Trypanosoma brucei Trypanothione Reductase Inhibitors: Discovery, Synthesis, and Characterization of their Binding Mode by Protein Crystallography

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei, the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR–ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

A comparative study of methylglyoxal metabolism in trypanosomatids

The glyoxalase system, comprising the metalloenzymes glyoxalase I (GLO1) and glyoxalase II (GLO2), is an almost universal metabolic pathway involved in the detoxification of the glycolytic byproduct methylglyoxal to d‐lactate. In contrast to the situation with the trypanosomatid parasites Leishmania major and Trypanosoma cruzi, this trypanothione‐dependent pathway is less well understood in the African trypanosome, Trypanosoma brucei. Although this organism possesses a functional GLO2, no apparent GLO1 gene could be identified in the T. brucei genome. The absence of GLO1 in T. brucei was confirmed by the lack of GLO1 activity in whole cell extracts, failure to detect a GLO1‐like protein on immunoblots of cell lysates, and lack of d‐lactate formation from methylglyoxal as compared to L. major and T. cruzi. T. brucei procyclics were found to be 2.4‐fold and 5.7‐fold more sensitive to methylglyoxal toxicity than T. cruzi and L. major, respectively. T. brucei also proved to be the least adept of the ‘Tritryp’ parasites in metabolizing methylglyoxal, producing l‐lactate rather than d‐lactate. Restoration of a functional glyoxalase system by expression of T. cruzi GLO1 in T. brucei resulted in increased resistance to methylglyoxal and increased conversion of methylglyoxal to d‐lactate, demonstrating that GLO2 is functional in vivo. Procyclic forms of T. brucei possess NADPH‐dependent methylglyoxal reductase and NAD+‐dependent l‐lactaldehyde dehydrogenase activities sufficient to account for all of the methylglyoxal metabolized by these cells. We propose that the predominant mechanism for methylglyoxal detoxification in the African trypanosome is via the methylglyoxal reductase pathway to l‐lactate.

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.

Synthesis and evaluation of 1-(1-(Benzo[b]thiophen-2-yl)cyclohexyl)piperidine (BTCP) analogues as inhibitors of trypanothione reductase.

Thirty two analogues of phencyclidine were synthesised and tested as inhibitors of trypanothione reductase (TryR), a potential drug target in trypanosome and leishmania parasites. The lead compound BTCP (1, 1‐(1‐benzo[b]thiophen‐2‐yl‐cyclohexyl) piperidine) was found to be a competitive inhibitor of the enzyme (Ki=1 μM) and biologically active against bloodstream T. brucei (EC50=10 μM), but with poor selectivity against mammalian MRC5 cells (EC50=29 μM). Analogues with improved enzymatic and biological activity were obtained. The structure–activity relationships of this novel series are discussed.

Assessing the Essentiality of Leishmania donovani Nitroreductase and Its Role in Nitro Drug Activation

ABSTRACT The nitroimidazole fexinidazole has potential as a safe and effective oral drug therapy for the treatment of visceral leishmaniasis. To date, nitroheterocyclics have not been used in the treatment of leishmaniasis, and relatively little is known about their mechanism of action. In African trypanosomes, nitro drugs are reductively activated by a type I nitroreductase (NTR), absent in mammalian cells. Modulation of nitroreductase levels in Trypanosoma brucei directly affected sensitivity to nitro compounds, with reduced concentrations of the enzyme leading to moderate nitro drug resistance. In view of the progression of fexinidazole into clinical development for visceral leishmaniasis, here we assess the essentiality of the nitroreductase in Leishmania donovani and the effect of modulating nitroreductase levels on susceptibility to fexinidazole. The failure to directly replace both endogenous copies of the NTR gene, except in the presence of an ectopic copy of the gene, suggests that the NTR gene is essential for the growth and survival of L. donovani promastigotes. Loss of a single chromosomal copy of the L. donovani NTR gene resulted in parasites that were mildly resistant (<2-fold) to the predominant in vivo metabolite of fexinidazole, while parasites overexpressing NTR were 18-fold more susceptible. These data confirm that Leishmania NTR plays a pivotal role in fexinidazole activation. Reliance on a single enzyme for prodrug activation may leave fexinidazole vulnerable to the emergence of drug resistance. However, the essentiality of the NTR in L. donovani promastigotes, combined with the limited resistance shown by NTR single knockout cells, suggests that the potential for the spread of NTR-based resistance to fexinidazole may be limited.

The R Enantiomer of the Antitubercular Drug PA-824 as a Potential Oral Treatment for Visceral Leishmaniasis

ABSTRACT The novel nitroimidazopyran agent (S)-PA-824 has potent antibacterial activity against Mycobacterium tuberculosis in vitro and in vivo and is currently in phase II clinical trials for tuberculosis (TB). In contrast to M. tuberculosis, where (R)-PA-824 is inactive, we report here that both enantiomers of PA-824 show potent parasiticidal activity against Leishmania donovani, the causative agent of visceral leishmaniasis (VL). In leishmania-infected macrophages, (R)-PA-824 is 6-fold more active than (S)-PA-824. Both des-nitro analogues are inactive, underlining the importance of the nitro group in the mechanism of action. Although the in vitro and in vivo pharmacological profiles of the two enantiomers are similar, (R)-PA-824 is more efficacious in the murine model of VL, with >99% suppression of parasite burden when administered orally at 100 mg kg of body weight−1, twice daily for 5 days. In M. tuberculosis, (S)-PA-824 is a prodrug that is activated by a deazaflavin-dependent nitroreductase (Ddn), an enzyme which is absent in Leishmania spp. Unlike the case with nifurtimox and fexinidazole, transgenic parasites overexpressing the leishmania nitroreductase are not hypersensitive to either (R)-PA-824 or (S)-PA-824, indicating that this enzyme is not the primary target of these compounds. Drug combination studies in vitro indicate that fexinidazole and (R)-PA-824 are additive whereas (S)-PA-824 and (R)-PA-824 show mild antagonistic behavior. Thus, (R)-PA-824 is a promising candidate for late lead optimization for VL and may have potential for future use in combination therapy with fexinidazole, currently in phase II clinical trials against VL.

The anti-tubercular drug delamanid as a potential oral treatment for visceral leishmaniasis

There is an urgent requirement for safe, oral and cost-effective drugs for the treatment of visceral leishmaniasis (VL). We report that delamanid (OPC-67683), an approved drug for multi-drug resistant tuberculosis, is a potent inhibitor of Leishmania donovani both in vitro and in vivo. Twice-daily oral dosing of delamanid at 30 mg kg-1 for 5 days resulted in sterile cures in a mouse model of VL. Treatment with lower doses revealed a U-shaped (hormetic) dose-response curve with greater parasite suppression at 1 mg kg-1 than at 3 mg kg-1 (5 or 10 day dosing). Dosing delamanid for 10 days confirmed the hormetic dose-response and improved the efficacy at all doses investigated. Mechanistic studies reveal that delamanid is rapidly metabolised by parasites via an enzyme, distinct from the nitroreductase that activates fexinidazole. Delamanid has the potential to be repurposed as a much-needed oral therapy for VL. DOI: http://dx.doi.org/10.7554/eLife.09744.001