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1. Introduction

Trypanosomatids are protozoans that belong to the order Kinetoplastidae, which includes Trypanosoma cruzi, T. brucei and different species of Leishmania. These parasites are transmitted by bite from tsetse fly, bed bug and sand fly, respectively (1). Trypanosomatids cause neglected tropical diseases, such as Chagas' disease, African sleeping sickness and leishmaniasis, which are debilitating and often fatal (2,3). These diseases affect >20 million individuals worldwide, primarily in rural and underdeveloped areas, resulting in significant suffering in these regions (4,5). For example, >60 million individuals in sub-Saharan African countries are at risk of sleeping sickness presenting with fever, headaches and neurological symptoms, with an annual infection rate of 300,000-500,000(6). Furthermore, ~350 million individuals in 92 countries are at risk of leishmaniasis which causes skin lesions and organ damage, significantly affecting social structures by exacerbating poverty, limiting economic productivity and straining healthcare systems in affected communities, with ~1.2 million new infections each year (7,8).

Current therapies for trypanosomatids, such as antimonials, Melarsoprol and pentamidine, have limited efficacy due to extreme toxicity, resistance, lack of cost efficiency and the need for parenteral delivery in some cases (6,9-11). Untreated trypanosomatid infections can be fatal (10). Furthermore, there are no effective vaccines for the prevention of trypanosomatid diseases, making rational drug design and the search for excellent therapeutic targets crucial (4,12). Extensive research has been conducted on the biochemical characteristics of the life stages of these parasites, leading to the identification of several potential targets for anti-trypanosomatid treatment (13,14). Several potential targets are proposed for anti-trypanosomatid treatment, such as trypanothione redox, sterol biosynthesis, purine uptake, folate metabolism, glycosomal enzymes and protein translocation to the glycosome (15-19). The present review explored rational drug design strategies targeting glycosomal enzymes and protein translocation in trypanosomatids. These targets are crucial because they play critical roles in the unique metabolic pathways of trypanosomatids, which are not found in the host organisms. Glycosomal glycolysis is the only source of energy production, so targeting these pathways is essential for developing effective and selective therapeutics against trypanosomatids.

2. Glycosomes

Glycosomes, which are membrane-bound cytoplasmic peroxisome-like organelles found in the protozoan parasites, Kinetoplastidae, play a vital role in the metabolism of trypanosomatids (8). These organelles have the unique feature of containing numerous enzymes from the glycolytic and gluconeogenic pathways (20).

Investigating energy metabolism in the amastigote stage of T. cruzi is challenging due to the complications arising from host metabolic activity. Nevertheless, studies have demonstrated that glycolysis serves as the primary energy source for the amastigote stage (21,22). Essential proteins and enzymes for parasite survival are synthesized in the cytosol and must be transported across the glycosomal membrane into the glycosome. Thus, inhibiting the transport and activity of these proteins and enzymes can adversely affect the parasites (23). The glycosome is a crucial organelle for trypanosomatid metabolism, containing enzymes essential for glycolysis and researching the energy metabolism of these parasites and their reliance on glycosomes is a promising potential therapeutic target.

Protein translocation across the glycosomal membrane

The formation of the glycosomal membrane, as well as the synthesis and importation of matrix proteins and certain membrane proteins, occurs in the cytoplasm. In some cases, membrane proteins may first be inserted into the endoplasmic reticulum before being transported via vesicles to developing organelles. This importation mechanism is conserved (24,25).

Following their synthesis during translation, glycosomal proteins are produced in the cytosol and then imported into glycosomes either as fully folded proteins or as complexes consisting of multiple subunits (26). The protein import mechanisms of glycosomes and peroxisomes share fundamental similarities and the process relies on a group of proteins called peroxins (PEX) for efficient importation of proteins into these organelles (27-29). In trypanosomatids, more than 10 PEX proteins have been described (28,30). PEX5 and PEX7, receptor proteins that recognize C- or N-terminal import signals in the polypeptide, are responsible for the importation of most glycosomal proteins (31).

Proteins designated for transport possess a peroxisomal targeting signal (PTS), which can be found either at the far end of the C-terminus (referred to as type-1 or PTS1) or in proximity to the N-terminus (known as type-2 or PTS2). While a small number of matrix proteins do not have these PTS signals, most proteins containing PTS1 are recognized by a cytosolic receptor called PEX5, while PEX7 recognizes PTS2-containing proteins. PEX5 functions as a co-receptor and also contains a binding box for PEX7(32) (Fig. 1). The receptors that carry the cargo bind to a loading complex consisting of PEX14, PEX13.1 and PEX13.2, facilitating their attachment to the glycosomal membrane (33,34). The interaction between PEX5 and PEX14 results in the creation of a transient import pore, allowing the translocation of the cargo into the glycosomal matrix (35).

Figure 1 Translocation of glycosomal enzymes from cytosol to glycosome. Blue: Peroxin protein; Yellow: inhibitor that blocks the binding of peroxisome-targeting signal and peroxin protein; Green: peroxisome-targeting signal. PTS1, peroxisome-targeting signal type 1; PTS2, peroxisome-targeting signal type 2; PEX 5, peroxin 5; PEX 7, peroxin 7; PEX 14, peroxin 14.

Once the cargo protein is internalized into the glycosomal matrix, the loaded PEX5 and PEX7 receptors are transported back to the cytosol through interactions with other peroxins associated with the glycosomal membrane (19,36) (Fig. 1).

In trypanosomes, the first seven enzymes involved in glycolysis possess a PTS, which guides them to the glycosomal matrix (32). These PTS sequences are specifically present in proteins intended for transport to the glycosomal matrix (Fig. 1). The glycolytic enzymes in trypanosomes are equipped with a PTS-1 at the COOH-terminus or a PTS-2 at the NH2-terminus. Studies conducted on T. brucei have shown that the cytosolic receptor proteins, TbPEX5p and TbPEX7p, recognize these newly synthesized enzymes (32). Subsequently, each complex, consisting of TbPEX5p with the PTS-1 protein or TbPEX7p with the PTS-2 protein, interacts with TbPEX14p, a component of the glycosomal membrane protein import machinery. This interaction facilitates the translocation of the enzyme complex into the glycosome. Research has shown that depleting PEX5p, PEX7p, or PEX14p levels using RNAi leads to mislocalization of the enzymes into the cytosol in T. brucei. This mislocalization significantly affects the growth of the parasite and often results in its death (32,37).

In trypanosomes, the primary pathway for energy generation is glycolysis, which takes place within the glycosomes instead of within the cytosol. As a result, the glycolytic enzymes produced in the cytoplasm exhibit a limited degree of sequence similarity to their human counterparts, which creates an opportunity for targeted drug development. The accurate localization of glycolytic enzymes is of utmost importance, as misplacement has been demonstrated to lead to the death of the parasite or hindered growth (32,37,38). However, this potential of being a target is tested in animal models (39). Understanding the mechanisms by which proteins are imported into glycosomes, especially the role of peroxins, is vital for designing drugs that disrupt protein importation.

Glycolysis in glycosomes

Glycolysis is vital for trypanosomes, as it is their main energy source. Any compound that effectively blocks glycolysis can be considered a potential antiparasitic lead. The damage caused by glycolysis inhibition will be much greater in trypanosomes than in the host. Furthermore, trypanosomal glycolysis differs from mammalian glycolysis in several ways (40,41). When comparing trypanosomatid glycolytic enzymes with their human counterparts, trypanosome enzymes exhibit changes in amino acid sequence, with only 40-50% sequence identity (42).

The significant evolutionary divergence between trypanosomatids and their mammalian hosts, coupled with the distinct structure of the glycolytic pathway of parasites, offers unique glycolytic characteristics that can be harnessed for the development of selective inhibitor targeting structures or catalytic mechanisms that do not disrupt host glycolysis (5,9). Targeting the glycolytic pathway appears promising for intervention in trypanosomatid. Specialized peroxisome-like organelles called glycosomes house seven glycolytic enzymes that catalyze the conversion of glucose to 3-phosphoglycerate (20,43,44).

Metabolic enzymes are attractive therapeutic targets because the growth and survival of pathogens rely on the availability of free energy and molecular building blocks provided by metabolism (45). Despite the high reliance of parasites on the oxidative degradation of fatty acids for adenosine triphosphate (ATP) production, glycolysis is an important mechanism for the stage of mammalian amastigotes of T. cruzi and Leishmania (46).

Due to the balance between ATP-consuming processes mediated by hexokinase and phosphofructokinase and the ATP-generating phase facilitated by phosphoglycerate kinase, there is no net ATP production within the glycosome. The glycosome contains a limited amount of ATP, which restricts the number of ATP-consuming processes until ATP is replenished by downstream reactions within the glycosome. Net ATP production occurs in the cytosol, where pyruvate kinase is located, with the resulting pyruvate being mostly expelled by the cells (47) (Fig. 2). Given that glycolysis is the primary source of ATP in the trypanosome lifecycle, it is critical for survival. Therefore, glycolysis represents a promising target for antitrypanosomal drugs, as even 50% inhibition can lead to trypanosome death (48). In summary, glycolysis is the primary energy source of trypanosomatides, with unique features that differs it from mammalian glycolysis. Targeting glycolysis offers a promising avenue for drug development.

Figure 2 Glycolytic pathways of trypanosomatids inside the glycosome. ATP, adenosine triphosphate; ADP, adenosine diphosphate.

Glycolytic enzymes

The bloodstream form of trypanosomatids contains ~14 enzymes involved in glycolysis. Among these enzymes, seven are located within the glycosome and catalyze the conversion of glucose to 3-phosphoglycerate, while the remaining enzymes are found in the cytosol and participate in the final steps of the process (20). The seven enzymes localized in the glycosome are hexokinase, glucose-6-phosphate isomerase, phosphofructokinase, aldolase, triosephosphate isomerase, glycerol-3-phosphate dehydrogenase and glycerol kinase. Trypanosomatids generate all their ATP by converting glucose to pyruvate, which is then released into the circulation of the host (49).

Hexokinase. Hexokinase is a carbohydrate kinase and it is only 37% identical to the human homolog, implying that selective inhibitor design is possible, so it has been targeted for the development of trypanosomatid inhibitor development (50). Hexokinase catalyzes glucose conversion to glucose-6-phosphate and various investigations have found that glucose analogs, such as glucosamine and 2-C-hydroxymethyl glucose derivatives, block the activity (51,52). Willson et al (53) tested numerous glucose-6-phosphate analogs against T. brucei hexokinase since glucose-6-phosphate possesses an affinity for the active site of T. brucei hexokinase. The hexokinase of T. brucei is inhibited by two glucose-6-phosphate derivatives, with a 75% inhibition at 3 mM for one derivative and a 60% inhibition at 0.2 mM for another derivative. The elimination of Tb HK1 RNA interference (RNAi) demonstrated that this protein was required for viability and this enzyme was blocked by lonidamine, a drug that effectively killed T. brucei parasites (54).

Glucose-6-phosphate isomerase. In glycolysis and gluconeogenesis, glucose-6-phosphate isomerase catalyzes the reversible aldose-ketose isomerization of D-glucose-6-phosphate to D-fructose-6-phosphate, as well as the recycling of hexose-6-phosphate in the pentose phosphate pathway, so this enzyme links glycolysis and the pentose phosphate pathway. When the crystallographic structure of the parasite glucose-6-phosphate isomerase is compared to the atomic structure of humans and other mammalian show that there are unique features of the enzyme of parasites (55).

Aldolase. The crystal structures of aldolases in trypanosomatids and mammals have been compared and it is found that the active site was largely conserved. However, by identifying minor differences and gaining detailed knowledge of the catalytic mechanism, the researchers were able to develop a specific inhibitor that binds quasi-irreversibly to the parasite enzyme. One such inhibitor is the compound 5-formyl6-hydroxy-2-naphthyl di-sodium phosphate, which can completely deactivate trypanosomatid enzymes with a Ki value of 23 µM. By contrast, this compound only has a weak effect on mammalian aldolase, even at concentrations that are 1,000 times higher than that used for parasites (56). When formulated as prodrugs to conceal the charged group, inhibitors derived from 5-formyl6-hydroxy-2-naphthyl disodium phosphate were able to effectively eliminate cultured trypanosomes with ED50 values in the low micromolar range. Importantly, these inhibitors did not have any impact on mammalian cell growth of mammalian cells (57).

Glycerol kinase. Glycerol kinase is responsible for the ATP-dependent conversion of glycerol into G3P, which is used for lipid synthesis. Furthermore, G3P can enter the glycolytic/gluconeogenic pathway by undergoing NAD+-dependent oxidation to dihydroxyacetone phosphate (DHAP) through the action of G3P dehydrogenase (G3PDH). However, the reverse reaction, which converts DHAP back to G3P, would only occur under thermodynamically favorable conditions characterized by high levels of adenosine diphosphate (ADP)/ATP and G3P/glycerol ratios. Achieving such conditions throughout the entire cell would be challenging, but they can potentially occur within a specialized compartment such as the glycosome (58). Therefore, this pathway that consumes glycerol is also important to maintain the balance of ATP/ADP and NAD+/NADH within the glycosome. In the bloodstream form of African trypanosomes, the localization of glycerol kinase (GK) within the glycosome serves as an additional mechanism to cope with periods of anaerobiosis when G3P cannot be oxidized through the shuttle mediated by mitochondrial GPO. During such periods, the inability to further metabolize G3P leads to the stoppage of glycolysis since glycosomal NADH cannot be oxidized. However, in trypanosomes where glycerol-3-phosphatase is absent, this problem is resolved by reversing the GK reaction, despite the fact that the equilibrium strongly favors phosphorylation (20).

Triosephosphate isomerase (TPI). TPI plays a crucial role in glycolysis by facilitating the conversion between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. This enzymatic activity ensures complete breakdown of the entire hexose unit, rather than just half of it. Notably, TPI in glycosomes lacks the PTS1 and PTS2 motifs, which are established peroxisome-targeting signals (59). Galland et al (60) conducted a study to identify the specific region within the TPI polypeptide responsible for its glycosome targeting. Surprisingly, the glycosomal TPI enzyme does not contain the recognized peroxisome-targeting signals. However, the researchers discovered a 22-amino acid fragment located internally within the polypeptide that can direct a reporter protein to glycosomes in transfected trypanosomes. This finding was confirmed by cell fractionation experiments and immunofluorescence studies. Notably, this internal routing information appears to be unique to the sequence of the trypanosome enzyme. When a reporter protein fused with a Saccharomyces cerevisiae peptide containing the corresponding sequence of the 22 residue fragment of the enzyme T. brucei was tested, it did not target glycosomes. In yeast and most other organisms, TPI is found exclusively found in the cytosol (60).

Phosphofructokinase (PFK). PFK is an enzyme that exists as a tetramer and uses ATP as a phosphate donor to facilitate the phosphorylation of fructose 6-phosphate, converting it into fructose 1,6-bisphosphate. This reaction is considered practically irreversible under normal physiological conditions. Notably, trypanosomatid PFKs share 20-30% of their genetic sequences with humans (61). However, the structure of PFK in trypanosomatids reveals the presence of two significant insertions that are unique to these parasites but absent in mammalian enzymes. These insertions are situated within and in close proximity to the active site of the enzyme. Consequently, this structural insight paves the way for conducting drug discovery experiments based on the structure of the enzyme, aiming to identify new lead compounds that specifically target the parasites responsible for diseases caused by the trypanosomatid family of protozoan parasites (62).

Glycerol-3-phosphate dehydrogenase (GPDH). NADH generated by glyceraldehyde-3-phosphate dehydrogenase during glycolysis is reoxidized by mitochondrial GPO using molecular oxygen. This electron transfer occurs via a glycosomal glycerol-3-phosphate dehydrogenase and the glycerol-3-phosphate:dihydroxyacetone phosphate (DHAP) shuttle. The resulting G3P is then transported across the glycosomal membrane and reoxidized through the G3P:DHAP shuttle, connecting with mitochondrial GPO (20). Inhibiting the activity of GPDH would hinder the ability of the trypanosome to produce ATP. Additionally, the inhibition of GPDH would result in the accumulation of DHAP within the glycosome, which could be highly detrimental to the trypanosome as DHAP is naturally converted to methylglyoxal, a toxic compound that disrupts protein function (63,64).

The unique structure of the glycosome creates an intraglycosomal environment that maintains the balance of redox and ATP. In the cytosol, ATP production occurs through the net action of pyruvate kinase. Under anaerobic conditions, G3P is converted to glycerol by the reverse action of a glycosomal glycerol kinase, accompanied by the production of ATP. This allows the glycosome to maintain the balance of ATP and NAD even in the absence of oxygen. However, instead of producing two molecules of pyruvate from one molecule of glucose, the glycosome generates one molecule each of glycerol and pyruvate, resulting in a reduced net ATP production of two to one. Due to this compartmentalization, a number of regulatory mechanisms that function in other cell types do not operate in trypanosomes. Glycosomal hexokinase and phosphofructokinase, for example, do not respond to activity-regulating substances that typically affect these enzymes in other cell types, reflecting the unique characteristics of trypanosomes (49,65-67).

In summary, the key glycolytic enzymes highlighted show promise as targets for selective inhibitors and the distinctions between trypanosomatid and human enzymes present opportunities for drug development, however, it is still on animal models only (68,69).

3. Conclusions and recommendations

Rational drug design targeting glycosomal enzymes and protein translocation in trypanosomatids offers significant promise for developing selective and effective treatments. However, current research faces limitations in elucidating the molecular interactions within these pathways. The glycosomal enzymes involved in glycolysis and the translocation of proteins across the glycosomal membrane are highly specific processes. These limitations arise mainly due to the complex structures of the enzymes and the dynamic nature of protein import mechanisms, which hinder the design of selective inhibitors that can differentiate between host and parasite proteins. This challenge is further compounded by the limited availability of high-resolution structural data, which is essential for understanding these molecular interactions in greater detail.

Future studies must expand our knowledge of the molecular mechanisms involved in glycosomal protein translocation and glycolysis to overcome these challenges. Advanced techniques such as cryo-electron microscopy, high-throughput screening and computational modeling should be employed to investigate these processes at a molecular level. By refining drug design approaches and conducting in vivo validation studies, the clinical translation of promising candidates can be ensured, ultimately leading to more effective treatments for neglected tropical diseases.

Acknowledgements

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Funding

Funding: No funding was received.

Availability of data and materials

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Authors' contributions

GA contributed to the conception and design of the present study, searching, analysis and writing the first draft of the manuscript. AA contributed to writing the first draft of the manuscript and YMW, AS and MG contributed to revising the manuscript. Data authentication is not applicable. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References