Mini review on tricyclic compounds as an inhibitor of trypanothione reductase.

Trypanosomiasis and leishmaniasis are two most ruinous parasitic infectious diseases caused by Trypanosoma and Leishmania species. The disease affects millions of people all over the world and associated with high morbidity and mortality rates. The review discuss briefly on current treatment of these parasitic diseases and trypanothione reductase (TryR) as potential targets for rational drug design. The enzyme trypanothione reductase (TryR) has been identified as unique among these parasites and has been proposed to be an effective target against for developing new drugs. The researchers have selected this enzyme as target is due to its substrate specificity in contrast to human analogous glutathione reductase and its absence from the host cell which makes this enzyme an ideal target for drug discovery. In this review we have tried to present an overview of the different tricyclic compounds which are potent inhibitors of TryR with their inhibitory activities against the parasites are briefly discussed.

The hemoflagellate protozoa of the family Trypanosomatidae are the causative agents of tropical diseases such as human African sleeping sickness (Trypanosoma brucei gambiense, T. brucei rhodesiense), Chagas’ disease (South American trypanosomiasis, Trypanosoma cruzi) and the visceral, cutaneous and mucocutaneous manifestations of leishmaniasis (e.g., Leishmania donovani, Leishmania tropica, Leishmania braziliensis). According to world health organization African trypanosomiasis was estimated to cause 48,000 deaths and a disease burden of 1.5 million disability-adjusted life years (DALYs) annually; Chagas’ disease, 14,000 deaths and a disease burden of 0.7 million DALYs annually; leishmaniasis, 51,000 deaths and a disease burden of 2.1 million DALYs annually. Recently drug discovery program directed toward leishmaniasis, malaria, Chagas disease and sleeping sickness has increased sharply and not only because they are major killing diseases, but also because disease control becomes more difficult due to a number of factors that limit the utility of current drugs such as high cost, poor compliance, drug resistance, low efficacy and poor safety. The development of new chemotherapeutic agents for the treatment of these parasitic diseases has been hindered due to lack of interest shown by top innovator pharmaceutical companies, which might be due to low profitability in this domain as poor are more sufferer of these disease.[1234]

The present review focuses on the major human diseases caused by trypanosomal and leishmanial infections and inhibitors of tryanothione reductase as potential targets for designing chemotherapeutic agents against these diseases. Table 1 gives an outline of the major human trypanosomiasis and leishmaniasis with their global annual disease burdens in terms of DALY The chemical structures of various antitrypnosomal and antileishmanial agents are presented as Figure 1.

Table 1

The major trypanosomiasis and leishmaniasis and their causative agents current treatments

Figure 1

Structures of various drugs used for the treatment of trypanosomiasis and leishmaniasis

There are several targets in these parasites through which drug or an investigational molecules act and some of these targets includes deoxyribonucleic acid (DNA) topoisomerases, Ergosterol biosynthesis, Purine salvage pathway, trypanothione reductase (TryR), microtubule assembly inhibitor etc.[5] Among all the targets known for trypanosomes and Leishmania, TryR has gained a lot of attention as a potential target for discovering a new antiparasitic drug for the treatment of human African sleeping sickness caused by T. brucei gambiense, T. brucei rhodesiense, Chagas’ disease (South American trypanosomiasis, T. cruzi) and the visceral, cutaneous and mucocutaneous manifestations of leishmaniasis (e.g., L. donovani, L. tropica, L. braziliensis).

As potential drug target in trypanosomes and Leishmania, TryR has been identified through the discovery of a fundamental difference between the redox defense system of the trypanosomal/leishmanial parasite and the infected host. The mammalian redox defense system is based on glutathione (l-g-glutamyl-l-cysteinylglycine) and glutathione disulfide reductase (glutathione reductase (GR); EC 1.6.4.2), this system is replaced in trypanosomatids by an analogous system based on trypanothione (N, N-bis [glutathionyl] spermidine) and trypanothione disulfide reductase (TryR; EC 1.6.4.8). The structures of the disulfide substrates for TryR and GR are illustrated in Figure 2. TryR is a nicotinamide adenine dinucleotide phosphate-oxidase-dependent flavoprotein oxidoreductase which maintains an intracellular reducing environment by the recycling of trypanothione disulfide T[S]2 to its dithiol T[SH]2 form. Trypanothione is oxidized back to T[S]2 following reaction with potentially damaging radicals and oxidants generated by aerobic metabolism and by host macrophages. By maintaining a high intracellular ratio of T[SH]2 the TryR redox cycle is a primary line of defense for these parasites against respiratory burst responses from the mammalian host. The trypanothione system is necessary for protozoan survival because the dithiol trypanothione is required for the synthesis of DNA precursors, the homeostasis of ascorbate, the detoxification of hydroperoxides and the sequestration/export of thiol conjugates. Moreover, the majority of peroxidases that eliminate the reactive oxygen species generated in the aerobic metabolism are trypanothione dependent. Disabling the function of TryR in Leishmania and T. brucei has been shown to markedly increase the parasites’ sensitivity to oxidative stress.

Figure 2

(a) Structure of Trypanothione and glutathione and their reduced form. (b) Mechanism of redox recycling of T[S]2 to T[SH]2 and GSSG to GSH in parasite and host cell respectively

T[S]2 differs from glutathione disulfide (GSSG) by the presence of a spermidine cross-link between the two glycyl carboxyl groups [compare GSSG and T[S]2 in Figure 2]. Due to structural and charge differences between T[S]2 and GSSG, TryR and GR are mutually exclusive with respect to substrate specificity. Thus the essential requirement of TryR in trypanosomal/leishmanial parasite and its absence in host metabolism make it an attractive therapeutic target for designing specific inhibitor. In the preceding section we have tried to compile various tricyclic compounds, which have shown potent inhibiting activity against TryR.[678]

Tricyclic trypanothione reductase (TryR) inhibitors

Knowing the fact that both TryR and GR has exclusive substrate specificity, various molecules have been explored as inhibitors of TryR which includes hydrophobic linear polyamine derivatives and the naturally occurring bis (tetrahydrocinnamoyl) spermine, Ponasik et al.[9]

To further address the need for new compounds and new compound classes, Richardson et al.[10] initiated screening of 1266 pharmacologically active compounds from the Sigma-Aldrich LOPAC1280 library. These compounds were screened against TryR and the top hits counter-screened against GR and live T. brucei parasites, yielding the IC values, selectivity for TryR over GR and antiparasitic activity. Among all the 1266 compounds from Sigma-Aldrich LOPAC1280 library, several tricyclic derivatives were identified as a potent inhibitor TryR [Figure 3].

Figure 3

Structures of various tricyclic drug having potent trypanothione reductase inhibiting activity and selectivity of human glutathione reductase

Recently, lunarine a spermidine-based macrocyclic alkaloid [Figure 4] has been identified as a competitive, time-dependent inhibitor of TryR. Lunarine is composed of a spermidine chain with the terminal nitrogen atoms forming amide linkages with two α, β-unsaturated carboxylic acid functions disposed upon an unusual 3-oxohexahydrodibenzofuranyl tricyclic scaffold.[11]

Figure 4

Structure of Kukoamine A and Lunarine

A study done by Hamilton et al.[12] presented a possible mechanism for this time dependent inhibition, which involves the covalent modification of a redox-active cysteine residue in the active site of TryR (C53) by conjugate addition to one of these unsaturated amide moieties in the lunarine macrocycle [Figure 5]. This was supported by both the requirement for the enzyme in its reduced form and the presence of a potential Michael acceptor unit in the inhibitor.

Figure 5

Proposed mechanism for time-dependent inactivation of trypanothione reductase by the reversible formation of a covalent adduct between an active site thiol (C53) and one of the α, β-unsaturated amide groups of lunarine

The Hamilton et al.[13] further explored this approach by preparing some benzofuranyl-based acyclic bis-polyamine analogues (1-5) of lunarine. In their approach they removed skew boat cyclohexanone moiety of lunarine leaving a planar bicyclic benzofuranyl scaffold. The acyclic bis-polyamine derivatives were chosen since bis-polyamine functionalized disulfides such as the naturally occurring N1-glutathionylspermidine disulfide and the synthetic bis-dimethylaminopropyl- and bis-N-methylpiperazinyl amides of Ellman's reagent (DTNB) are known TryR substrates. These three polyamine chains were chosen for functionalisation of a 3, 5-disubstituted benzofuranyl template to give potential inhibitors (1-5).

In their series of compounds the bis-polyaminoacrylamide derivatives (1-3) were shown to be competitive inhibitors, but only the bis-4-methyl-piperazin-1-yl-propylacrylamide derivative 3 displayed time-dependent activity. Analysis of in vitro activity showed that these compounds were simple competitive inhibitors of TryR, with respect to T[S]2. The starting material 4, 5 and 6 which do not have polyamine side chain were also evaluated for enzyme inhibitory activity against TryR. It was observed that due to the absence of any polyamine side chains, neither the diester 6 nor the diacid 4 showed any inhibitory activity towards TryR at 100 μM concentrations.

Bonnet et al.[14] designed and synthesized a series of symmetrical substituted 1,4-bis (3-aminopropyl) piperazines derivatives. The compounds were prepared by reacting 1,4-bis (3-aminopropyl) piperazine with various aldehydes via reductive amination. All compounds were tested for their inhibitory potency towards TryR from T. cruzi and their trypanocidal effects upon T. cruzi trypomastigote as well as for their trypanocidal effect upon T. brucei trypomastigote stage. They also studied cytotoxicity toward human MRC-5 cells (diploid embryonic lung cell line). Their study revealed that in the aromatic series the most potent TR inhibition was observed for polyphenyl derivatives 7 and 8 (IC50 of 32 and 28 μM) respectively. These two compounds showed 100% Inhibition on T. brucei at a concentration of 6.3 and 3.1 μM.

A study done by Chibale et al.[15] reported design and synthesis of 9, 9-dimethylxanthene derivatives (9-14) as potential inhibitor of TR. They designed target compounds in which 9, 9-dimethylxanthene ring was exploited as an aromatic hydrophobic tricyclic moiety that bears resemblance to the aromatic hydrophobic tricyclic moieties found in other tricyclic compounds already reported as competitive inhibitors of TR, where the tricyclic moiety binds in the hydrophobic pocket involved in recognition of the spermidine moiety of trypanothione disulfide, the substrate for TR. Moreover, in 9, 9-dimethylxanthene system potential multiple sites are provided by chemically reactive 2, 7 and 4, 5 positions for introducing chemical diversity. Apart from these functions, they also introduced terminal tertiary amino group (exemplified by the dimethylamino group) into compounds (9-14) to provide a positive charge which has been shown to favor TryR over GR, the closest related host enzyme. Thorough analysis of results of TryR inhibitions study [Table 2], it was found that compounds (9-11) bearing either one (10) or no methylene spacer (9 and 11) between the tricyclic moiety and the secondary nitrogen atom generally show weaker inhibition of TR compared to derivatives with two or three carbon methylene spacer (12, 13 and 14) [Figure 6].

Table 2

Percentage inhibition of TryR and ED50 of compounds (9-14)

Figure 6

Potential compounds from different literature

They concluded that within the series of compounds (9-15), there is no clear correlation between potency as inhibitors of TR and the in vitro antiparasitic activities and that there is no apparent single structural feature controlling in vitro antiparasitic activities.

In another study by Chibale et al.[16] had shown that chemical structures of agents which increase accumulation and/or reverse chloroquine (CQ) resistance reveals the importance of a hydrophobic group and a protonatable nitrogen and incidentally these are the same chemical features known to be important for potent and selective against TR inhibitor. This was exemplified in the antimalarial acridine quinacrine and tricyclic antidepressants promazine and clomipramine. A wide variety of structurally diverse drugs (including Mepacrine, Promazine and clomipramine) have also been described as CQ resistance reversal agents.

Based on this observation it was envisaged by Chibale et al. that these tricyclic could be utilized as dual purpose scaffolds for the discovery and development of CQ resistance reversal agents and inhibitors of TryR. They designed and developed a series of xanthenes derivatives as potential TR inhibitor. All the tested compounds showed weak TR inhibitory activity against T. cruzi TryR. Among the all derivatives of xanthenes, compound 16 showed highest TryR inhibitory activity of IC50 of 35.7 μM and intrinsic antimalarial activity of IC50 = 1.748 μM.

A series of sulfonamide and urea derivatives of quinacrine with varying methylene spacer lengths have been tested for inhibition of TryR and for activity in vitro against strains of the parasitic protozoa Trypanosoma, Leishmania and Plasmodium by Kelly et al.[17] The results of the studies revealed [Table 3] that these derivatives were superior inhibitors of TryR relative to quinacrine with the best compound being 40 times more potent. Results of their studies revealed that sulfonamide derivatives were more active than urea in inhibiting TryR and this trend of activity did not correlate with the in vitro activities against L. donovani, T. cruzi, and T. brucei.

Table 3

In vitro sensitivity of the parasite to quinacrine analogue (17-19) and (20-23)

Girault et al.[18] designed and optimized various bis (2-aminodiphenylsulfides) derivatives and tested for inhibitory activity against TryR from T. cruzi. In the series bis (2-aminodiphenylsulfides) compounds possessing three side chains were synthesized and various moieties were introduced at the end of the third side chain, including acridinyl or biotinyl moieties for fluorescent labeling studies. The results of the TR inhibition screening showed that most potent inhibitor (24) with IC50 = 200 nM, whereas the tricyclic derivatives (25) exhibited IC50 of 250 nM. All the compounds were also tested in vitro upon T. cruzi and Leishmania infantum amastigotes, upon T. brucei trypomastigotes.

In order to provide improved tricyclic derivatives as an inhibitor of TryR, Richardson et al.[10] have reported structural modification of Prochlorperazine which shown IC50 of 7.46 μM against TryR. They prepared a derivative having additional propylbenzene ring on piperazine moiety of Prochlorperazine and found 10 fold increase in IC50 against TryR (IC50 = 0.75 μM).

Conclusion

The enzyme TryR from trypanosomal and leishmanial parasites meets most of the ideal features as a drug target needed for developing a potent and specific inhibitor for treating infections caused by trypanosomal and leishmanial parasites. Of various class of compounds developed as an inhibitor of TryR, tricyclic derivatives have come up with potential to be further exploited as the drug candidate for the treatment of trypanosomiasis and Leishmaniasis. Although a lot research work has been done to provide tricyclic based derivatives as future therapeutic agents against these parasitic diseases, but yet none of tricyclic agent is able to reach at the level of approval as an effective therapy for trypanosomiasis and Leishmaniasis.