2021 年 3 巻 2 号 p. 43-50
African trypanosomiasis is a parasitic zoonosis that is fatal without treatment and has been recognized as a neglected tropical disease. In humans, this disease is referred to as human African trypanosomiasis (HAT) or sleeping sickness, which is characterized by severe sleep disorders. An overwhelming number of African people have experienced HAT without adequate treatment, as some currently available drugs remain unsuitable to the needs of endemic areas. Since 2001, efforts to eliminate HAT have been reinforced worldwide, thus reducing the number of new HAT cases. In addition, this has led to the discovery of several drugs that are easily applicable for therapy; however, additional chemicals and drug targets need to be explored. In the present review, we summarize the symptoms and epidemiology of HAT, the biology of the causative parasitic protozoa Trypanosoma brucei, and therapeutics used in the present treatments. Lastly, we introduce two representative drug discovery studies that are ongoing.
• HAT or sleeping sickness is a neglected tropical disease and is fatal without treatment.
• Only a few HAT treatments are available in clinics due to serious adverse events, high cost, or cumbersome management.
• Worldwide efforts in recent years to eliminate HAT have successfully reduced new disease cases and discovered two promising drugs, fexinidazole and acoziborole.
• Further development of HAT drugs is ongoing worldwide.
African trypanosomiasis is a parasitic zoonosis known to occur in sub-Saharan Africa, often fatal without treatment. The causative protozoa Trypanosoma brucei (T. brucei) is transmitted by the tsetse fly (genus Glossina) as the vector insect and is transmitted via fly bites to a wide variety of mammals, such as humans, cattle, horses, sheep, goats, dogs, and cats; the disease is termed human African trypanosomiasis (HAT) or sleeping sickness in humans, and animal African trypanosomiasis or nagana in animals. In human infection, the parasites proliferate in the bloodstream and lymphatic system, and the patients predominantly experience intermittent fever, headache, and lymphadenopathy [1], which are major symptoms of stage 1 HAT. In the later stage (stage 2), the parasites cross the blood-brain barrier (BBB) and enter the central nervous system [2]. Patients exhibit overwhelming daytime sleepiness and disrupted sleep-wake cycles, hence termed “sleeping sickness”, with other neurological symptoms of movement and mental disorders [1]. Subsequently, the symptoms may progress to meningoencephalitis and coma, and most infected individuals eventually die without treatment [3].
T. brucei is grouped into three subspecies, T. brucei brucei (T. b. brucei), T. brucei gambiense (T. b. gambiense), and T. brucei rhodesiense (T. b. rhodesiense). T. b. brucei is a parasite of non-human mammals, while the remaining two variants are infective to humans and cause HAT. T. b. rhodesiense causes an acute disease; progression to stage 2 may occur within weeks, leading to death within 6 months [1, 4]. Infection with T. b. gambiense results in chronic disease and progresses to stage 2 more slowly, from months to several years after infection [1, 5].
T. brucei completes its life cycle between the tsetse fly and mammalian hosts (Fig. 1). During infection in mammals, parasites exist in two forms: long slender and short stumpy bloodstream forms. Once trypanosomes infect mammals by fly bites, they then differentiate into slender cells and proliferate in the host bloodstream [6]. As the parasitemia reaches the maximum level, some slender cells begin to appear in the cerebrospinal fluid by disturbing the BBB; however, most remaining cells irreversibly differentiate into non-proliferative stumpy cells [6], triggered by an unknown quorum-sensing mechanism [7]. The stumpy, but not slender cells fed by flies, can survive in the host midgut by differentiating into procyclic trypomastigotes [7]. Procyclic cells can proliferate and migrate in the midgut and then further differentiate into epimastigotes at the proventriculus [6, 8]. Eventually, they reach the salivary glands and differentiate into metacyclic trypomastigotes, which are infectious to mammalian hosts when the tsetse fly sucks blood [6, 8].
The life cycle of Trypanosoma brucei. T. brucei is a flagellate protozoan approximately 20 µm in length. Trypanosomes are infective to both human and other mammalian organisms and transmitted by tsetse flies via blood-feeding. They successively undergo irreversible differentiation during their life cycle to adapt to the different environments among the host organisms.
In 1995, approximately 25,000 cases were detected, and 60 million people were estimated to be at risk of HAT infection. Thereafter, the World Health Organization (WHO) and co-workers have worked toward eradicating HAT as a neglected tropical disease. In 2001, the WHO launched an initiative to reinforce the control and surveillance of HAT, resulting in a significant decline in the ensuing years. Recently, the WHO reported that the annual number of HAT cases dropped to less than 1,000 in 2019. However, only a few drugs, with severe adverse reactions and/or restrictions, are available for HAT treatment; accordingly, during 2014–2018, approximately 54 million people were estimated to be at various risk levels for developing HAT [9].
Traditional therapeutics for HAT treatmentDrug discovery began in the early 1900s; however, only four drugs have been available for HAT treatment until recently, including suramin, pentamidine, melarsoprol, and eflornithine (Fig. 2A, Table 1). Suramin (synthesized in 1917) [10] disrupts glycolysis, an energy source for bloodstream trypanosomes [11,12,13]. Suramin is used to treat rhodesiense HAT (rHAT) stage 1, but not stage 2, as it does not penetrate the BBB. Pentamidine (discovered in 1937) [14] is thought to bind to nucleic acids and disrupt the mitochondrial genome [15, 16]. It is used to treat T. b. gambiense HAT (gHAT) but not rHAT and is available only for stage 1 owing to its inefficiency in penetrating the BBB and the clearance mechanism from the brain [17]. Melarsoprol was first introduced in 1949 as an effective agent against both gHAT and rHAT [18]. It is an arsenic compound available for stage 2 HAT treatment; however, the treatment is extremely painful and often exhibits severe life-threatening adverse effects; 5–10% of melarsoprol-administered patients reportedly develop reactive encephalopathy, which is fatal in 50% of the cases [18]. Furthermore, T. b. gambiense is thought to increase resistance to melarsoprol [10, 19]. Eflornithine was first developed in the 1970s as a cancer chemotherapeutic [20]; the trypanostatic action, with less toxicity than melarsoprol, was observed in 1980 [14, 21]. Eflornithine is effective in both stages of gHAT by inhibiting ornithine decarboxylase in T. b. gambiense but not in T. b. rhodesiense [18]; thus, it has been used for the treatment of stage 2 gHAT since 1990 [22]. However, eflornithine has a relatively short half-life (1.5–5 hr) and low efficacy in crossing the BBB, which may explain the necessity for a long-term, intense regimen, with high dose (100 mg/kg) infusions every 6 hr, for a total of 7 or 14 days [14, 23].
Compounds against human African trypanosomiasis (HAT) that are in use for treatment (A), in clinical trials (B), and in preclinical research reviewed in the present review (C).
| Drug | Deve-loped | Target | Stage | Mechanism of action | Regimena,b) | Major adverse reaction | Issues |
|---|---|---|---|---|---|---|---|
| Suramin | 1916 | rHAT | Stage 1 | Inhibiting a series of glycolytic enzymes | 1 g on days 1, 3, 7, 14, and 21, intravenous | Mild andreversible nephrotoxicity | Needing injections.Unavailable in treatment of the second stage. |
| Pentamidine | 1937 | gHAT | Stage 1 | Disrupting mitochondrial genome | 4 mg/kg/day for 7–10 days, intramuscular or intravenous | Hypotension | Needing injections.Unavailable in treatment of second stage.Emergence of drug-resistant parasites |
| Melarsoprol | 1949 | gHATrHAT | Stage 2 | Mechanism of action is unknown, although acquiring resistance relies on P2 purine transporter and/or aquaglyceroporin 2 | 2.2 mg/kg/day for 14 days, intravenous. | Severe encephalopathy, sometimes life-threatening | Emergence of drug-resistant parasites. |
| NECT | 2009 | gHAT | Stage 2 | Nifurtimox: unknown, but seemed to generate ROS.Eflornithine: irreversible inhibition of ornithine decarboxylase | Nifurtimox: 15 mg/kg/day orally for 10 days.Eflornithine, 400 mg/kg/day, intravenous 2-hr infusions for 7 days. | Diarrhea, nausea/vomiting, headache | Decreased risk of major adverse effects but needs long-term, complicated treatment with hospitalization.High cost. |
| Fexinidazole | 2018 | gHAT(rHAT)c) | Stage 1Stage 2 | Activated by NADH-specific nitroreductase, though the fate of the active metabolite is unknown | Adults (≥35 kg): 3 tabletsd)/day for 4 days, followed by 2 tablets for 6 days.Aged ≥6 years (20–35 kg): 2 tablets/day for 4 days, followed by 1 tablet for 6 days. | Decreased risk of major adverse effects | Orally available. |
a) "WHO Interim Guidelines for the Treatment of Gambiense Human African Trypanosomiasis (2019)". b) "Control and surveillance of human African trypanosomiasis, WHO technical report series; no. 984 (2014)". c) Availability for rHAT is in phase II/III trials (https://dndi.org/research-development/portfolio/). d) One tablet is equivalent to 600 mg of fexinidazole. rHAT, rhodesiense HAT; gHAT, gambiense HAT.
In 2003, the Drugs for Neglected Disease initiative (DNDi) promoted drug discovery for the treatment of neglected tropical diseases, and in 2009, collaborators introduced a nifurtimox and eflornithine combination therapy (NECT) as a regimen that enables the simpler and safer application of eflornithine against stage 2 gHAT [21, 24]. In addition, NECT reduced the hospitalization period; however, it still requires 7 days of serial infusions that cost $357 per treatment, approximately 10 times higher than the ideal cost proposed by DNDi.
DNDi has continued its efforts, and very recently, they won a significant outcome in discovering new HAT therapeutics that may solve challenges associated with the former drugs listed above. The nitroimidazole analog fexinidazole (Fig. 2A), which DNDi rediscovered in 2010 during a screening campaign of nitroimidazoles, was effective against both stages of rHAT and gHAT following oral administration (Table 1) [25]. Clinical trials have been conducted since 2011, and oral administration of fexinidazole provides excellent therapeutic efficacy with few adverse events during gHAT treatment [26]. Since 2018, the European Medicines Agency has recommended the use of fexinidazole to treat stage 2 gHAT, which was first approved in 2019 in the Democratic Republic of Congo. Trials for assessing efficacy against rHAT have already been initiated and rolled out in Uganda and Malawi in 2017. Fexinidazole is the first oral treatment effective against stage 2 HAT without severe adverse reactions and can be administered in a home setting [27].
Clinical trials of another novel candidate for HAT therapy are ongoing. Acoziborole (SCYX-7158/AN5568, Fig. 2B), a benzoxaborole with a long half-life of 400 hr was found to be effective after a single oral administration. Acoziborole entered phase I efficacy trials in 2015; thereafter, DNDi has conducted a phase II/III trial of single-dose acoziborole for stage 2 gHAT in the Democratic Republic of Congo since 2016 [28].
The discovery of fexinidazole and acoziborole is expected to facilitate the elimination of HAT; however, the search for novel therapeutics against HAT needs to continue, as these new drugs might be inefficacious in some patients. Importantly, new drug development can help combat drug resistance in trypanosomes. Numerous studies have sought new targets in trypanosomes and compounds attacking them on a global scale. Herein, we introduce two representative studies that present novel drug targets, as well as the mechanism of action of inhibitors targeting them to exhibit trypanostatic effects.
Trypanosome alternative oxidase (TAO)Bloodstream trypanosomes possess a unique energy metabolism; they solely depend on their energy source for glycolysis [29], which occurs in trypanosomatid-specific organelles called glycosomes enclosing high concentrations of glycolytic enzymes (Fig. 3) [30]. Therefore, the mitochondrial respiratory chain in bloodstream trypanosomes is not directly involved in ATP synthesis; however, the respiratory chain is still essential for the reoxidation of co-enzymes accumulated during glycolysis to maintain glycolytic ATP production (Fig. 3) [31]. Subspecies of T. brucei commonly possess the mitochondrial inner membrane protein TAO, a member of cyanide-insensitive alternative oxidases that are present in the mitochondria of higher plants, algae, fungi, and nematodes [32]; TAO functions as a terminal oxidase in the glycerol-3-phosphate oxidation to oxidize reduced ubiquinone (ubiquinol), produced by glycerol-3-phosphate dehydrogenase (G3PDH) using oxygen molecules [32]. Importantly, TAO is an entirely different enzyme from mammalian cyanide-sensitive cytochrome c oxidases and is involved in the glycerol-3-phosphate oxidation system, which is absent in mammalian cells. Therefore, it is considered a promising target for HAT chemotherapy [32, 33].
Energy metabolism in Trypanosoma brucei bloodstream forms. Bloodstream trypanosomes depend on glycolysis for energy synthesis, which predominantly occurs in the glycosomes. A part of dihydroxyacetone-3-phosphate (DHAP) is metabolized to glycerol-3-phosphate (Gly3P); Gly3P is subsequently recycled to DHAP by mitochondrial G3PDH (mG3PDH). Ubiquinol (UQH2) produced in this recycling reaction is oxidized to ubiquinone (UQ) by TAO. Therefore, TAO inhibition by ascofuranone results in dysregulation of energy metabolism in the bloodstream trypanosomes. FBP, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; BPG, 1,3-bisphosphoglyceric acid; PEP, phosphoenolpyruvate; TAO, Trypanosome alternative oxidase.
Minagawa et al. observed that ascofuranone (Fig. 2C), an antibiotic isolated from Ascochyta visiae, potently inhibited glucose-dependent cellular respiration and glyceraldehyde-3-phosphate-dependent mitochondrial O2 consumption of long slender bloodstream forms of T. b. brucei (Fig. 3) [34]. Furthermore, a combination treatment of ascofuranone and glycerol showed potent anti-trypanosomal activity in infected mice; trypanosomes in the blood disappeared immediately after intraperitoneal or oral treatment [35]. Cytotoxicity is highly selective to bloodstream trypanosomes, as ascofuranone specifically inhibits TAO, which is absent in mammalian metabolism [36]. Currently, the inhibitory mechanisms of TAO and the structure-activity relationship of the ascofuranone analogs are being clarified by using X-ray crystal structures of complexes with TAO to develop promising drugs against sleeping sickness [36,37,38].
Guanine nucleotide metabolismIn addition to glycolysis, differences between trypanosomes and the host mammals have been found in the metabolism of purine nucleotides. Despite the absence of a de novo synthesis mechanism, trypanosomes can produce purine nucleotides via the salvage pathway using nucleobases and nucleosides acquired from the host (Fig. 4), which predominantly occurs in glycosomes [39, 40]. In addition, the salvage pathway acts as a channel for the interconversion of guanine and adenine nucleotides to maintain the balance of their intracellular concentrations; therefore, the enzymes in this pathway are considered good candidates to develop drugs targeting HAT [40]. Recently, the enzymes involved in purine nucleotide salvage of T. brucei were characterized, i.e., guanosine 5ʹ-monophosphate reductase (GMPR) [41] and inosine 5ʹ-monophosphate dehydrogenase (IMPDH) [42] (Fig. 4). The former catalyzes the conversion of guanosine 5ʹ-monophosphate (GMP) to inosine 5ʹ-monophosphate (IMP), whereas the latter utilizes IMP to produce xanthosine 5ʹ-monophosphate (XMP) as an intermediate for GMP synthesis.
Schematic diagram of the salvage pathway for adenine and guanine. Trypanosome survival depends on the salvage pathway to recycle purine nucleosides and bases acquired from the hosts. Both guanosine 5ʹ-monophosphate reductase (GMPR) and inosine 5ʹ-monophosphate dehydrogenase (IMPDH) contribute to maintaining the intracellular concentrations of adenine and guanine nucleotides. SAM, S-adenosylmethionine;
Trypanosomatid GMPRs possess a unique structure with a cystathionine-β-synthase (CBS) or Bateman domain, which is absent in the homologous enzymes of mammals, including humans [41, 43]. Some enzymes with the CBS domain are known to regulate activity through purine nucleotide binding to this domain [44]. A recent study demonstrated that TbGMPR has an oligomeric conformation with allosteric regulation via the CBS domain [45]. The binding of guanine nucleotides (GTP or GMP) and adenine nucleotides (ATP) to an allosteric regulatory site, formed at the cleft between the catalytic and CBS domains, leads to positive and negative regulation, respectively, accompanied by changes in the multimeric enzyme structure (Fig. 5) [45]. Specifically, TbGMPR with a “relaxed octamer” conformation in the apo form is transformed into an active “twisted octamer” by binding to GTP or GMP, while the binding of ATP disrupts the octamer structure to dissociate into two inactive tetramers. These findings indicate that alterations in the oligomeric states of TbGMPR are responsible for allosteric regulation by purine nucleotide binding to the CBS domain. The GMPR of T. brucei is distinct from that of the host organisms due to the presence of the CBS domain [41, 45]; therefore, the allosteric site in the CBS domain of TbGMPR could be a suitable therapeutic target for African trypanosomiasis. The purine nucleotide analog ribavirin 5′-monophosphate (RMP) and its nucleoside form ribavirin (Fig. 2C) may provide valuable information for developing novel inhibitors. Ribavirin is widely employed for the treatment of hepatitis C. Moreover, it has been shown to exhibit anti-trypanosomal effects in culture by inhibiting both TbGMPR and TbIMPDH through phosphorylation to RMP; however, its IC50 (~25 µM) has been deemed insufficient for clinical studies without improvement [41]. Ribavirin reportedly penetrates the BBB after peripheral administration [46,47,48], implicating that a purine nucleoside analog inhibiting TbGMPR and/or TbIMPDH could be effective in stage 2 HAT treatment. Further structural studies of TbGMPR complexed with other nucleotides or their synthetic analogs should be undertaken to identify compounds that specifically inhibit TbGMPR with high and selective trypanocidal activities.
Crystal structures of guanosine 5ʹ-monophosphate reductase of Trypanosoma brucei (TbGMPR) in the absence (top, PDBID:6JL8) or presence of GMP (bottom, 6JIG). TbGMPR exists as an octamer composed of a pair of tetramers (Tetramer 1 and 2) in the presence or absence of guanine nucleotides. The binding of guanine nucleotide to the allosteric site (designated in green) at each cystathionine-β-synthase (CBS) domain extruding from the catalytic domain induces a ‘twisted’ conformation with some compression in size. The dashed square indicates the position of Tetramer 1 in the ‘relaxed’ apo form, while the red square indicates the position after binding of GMPs (yellow). The alphabets A–D and A’–D’ indicate the subunits in Tetramer 1 and 2, respectively.
Patients in endemic areas of sleeping sickness have been neglected for a long time; however, increasing efforts to expand HAT therapy have delivered several new treatment strategies in recent years. However, practical therapeutics for the complete elimination of HAT are limited, emphasizing the necessity to continue exploring additional agents while encouraging preventative and control activities in endemic areas.
The authors declare no conflict of interest.
This work was supported by the Grants-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant Nos. 25660231, 25242046, and 17K19329 to T.I.).
The author would like to thank Ms. Kasumi Minemura for creating the figures.
