Targeting Trypanothione Synthetase and Trypanothione Reductase: Development of Common Inhibitors to Tackle Trypanosomatid Disease

Abstract

Neglected Tropical Diseases (NTDs) encompass a range of disorders, including infectious diseases caused by viruses, bacteria, parasites, fungi, and toxins, mainly affecting underprivileged individuals in developing countries. Among the NTDs, those caused by parasites belonging to the Trypanosomatidae family are particularly impacting and require attention, since the lack of financial incentives has led to constraints on the development of novel drugs to tackle them effectively. To circumvent the minor advances in drug discovery in this area, academic research emerges as a crucial player, namely through the identification and validation of new drug targets, thereby contributing to the development of more efficient, safe, and less expensive therapies against Trypanosomatidae infections. Noteworthy, this is a matter of utmost urgency since these diseases are endemic in countries with low socioeconomic standards. This review provides a comprehensive understanding of the current paradigm of NTDs caused by parasites belonging to the Trypanosomatidae family, addressing the ongoing limitations and challenges associated to the current chemotherapy solutions for these diseases and discussing the opportunities unravelled by recent research that led to the identification of new biomolecular targets that are common to Trypanosomatidae parasites. Among these, the unique properties of Trypanothione Synthetase (TryS) and Trypanothione Reductase (TryR), two key protozoan enzymes that are essential for the survival of Trypanosoma and Leishmania parasites, will be emphasised. In addition to a critical analysis of the latest advances in the discovery of novel molecules capable of inhibiting TryS and TryR, the possibility of dual targeting through a combination of TryS and TryR inhibitors will be addressed.

Keywords: inhibitors; leishmaniases; neglected tropical diseases; trypanosomiasis; trypanothione reductase; trypanothione synthetase.

Figure 2

Biosynthesis of trypanothione [T(SH)₂], occurring in two consecutive steps. In the first, driven by glutathionyl-spermidine synthetase (GspS) or TryS, the glycine carboxylate group of glutathione (GSH) is covalently bound to one of the terminal amino groups of spermidine; the second can only be catalysed by TryS, in which the glycine carboxylate group of another glutathione is covalently linked to the free terminal amino group of N-glutathionylspermidine. The whole process consumes two molecules of adenosine triphosphate (ATP) to produce one molecule of T(SH)₂. Abbreviations: ADP, Adenosine Diphosphate; P_i, Inorganic Phosphate. Adapted from [112,113].

Figure 3

General scheme depicting trypanothione-dependent redox reactions. The process of T(SH₎₂ reduction is depicted in light blue: Whenever a trypanothione molecule complexes with an oxidative substance (e.g., metals, drugs, and oxoaldehydes), there is formation of a disulphide bridge between the two sulphurs present in each glutathione molecules (cysteine residues), with formation of a trypanothione disulphide (TS₂). It is possible to recover the initial reduced form of trypanothione [T(SH)₂] by breaking the disulphide bond, a reaction catalysed by TryR (red). Thus, T(SH)₂ assumes five functions, depicted in dark blue: (1) depletion of hydrogen peroxide (H₂O₂) via ascorbate-dependent detoxification; (2) neutralisation of hydroperoxides (ROOH) through the complex tryparedoxin/tryparedoxin peroxidase I (TXN/TXNPx); (3) reduction of ribonucleotides into deoxyribonucleotides as essential precursor subunits of DNA; (4) elimination of metals and drugs by complexing with them and forming thiol-conjugates that are then exported/sequestered; and, finally, (5) direct reduction of disulphides into their respective thiols by the dithiol. The solid arrows represent all processes involving H₂O₂, the bold solid arrows indicate all initial/final substrates, and the dashed arrows illustrate all oxidation/reduction reactions. Abbreviations: A, one-electron oxidant; APX, Ascorbate-dependent Peroxidase; Asc, Ascorbate; dhAsc, Dehydroascorbate; FeSOD, Iron-Superoxide Dismutase; GPx-I, GPx-II, Glutathione Peroxidase-like tryparedoxin peroxidases I and II; GSSG, Glutathione Disulphide; GSH, Glutathione; RR, Ribonucleotide Reductase; TXNPx, Tryparedoxin Peroxidases. Adapted from [115].

Figure 4

Structural representation of paullone derivatives 1 and 2, with anti-TryS activity [128,129,130,131]. Legend: Lb, L. braziliensis; Li, L. infantum; LiTryS, L. infantum TryS; SI, Selectivity Index; Tb, T. brucei; TbTryS, T. brucei TryS; TcTryS, T. cruzi TryS.

Figure 5

Structural representation of compounds 3, 4, and 5, with anti-TryS activity [132,133]. Legend: Tb, T. brucei; TbTryS, T. brucei TryS.

Figure 6

Structural representation of compound 6, with anti-TryS and anti-TryR activity [134]. Legend: Ld, L. donovani.

Figure 7

Structural representation of compound 7, with anti-TryS activity [129]. Legend: LiTryS, L. infantum TryS; TbTryS, T. brucei TryS; TcTryS, T. cruzi TryS.

Figure 8

Structural representation of compounds 8 and 9, with anti-TryS activity [135]. Legend: LiTryS, L. infantum TryS; TbTryS, T. brucei TryS; TcTryS, T. cruzi TryS.

Figure 9

Structural representation of compound 10, with anti-TryS activity [136]. Legend: Ld, L. donovani; Lm, L. major; LmTryS, L. major TryS; Tb, Trypanosoma brucei.

Figure 10

Structural representation of compounds 11, 12, 13, 14, 15, 16, and 17, identified from in silico studies as potential TryS inhibitors [137,138,139]. Legend: Li int, Intracellular L. infantum; LiTryS, L. infantum TryS; SI, Selectivity Index; ΔG bind, Change in Gibbs free energy on binding to the target.

Figure 11

Structural representation of mepacrine (compound 18), an antimalarial drug with anti-TryR activity [142,143]. Legend: K_i,apparent, Apparent Inhibition Constant; TcTryR, T. cruzi TryR.

Figure 12

Structural representation of diarylsulphide derivatives 19 and 20, with anti-TryR activity [144,145]. Legend: hGR, Human Glutathione Reductase; K_i, Inhibition Constant; Li, L. infantum.

Figure 13

Structural representation of leads with anti-TryR activity; 21, 22, and 23 [146,149,150]. Legend: hGR, Human Glutathione Reductase; K_i, Inhibition Constant; LiTryR, L. infantum TryR; Tb, T. brucei; Tb endTryR, Endogenous T. brucei TryR; TbTryR, T. brucei TryR; TcTryR, T. cruzi TryR.

Figure 14

Structural representation of leads with anti-TryR activity; 24, 25, 26, and 27 [141,151]. Legend: hGR, Human Glutathione Reductase; Li, L. infantum; Li ax amastigotes, L. infantum axenic amastigotes; Li int, Intracellular L. infantum; LiTryR, L. infantum TryR; SI, Selectivity Index.

Figure 15

Structural representation of compounds 28, 29, 30, and 31, with proven anti-TryR activity [158,159,160]. Legend: Lb, L. braziliensis; Ld, L. donovani; Li, L. infantum; SI, Selectivity Index; Tc, T. cruzi; TcTryR, T. cruzi TryR; Mϕ, macrophages.

Figure 16

Structural representation of compounds 32 and 33, with anti-TryR activity [161]. Legend: hGR, Human Glutathione Reductase; Li, L. infantum; LiTryR, L. infantum TryR.

Figure 17

Structural representation of compounds 34, 35, 36, 37, 38, and 39, with proven anti-TryR activity [163,164,165,166]. Legend: α, Inhibitor’s binding affinity to the free enzyme compared to its binding affinity to the enzyme-substrate complex; act. LiTryR IC₅₀, Inhibitory activity against L. infantum TryR oxidoreductase; β, Catalytic activity fraction between the enzyme–substrate–inhibitor complex to the enzyme–substrate complex; dim. LiTryR IC₅₀, Inhibitory activity through monomer displacement of L. infantum TryR; hGR, Human Glutathione Reductase; K_i, Inhibition Constant; k_n, First-order constant number n; Li, L. infantum; Li ax. amastigotes, L. infantum axenic amastigotes; Li int. amastigotes, L. infantum intracellular amastigotes; LiTryR, L. infantum TryR; SI, Selectivity Index; TbTryR, T. brucei TryR; TcTryR, T. cruzi TryR; TcoTryR, T. congolense.

Figure 18

Structural representation of compounds 40, 41, and 42, with anti-TryR activity [169,170,171]. Legend: FEB, Free Energy of Binding; hGR, Human Glutathione Reductase; K_i, Inhibition Constant; Li ax. amast, L. infantum axenic amastigotes; Li int. amast, L. infantum intracellular amastigotes; LiTryR, L. infantum TryR; SI, Selectivity Index; TbTryR, T. brucei TryR; Trypo, Trypomastigotes.

Figure 19

Structural representation of compounds 43, 44, and 45, with anti-TryR activity [176,178,179]. Legend: k_cat, Turnover number; K_m, Michaelis-Menten Constant; Ld, L. donovani; Li, L. infantum; SI, Selectivity Index; Tb, T. brucei; Tc, T. cruzi; TcTryR, T. cruzi TryR.

Figure 1

Representation of the chemical structures of the available drugs used in the treatment of Human African Trypanosomiasis (highlighted in dark blue), Chagas Disease (highlighted in red), and leishmaniases (highlighted in light blue). Molecular structures are of public domain and were drawn using the available software Chemdraw Ultra 12.0.