Perspectives on the Drug Discovery of Intestinal Protozoan Parasites

Abstract

The intestinal protozoan parasites pose serious health concerns, infecting more than one billion individuals every year and mainly causing diarrhea in infants and adults. Main pathogens include Giardia intestinalis, Entamoeba histolytica, Cyclospora cayetanensis, and Cryptosporidium spp. causing giardiasis, amoebiasis, cyclosporiasis, and cryptosporidiosis, respectively. The drug arsenal to treat these diseases is limited (<25 drugs are in clinical use) for the treatment of all protozoal infections. The existing treatment options are decades of years old (discovered in 1930–1980s) and have limitations such as low therapeutic index, toxic side effects during long-term treatment, and drug resistance. Therefore, urgent renewed drug discovery efforts are needed to tackle these neglected protozoal diseases. This chapter discusses the current status of treatment options and their limitations, along with current drug discovery efforts. We conclude that the knowledge gained in the genomic and post-genomic era should be appropriately harnessed to accelerate the futuristic drug discovery process in this field.

1. Introduction

Worldwide, around 1.7 billion incidents of diarrheal illness are recorded annually [1]. According to the WHO report 2024, every year, diarrheal disease kills approximately 443,832 children and is the third leading cause of death among children aged below 5 years. A diverse group of pathogenic microbes, including bacteria, viruses, and parasitic organisms, can cause diarrheal disease. Of these pathogens, protozoan parasites are the major contributors [2]. These intestinal protozoans are a divergent group of unicellular organisms that reaches the intestine through ingestion of contaminated food or drinking water [3], particularly in the tropics and sub-tropics, causing millions of cases of diarrhea annually.

The intestinal infections are caused by different protozoan parasites such as Dientamoeba fragilis, Giardia lamblia, Isospora belli, Microsporidia (Septata intestinalis, Enterocytozoon bieneusi), Entamoeba histolytica, Balantidium coli, Blastocystis hominis, Cyclospora cayetanensis, Cryptosporidium spp., etc. [4]. However, this manuscript focuses primarily on Giardia intestinalis, Entamoeba histolytica, Cyclospora cayetanensis, and Cryptosporidium spp. causing giardiasis, amoebiasis, cyclosporiasis, and cryptosporidiosis, respectively [5]. C. cayetanensis, G. intestinalis, and C. pavrum are the small intestinal parasites that affect children and elderly persons, whereas Entamoeba, the large bowel parasite, infects all age groups but mainly adults [6]. They are transmitted through the oral-fecal route via indirect or direct contact with the transmittable phase, leading to cases of mortality and morbidity [7].

These intestinal infections can be managed by both preventive measures as well as by appropriate drug treatment. For treating different protozoan parasites, only 25 drugs are used in clinical settings [8]. However, even after the latest advancements in the field, such as the availability of the complete genomes and a comprehensive understanding of the life cycles of these pathogens, there are limited translational breakthroughs from the lab to the clinic, and no major new classes of antiprotozoal drugs have been developed. Moreover, the efficacy of currently available drugs is insufficient, and the emergence of drug resistance is another challenge resulting in clinical treatment failures [9]. Therefore, due to the lack of effective, safe, and reasonable drugs, the impact of protozoan infections on humans has been amplified. Understanding the molecular basis of resistance facilitates the identification of novel drug targets and helps in the advancement of therapeutic agents, thereby necessitating further research to ensure a sustainable discovery of effective compounds. Moreover, the lack of vaccines against these parasites further demands renewed efforts toward the development of novel drugs, especially in the post-genomics era.

This chapter discusses current treatment options for different human intestinal protozoa to understand the scenario of drug-resistant strains, available alternatives, and future therapeutic strategies for the effective management and cure of these infections.

2. Giardiasis

Giardiasis is a small intestinal disease caused by Giardia intestinalis, known as G. lamblia or G. duodenalis. Giardiasis is a global public health concern, with approximately 280 million cases per annum [10]. A huge number of cases of parasitic infections (2-30%) while in developed countries the infection frequency is in the range of 2-7% [11].

G. intestinalis is a human intestinal parasite, that also affects domestic and wildlife animals, particularly mammals. The latest study has revealed the existence of trophozoites similar to Giardia in the gut of invertebrate Heterotermes tenuis [12]. It was initially elucidated by Antonie van Leeuwenhoek while examining his own diarrheal stool in 1681. Subsequently, Vilém Dušan Lambl discovered Giardia in 1859 and explained it in detail, and therefore, the protozoan was named after him. The parasite is transmitted through contaminated water, either freshwater or public water supply. G. intestinalis exists in two forms: trophozoite and cyst. It is transmitted in cyst form either through contaminated water or direct oral-fecal route [13]. After consumption, excystation takes place, followed by the release of trophozoites. The trophozoites infect the duodenum and upper intestine, taking advantage of the favorable alkaline pH, resulting in the development of the disease [14].

2.1 Current drug regimen for giardiasis

The therapy administered for giardiasis includes nitroimidazole compounds, which are potent against various bacterial as well as parasitic infections, especially a synthetic 5-nitroimidazole derivative called metronidazole primarily used for the treatment of trichomoniasis [15]. Eventually, Darbon et al. proclaimed that metronidazole can be used against Giardia infections [16]. Since the 1960s, it remains the predominant drug used worldwide for the treatment of giardiasis [17]. Its success rate varies from 60 to 100%, and thus is the first-line drug used for the treatment [17, 18]. Metronidazole is a pro-drug that, after entering the trophozoite, is reduced by the parasite’s electron transport proteins (ferredoxins) that donate the electrons to the nitro group [19]. This drug builds oxidative stress through binding with thiol groups, resulting in the cysteine adducts and distorting the DNA by developing a double-strand rupture [20]. The activation of metronidazole is intervened by the enzyme nitroreductase-1 (NRT-1) as well as by the reduction pathway mediated by ferredoxin. Other enzymes that result in the reduction of metronidazole are pyruvate: ferredoxin oxidoreductase (PFOR) and thioredoxin reductase (TrxR) [21]. The reduction of the drug creates a gradient that helps in the intracellular transport of the drug, which primarily destructs the replication phase of G. intestinalis cell cycle and further results in the DNA fragmentation and failure of its functions, leading to the death of trophozoite (Figure 1) [20]. Even though metronidazole is the first-line drug used against various parasites, it has been pointed out to be carcinogenic by the US Department of Health, stating that it created tumors in mice at higher doses, but no cancer-related symptoms have been found in humans (Table 1) [34]. Another compound that is extensively used for the treatment of giardiasis is albendazole, a benzimidazole class drug. This compound has been utilized as an anthelmintic for humans since the 1960s in veterinary medicine and as an antifungal in agriculture. Their inhibitory efficiency as anti-protozoan compounds was discovered in the 1980s as they inhibited the growth of Trichomonas vaginalis. Its efficiency against G. intestinalis was also discovered afterwards [35]. Due to the low cure rate of this drug, it is observed as a secondary option for the treatment. Also, this drug is not recommended during pregnancy due to teratogenic effects in animals and may be in humans as well [17]. Trophozoites metabolize albendazole and result in the formation of toxic intermediates: albendazole sulfone and albendazole sulfoxide with the help of flavohemoglobin, a NADH oxidase (gNADHox) present inside the parasite (Figure 1) [36]. It has been established that the albendazole binds to the beta-tubulin of the parasite, and the presence of two amino acids (Glu-198 and Phe-200) in the beta-tubulin sequence renders this protozoan susceptible to benzimidazoles (Table 1). However, E. histolytica displays resistance due to the presence of different amino acids in this particular region [37]. Other drugs that are employed include furazolidone [38], acridine (quinacrine) [39], aminoglycoside (paromomycin) [40], and nitazoxanide (Table 1) [25]. Nitazoxanide, a nitrothiazolide derivative, shows inhibitory activity against bacteria, protozoa, and helminths. It was approved for giardiasis treatment in 2004. It is hypothesized that this drug inhibits enzymes such as PFOR and nitroreductases [41]. Moreover, nitazoxanide destroys the cyst by disrupting the protozoan’s cyst wall [42]. Furazolidone is another drug used for the treatment of giardiasis, particularly for pediatric cases [29]. However, due to evident genotoxicity and activation of neoplastic processes, the use of this drug is prohibited in the United States [17]. Even though the mechanism of action of this drug is similar to metronidazole, it is reduced by NADH oxidase instead of PFOR [43]. Quinacrine, another drug used for the treatment of giardiasis, is an acridine derivative with broad-spectrum activity. The usage of this drug is limited due to side effects like vomiting and psychosis, while in some countries, it is used when patients do not respond to metronidazole.

Figure 1.

Schematic representation of the existing and novel drug targets in G. intestinalis. The figure also illustrates the anti-giardia drugs in the current therapy (mentioned in blue) as well as the drugs under various phases of research (mentioned in red). Moreover, auranofin (mentioned in violet) is the only novel drug undertaken for clinical trial. Created with BioRender.com.

S. No.	Drug	Parasite	Mechanism	References
1	Metronidazole	Entamoeba histolytica	Reduction of nitro groups; DNA fragmentation and cell cycle arrest.	[22, 23]
		Giardia intestinalis
2	Albendazole	Giardia intestinalis	Binds to β-tubulin; cell cycle arrest.	[24]
3	Nitazoxanide	Giardia intestinalis	Inhibits PFOR, nitroreductase, disrupts plasma membrane	[25, 26, 27]
		Entamoeba histolytica
		Cyclospora cayetanensis
		Cryptospordium parvum
4	Furazolidone	Giardia intestinalis	Depletes cytoplasm, produces toxic intermediates	[22]
5	Paromomycin	Giardia intestinalis	Inhibits protein synthesis	[28]
		Entamoeba histolytica
		Cryptospordium parvum
6	Quinacrine	Giardia intestinalis	Inhibits DNA production	[29]
7	Trimethoprim-Sulfamethoxazole	Cyclospora cayetanensis	—	[30, 31, 32]
8	Ciprofloxacin	Cyclospora cayetanensis	—	[33]
9.	Roxithromycin	Cryptospordium parvum	Disrupt bacterial protein synthesis	[26]
10.	Rifabutin and rifaximin	Cryptospordium parvum	Inhibits RNA synthesis	[26]

Table 1.

Currently used drugs for the intestinal parasites and their mode of action.

Resistance to the drugs metronidazole and albendazole used for the treatment of giardiasis is a major emerging clinical concern with unspecified consequences. Human giardiasis has been primarily treated with metronidazole for the past 60 years, but the efficiency of this antibiotic has been weakened due to an increase in resistance against metronidazole, a first-line drug. This treatment failure has turned into a major health concern in the last 15 years [44]. Moreover, a large number of giardiasis cases refractory to metronidazole have been documented in low Giardia frequency settings. A study conducted in 2010 revealed that the regimen containing one or more nitroimidazoles did not succeed in curing 5.8% of patients out of a group of 170 [45]. Other data from 2008 to 2013 from the Hospital for Tropical Diseases in London reported a sharp rise in metronidazole-refractory cases from 15% to >40% of all the cases [46]. Moreover, it has also been discovered that metronidazole-refractory patients are difficult to treat with other antibiotics, such as nitazoxanide and albendazole. Furthermore, a sharp rise in refractory patients has also been documented in India [47]. In vitro studies have illustrated that the induction of resistance to benzimidazole is correlated with β-tubulin mutation, degradation of enzymes involed in glycolysis and arginine metabolism, and reduced mRNA expression of flavohemoglobin [36]. The detailed molecular mechanisms of resistance that lead to 5-nitroimidazole refractory Giardia infection as well as other types of resistance is not completely understood despite substantial efforts with laboratory as well as clinical strains. Therefore, it warrants the search for new compounds through screening of libraries or drug repositioning. The drugs commonly used to treat giardiasis are listed in Table 1.

2.2 Recent drug discovery efforts against giardiasis

Giardiasis has been overlooked for years, typically in underdeveloped countries. The search and development of novel drugs have been poorly researched, mainly due to economic reasons. The genome sequencing demonstrated that G. intestinalis comprises a compact genome of approximately 12 Mb with around 4800 expressed genes [48]. The completion of the Giardia genome project and advanced molecular tools helped in the identification of new inhibitors for almost one-tenth of the total potent drug targets in the parasite. High-throughput technologies have also benefited in screening the repurposed drugs as well as new pharmacophores, escalating the depository of anti-giardia compounds, mostly exhibiting activity against metronidazole or albendazole-resistant Giardia [49]. The drugs under investigation against giardiasis are highlighted in Figure 1.

Auranofin, a gold (Au) containing anti-rheumatic compound, was repurposed against the parasite in 2013. The clinical phase trial IIa (NCT02736968) revealed that it decreases the load of Giardia trophozoites and is also safe to use at a dose of 6 mg/day. Even though auranofin has been described as a TrxR activity inhibitor, the mode of action is not fully understood and requires further research [39]. Orlistat, an antiobesity drug, also exhibited anti-giardia activity in vitro both against susceptible and metronidazole-resistant Giardia, at lower concentrations than metronidazole (4.3 μM and 11.0 μM) [50]. Robenidine, an anticoccidial drug classified as a guanidine derivative, showed better and faster activity than metronidazole against G. intestinalis with minimum lethal concentration (MLC) as low as 2.8 μM. Several analogs of this drug are developed, researched, and patented [51].

Azidothymidine (AZT), an anti-retroviral drug, also exhibits inhibitory activity in-vivo against Giardia, even against the metronidazole-resistant strains [52]. Disulfiram, a thiuram disulfide drug used to treat alcohol abuse disorder, also exhibited in vitro anti-giardia activity with a minimum lethal concentration of 1.23 μM and modest activity in vivo with a 21% cure rate in mice against Giardia [53]. In addition, mavelertinib, an EFGR-TKI (tyrosine-kinase inhibitor), has also displayed effective activity in mice infected with G. intestinalis [54]. Several anticancer drugs have also been suggested to be repurposed against protozoa including G. intestinalis. Trichostatin A, tubastatine A, and nicotinamide have inhibitory effects on the growth of trophozoites [55]. Moreover, a new compound, KH-TFMDI, a 3-arylideneindolin-2-one derivative, and KV-46, a 4-[(10H-phenothiazin-10-yl) methyl]-N-hydroxy benzamide, displayed growth inhibitory activity, with IC₅₀ less than 1 μM. While these compounds primarily inhibit histone acetylase, there is a possibility that they affect other pathways as well (Figure 1) [56].

The cytoskeleton of G. intestinalis serves as a potent target for novel drug discovery as existing as well as proposed drugs affect the components of the parasitic cytoskeleton. Giardia depends on substrate-level phosphorylation due to the lack of typical mitochondria. Therefore, targeting the enzymes of carbohydrate metabolism is a potential strategy. Various compounds that target metabolic pathways associated with sterol synthesis have been examined in vitro. For example, azasterol and epiminolanosterol that suppress the activity of ∆24(25) sterol methyltransferase (24-SMT) have exhibited activity against trophozoites of Giardia, although the parasite does not produce 24-alkyl sterols or ergosterol [57]. The enzymes involved in this process can be different from mammalian cells and thus can be potential targets in drug discovery (Figure 1) [58]. Few kinase inhibitors against Giardial NEK kinases are under investigation. The screening of kinase inhibitors provided five compounds that hinder the growth of G. intestinalis. One compound (BKI1213) particularly impacted cytokinesis [59], whereas another notable compound (GW843682X) hinders flagellar length and thus alters cytokinesis [60]. Moreover, a recently discovered nucleolus has also emerged as a potential target [61]. This structure would constitute several transcription initiation features that are unique to the parasite [62], making it a strong candidate for drug discovery (Figure 1). Natural compounds such as curcumin, berberine, garlic extract, and grapefruit seed extract have been investigated to demonstrate anti-Giardia activity [63]. Curcumin inhibits Giardia lamblia trophozoite adhesion and growth by altering the microtubule cytoskeleton and preventing parasite multiplication [64]. These chemicals have the potential as alternative therapeutics and represent a viable avenue for the development of novel anti-giardia medicines.

3. Amoebiasis

Amoebiasis, also known as amebiasis or amoebic dysentery, is a condition caused by the infection of Entamoeba spp., which are protozoan parasites that typically reside in the intestine. Most of the infections are asymptomatic. However, the invasive disease may develop, showing symptoms of abdominal cramps, acute diarrhea, dysentery, and amoebic colitis (bloody diarrhea with mucus). Amoebic liver abscesses (ALA) develop if the parasite reaches the liver through the bloodstream. The symptomatic manifestation of amoebiasis has been attributed mainly to Entamoeba histolytica, but other species such as E. dispar, E. moshkovskii, E. hartmanni, E. polecki, and E. bangladeshi are also implicated [65]. However, further research is required to establish the pathogenicity of E. dispar and E. bangladeshi. Amoebiasis has been responsible for more than 100,000 annual deaths worldwide [66] and ranks third in the category of parasitic diseases (after malaria and schistosomiasis) responsible for mortality across the globle [67]. The disease is more prevalent in developing countries of Asia, Africa, and Central and South America. It represents the highest disease burden in Asia under the category of neglected tropical diseases. South Africa has a disease prevalence of 12.4% in 2006 [68] while in 2011, India reported the prevalence of intestinal amoebiasis to be ~11.7% [69]. The most affected countries include Bangladesh, China, India, Mexico, Columbia, and Brazil [70]. Although it is considered a tropical disease, it is also being reported in developed countries, especially among travelers returning from endemic places.

The life cycle of E. histolytica is a simple two-stage cycle compared to the other protozoan parasites. It consists of an infectious cyst that can survive outside its host for sufficient time to be able to cause transmission [71]. The transmission of the disease occurs by ingestion of the cysts from contaminated food and water [71]. Once the cysts reach the small intestine, the process of excystation starts with the action of intestinal trypsin enzymes, resulting in the formation of eight vegetative trophozoites per cyst that colonize the caecum and colon. The motile trophozoites are responsible for invasive disease as they divide and multiply by binary fission, and can migrate to other tissues. It may cause extraintestinal amoebiasis and also convert back to cysts in a process called encystation to be excreted out in feces. Hence, a new cycle of fecal-oral transmission starts [72]. E. histolytica remains in the human intestine, causing asymptomatic disease in about 90% of the cases, and only a minority of cases develop invasive disease. However, the reasons behind this selectivity are poorly understood. It is now established that the pathogenesis of E. histolytica is multifactorial, involving interactions of parasitic virulent factors, host cells, and components of the immune system and microbiota of the intestine [72]. The trophozoites adhere to the intestinal epithelium and interact with the TJ junctions, resulting in paracellular permeability and destroying the membrane integrity. The parasite ingests the host target cells and degrades them, leading to the development of ulcers [73].

3.1 Current drug regimen for amoebiasis

Feder Losch, in 1875, first detected amoebae in fecal samples. In 1903, Fritz Schaudinn was the first to distinguish between E. coli and E. histolytica and named the latter because of its tissue lysing ability. Maintaining basic hygiene practices can easily control the spread of the disease. Prohibiting open defecation, access to toilets, effective sewage water treatment, and improved water purification systems could be crucial measures in the prevention of amoebiasis [74].

Amoebiasis, despite being a significant global public health concern, currently lacks vaccinations or prophylactic drugs for prevention. The WHO recommends that all cases of amoebiasis including asymptomatic patients should be treated [75]. The treatment of asymptomatic cases is necessary not only to stop the invasive disease but also to inhibit the spread and further transmission through excreted cysts. The current lines of drugs that are used in the treatment of this infection have been categorized into two types. Drugs that are used to treat non-invasive colitis are referred to as luminal agents (paromomycin, diloxanide furoate, iodoquinol, and nitazoxanide) that kill the intraluminal cysts. Invasive amoebiasis and extraintestinal disease have been treated with chloroquine, emetine, tinidazole, and metronidazole, which are the elected treatment in patients with symptomatic intestinal amoebiasis (Table 1) [72]. These organic compounds have been the mainstay therapy for this disease since the 1960s and are active only against trophozoites [76]. In particular, metronidazole is used widely as a first line of treatment for amoebic colitis for at least 5 days. The other nitroimidazoles with longer half-lives have also been successfully used for shorter durations such as ornidazole, tinidazole, and secnidazole [77]. Another relatively newer drug, nitazoxanide, successfully cures 80–90% of patients with intestinal amoebiasis in 3 days [78]. The nitroimidazoles are not effective against luminal stages. Therefore, an effective therapy should include a course of luminal agents such as paromomycin following metronidazole treatment [28]. Despite its side effects, metronidazole still remains the chief drug being used in the treatment of amoebiasis, and so far, there is no clear evidence for the clinically resistant strains of E. histolytica [72]. However, cases of treatment failure, especially with ALA patients, have been reported [79]. Other studies have described the increase in MIC values by E. histolytica when exposed to increased metronidazole concentrations in vitro [80]. The drugs commonly used to treat amoebiosis are listed in Table 1.

3.2 Recent drug discovery efforts against amoebiasis

Auranofin is the latest drug with potential against E. histolytica, even for metronidazole-resistant parasites, and is currently in clinical phase II trials (NCT02736968). In the era of drug repurposing, several compounds have been suggested to be explored against this parasite. Disulfiram or antabuse, used to treat alcohol dependence, has been explored against E. histolytica but showed negligible impact on parasite viability [81]; however, its metabolite, ZnDTC, showed efficient antiparasitic effects in a mice model at low nanomolar concentration by inhibiting the ubiquitin-proteasome pathway (Figure 2A) [82]. A recent study has identified 26 potential compounds after screening ~12,000 compounds in cell culture, which could be further developed into anti-amoebiasis drugs [83]. Out of them, ponatinib, a multikinase inhibitor, can be the most effective for repurposing strategy. It was found to be 30X more potent than metronidazole in vitro [83]. Beloranib and TNP470 (analogs of fumagillin), are a class of MetAP2 inhibitors that can prevent encystation, block reoccurrence, and are most effective against metronidazole-resistant E. histolytica parasites. However, the use of beloranib is associated with thromboembolism. This warrants further research and development of newer and safer derivatives of MetAP2 inhibitors [84]. Another class of mTOR/PI3K inhibitors, including dactolisib, sapanisertib, omipalisib, and a few others, have shown inhibitory potential against metronidazole-resistant parasites in vitro. The drugs under investigation against amoebiasis are highlighted in Figure 2A.

Figure 2.

Schematic representation of recent drug discovery efforts for Entamoeba histolytica and Cryptosporidium parvum. A. Potential drug disulfiram’s metabolite, ZnDTC inhibits COP9 signalosome subunit 5 (CSN5) activity in Entamoeba histolytica. The mode of action of auranofin is not completely understood. B. Illustration of putative drugs and their targets within Cryptosporidium parvum.

In addition, exploring the molecular pathways and cellular components of E. histolytica for developing novel therapeutics holds an enormous potential that necessitates rigorous efforts in the research and development in the field. The update and information on promising drug targets for amoebiasis have been compiled and summarized elsewhere [85].

4. Cyclosporiasis

Cyclosporiasis is an intestinal parasitic disease caused by the coccidian parasite Cyclospora cayetanensis [86], first described in 1870. It is the only species known to infect humans, with the first recorded instance in Papua New Guinea in 1979 [87]. The attention toward the parasite has increased due to its capacity to induce gastrointestinal distress among human populations, transcending geographical boundaries and socioeconomic disparities and surge in clinical cases globally [88]. Despite efforts to curtail its proliferation, C. cayetanensis infections persist as a significant concern, underscoring the complexity of managing this pathogen in contemporary society [89]. Endemic hotspots for C. cayetanensis include tropical and subtropical regions, where poor sanitation results in environmental contamination with human feces [90]. Contaminated food and water sources play a pivotal role in Cyclospora transmission, necessitating stringent sanitation measures and enhanced surveillance efforts.

The clinical outcomes of cyclosporiasis are multifactorial and influenced by various factors, including the age and immune status of the host [91]. It is a significant challenge, especially for vulnerable populations such as immunosuppressed individuals, children, and the elderly that are more susceptible to severe forms of the disease, including extraintestinal complications like biliary disease, Guillain-Barré syndrome, reactive arthritis syndrome, and ocular inflammation [92]. C. cayetanensis has a complex life cycle involving both sexual and asexual stages. It begins when an individual ingests food or water contaminated with oocysts, which develop into merozoites that invade new epithelial cells [93]. The merozoites then undergo sexual reproduction in the gastrointestinal tract, developing into microgametocytes and macrogametocytes, which are released into the environment through feces [90]. The fertilization occurs in outside environment. The zygote forms sporulated oocysts, capable of infecting new hosts (Figure 3). The transmission of C. cayetanensis is facilitated by factors like globalization of food supply chains and increasing international travel [90]. Public health interventions should focus on improving food safety standards, enhancing water sanitation infrastructure, promoting hygiene practices, and strengthening surveillance and outbreak response systems [94].

Figure 3.

Life cycle and transmission of Cyclospora cayetanensis. The infection starts with the ingestion of the sporulated oocysts. It shows both the asexual and sexual reproduction. Fertilization occurs in the intestine. Unsporulated oocysts are excreted out in the environment where they sporulate and start the cycle again. It was noted that no drugs for cyclosporidiosis are in the clinical trials. Created with BioRender.com.

4.1 Current drug regimen for cyclosporiasis

Cyclosporiasis, is a highly prevalent disease, particularly among vulnerable population like those with HIV/AIDS, organ transplant recipients, and those undergoing immunosuppressive or anticancer regimens [6]. The primary treatment option for cyclosporiasis is trimethoprim-sulfamethoxazole (TMP-SMX), also known as co-trimoxazole, which is a combination antibiotic that inhibits Dihydrofolate reductase (DHFR) in C. cayetanensis (Table 1). This combination therapy disrupts folate metabolism, hindering DNA synthesis and thereby leading to parasite death. TMP-SMX has shown efficacy in both immunocompetent and immunocompromised patients (HIV/AIDS), with cure rates exceeding 90% in some studies [95]. However, prolonged use can result in antibiotic resistance, hence resulting in drug-resistant strains [96]. The extended use of TMP-SMX has been associated with a wide range of harmful effects on organs, including hematologic problems, liver damage, and skin responses [97].

For patients who cannot tolerate TMP-SMX due to sulpha allergy or treatment failure, alternative antibiotics like ciprofloxacin may be considered. Ciprofloxacin, although less effective than TMP-SMX, can be used as an alternative [33]. However, there have been situations where ciprofloxacin has been reported to be ineffective in treating certain condition, although it is generally acknowledged to be therapeutic in specific cases. Nitazoxanide is another drug with promise in treating cyclosporiasis [98], especially in patients with sulpha allergy (Table 1) [99]. Although the drug’s exact mode of action is yet unknown, it is believed to function by inhibiting their energy metabolism, damaging cell membranes, and compromising mitochondrial function. It interferes with the pyruvate: ferredoxin/flavodoxin oxidoreductase (PFOR) cycle, essential for energy production, and induces lesions in cell membranes. Additionally, it disrupts mitochondrial membrane potential and inhibits key enzymes, leading to parasite death [100]. Furthermore, it has been observed that nitazoxanide exhibits a direct inhibitory effect on coccidian oocysts, resulting in a reduction in their viability, as evidenced by the ultrastructural analysis [101]. Studies have reported cure rates ranging from 71 to 87% with nitazoxanide treatment, and the drug has been well-tolerated with no serious adverse effects [99]. The drugs commonly used to treat cyclosporiasis are listed in Table 1.

However, it is important to note that some antibiotics, such as norfloxacin, metronidazole, tinidazole, and quinacrine, have been shown to be ineffective against C. cayetanensis infections, rendering the treatment of C. cayetanensis infections highly challenging [90]. The research efforts are underway to explore novel drug targets and therapeutic strategies for C. cayetanensis infections using high-throughput screening assays, structure-based drug design, and drug-repurposing approaches. Advances in molecular biology and genomics have provided insights into the molecular mechanisms of Cyclospora pathogenesis, paving the way for the development of targeted therapies [1].

4.2 Recent drug discovery efforts against cyclosporiasis

The occurrence of adverse effects linked to trimethoprim-sulfamethoxazole (TMP-SMX) and ciprofloxacin drugs, coupled with the increase in Cyclospora resistance, hinders the efficiency of treatment [96]. This emphasizes the urgent requirement for innovative therapeutic approaches to tackle the issue of multidrug-resistant phenotypes. Recent research has highlighted the promising efficacy of curcumin and curcumin nanoemulsion (CR-NE) as novel therapeutic agents [102]. Studies conducted on mice models have demonstrated that CR-NE exhibits increased efficacy compared to the standard treatment of trimethoprim-sulfamethoxazole (TMP-SMX). It is believed that the anti-protozoal activity of CR is due to its ability to regulate transcription pathways and induce cellular death. They can also trigger apoptosis in injected cells by the activation of intracellular calcium release and mitochondrial membrane depolarization. It also affects cellular signaling pathways by targeting growth factors, receptors, transcription factors, cytokines, enzymes, and genes that are involved in the regulation of apoptosis. Its interference with the ability of Cyclospora to survive and proliferate within host cells could potentially lead to new treatments for cyclosporiasis [102].

These findings underscore the potential of curcumin and curcumin nanoemulsion as promising therapeutic options for cyclosporiasis. The enhanced efficacy of CR-NE, as demonstrated by decreased oocyst burden and improved antioxidant biomarkers, suggests its potential superiority over conventional therapies such as TMP-SMX. Further research and clinical studies are warranted to validate these findings and explore the clinical utility of CR-NE in treating cyclosporiasis in humans [102].

Modulating the host immune response represents a novel approach to combat Cyclospora infections. Recent research has focused on elucidating the immunopathogenesis of C. cayetanensis and identifying host immune factors associated with protection or susceptibility. Immunomodulatory agents, such as cytokine inhibitors or immune checkpoint inhibitors, hold promise for enhancing host defenses against the parasite [95]. Nanoparticle-based drug delivery systems hold promise for targeted drug delivery to the site of infection, minimizing systemic toxicity and enhancing therapeutic outcomes. Another study on magnesium oxide (MgO) nanoparticles reveals their effectiveness against C. cayetanensis oocysts, suggesting their potential as a new treatment approach. MgO nanoparticles show promise in inhibiting the viability and infectivity of C. cayetanensis, offering a novel strategy for combating this parasitic infection [103]. Further investigation into parasitic-specific research is required in order to acquire a more comprehensive understanding of the molecular pathways, with the aim of identifying novel therapeutic targets (Figure 3).

The apicoplast of C. cayetanensis, a non-photosynthetic plastid with its own genome, presents a potential focal point for developing novel therapeutic agents to combat the disease [104]. The ferredoxin-NADP+ reductase/ferredoxin redox system, also a key metabolic agent in apicomplexan human parasites, has potential as a therapeutic target due to its involvement in electron transfer reactions. Targeting this system could interfere with crucial metabolic processes essential for parasite survival, offering a promising opportunity for developing new antiparasitic medications [105]. C. cayetanensis also harbors a diverse array of proteases from distinct families such as cysteine, serine, and metalloproteases. Understanding the involvement of proteases in the biological processes of C. cayetanensis could provide valuable knowledge about its pathogenesis and aid in identifying potential therapeutic targets [106].

5. Cryptosporidiosis

Cryptosporidiosis is a gastrointestinal, watery diarrheal disease of humans, affecting children younger than 2 years and immunocompromised individuals more severely [107]. Cryptosporidiosis is caused by Cryptosporidium spp. which is an intracellular, intestinal protozoan parasite. It was first identified by Ernest Tyzzer in 1907 in a common mouse’s gastric glands [108]. Cryptosporidium hominins and Cryptosporidium parvum are the main species responsible for 90% of the cases in humans, causing the largest known outbreak of waterborne disease in the United States in 1993, affecting ~403,000 people [109]. The parasite is transmitted through the fecal-oral route via contaminated food and water [110]. Cryptosporidium, a Coccidia parasite, has a distinctive life cycle consisting of asexual and sexual stages within a single host [111]. It begins with the ingestion of infective oocysts containing four sporozoites, which then invade intestinal epithelial cells, transforming into trophozoites [112]. Trophozoites undergo multiple rounds of asexual replication, which amplifies the infection [113]. The sporozoites are excreted in feces, contributing to environment contamination and transmission to new hosts [114]. The adaptation of Cryptosporidium to a monoxenous life cycle and persistence in the environment pose significant public health challenges.

5.1 Current drug regimen for cryptosporidiosis

Nitazoxanide (NTZ), a nitrothiazole benzamide derivative, is the only FDA-approved drug used for the treatment of cryptosporidiosis [115]. NTZ treats and reduces diarrhea and oocyst shedding in children and adults [116] but does not show efficient results against HIV-infected or immunocompromised patients [117]. Paromomycin, an aminoglycoside, is another drug used against cryptosporidiosis [103]. Roxithromycin, a macrolide, has been found effective in patients having cryptosporidium infections along with AIDS, but only 50% of the medicated individuals had full clearance of parasites [118].

Rifamycin derivatives are the class of drugs that mainly affect mycobacterial infections by binding to DNA-dependent RNA polymerase and inhibiting RNA synthesis [119]. Its derivatives such as rifabutin and rifaximin are shown to be effective in treating cryptosporidiosis in individuals infected with HIV. Additionally, rifabutin was found to be more effective than other derivatives [120]. Letrazuril, a benzene acetonitrile derivative, has shown modest efficacy against advanced AIDS-related cryptosporidial diarrhea (Table 1) [121].

Additionally, different combinational therapies have also been used that show promising effectiveness for the treatment of cryptosporidiosis. When azithromycin/paromomycin combination therapy is used to treat cryptosporidial infections, there is a significant reduction in the amount of cryptosporidial oocysts excreted in feces [122]. A combination of azithromycin and nitazoxanide has been found to effectively treat cryptosporidiosis resulting in the total eradication of both diarrhea and parasites [123]. A combination of azithromycin, nitazoxanide, paromomycin, or rifaximin used in triple treatment resulted in a complete clinical parasitological recovery in kidney transplant recipients, with no instances of the recurrence [124]. The drugs commonly used to treat cryptosporidiosis are listed in Table 1.

5.2 Recent drug discovery efforts against cryptosporidiosis

Till now there has been no permanent treatment against cryptosporidium in humans as well as in animals. Due to the limited genetic tractability of Cryptosporidium, the absence of conventional targets, unique host cell location, and insufficient cell culture platforms, the progress in understanding host-parasite interactions is hampered [107], and subsequent drug discovery or development efforts are affected. However, recently, there have been advancements in drug discovery for the treatment of cryptosporidial infections due to improvements in the methods of its culture and genetic manipulation [125]. Currently, certain drugs such as bump kinase inhibitors (BKIs), pyrazolopyrimidine-based KDU731, triazolopyradizine MMV665917, benzoxaborole AN7973, and compound 2093 have demonstrated potential in treating cryptosporidiosis in animals (Figure 2B) [126]. Several studies have reported that BKI-1294 significantly reduces the shedding of oocysts and improves clinical symptoms by inhibiting C. parvum calcium-dependent protein kinase 1 (CpCDPK1) [127]. This enzyme is necessary for invading host cells [126]. However, BKI-1294 does not completely eliminate diarrhea and dehydration in infected bovines and neonatal calves [127]. Alternatively, BKI-1369, another inhibitor of CpCDPK1, shows its efficacy as a primary agent in combating Cryptosporidium infections in animals [126]. Specifically, it has shown effectiveness in treating neonatal calves infected with C. parvum [128]. However, these compounds cause cardiotoxicity and long QT syndrome in humans; thereby, more research efforts are needed to obtain better and safer derivatives of BKIs. Another potential anti-cryptosporidium drug molecule can be pyrazolopyrimidine derivative KDU731, which inhibits cryptosporidial lipid kinase PI (4)K (phosphatidylinositol-4-OH kinase) enzymatic activity [129].

The compound MMV665917, a piperazine derivative, showed inhibitory activity against both C. parvum and C. hominis in vitro as well as in mouse models [130]. A study conducted on the gnotobiotic piglet model strongly indicated that the use of MMV665917 resulted in the presence of C. hominis parasites in fecal matter, as well as a decrease in the shedding of oocysts, intestinal damage, and the severity of diarrhea [131]. However, like BKIs, MMV665917 also shows partial hERG inhibition and cardiotoxicity in humans [26]. Hence, further studies and research are needed to establish this molecule as an anti-cryptosporidial agent. The 6-carboxamide benzoxaborole AN7973 is also capable of inhibiting the growth of cryptosporidium and is termed safe and stable with good pharmacokinetic characteristics, but its mechanism of action and molecular targets in the parasite have not yet been identified [132]. The drugs under investigation against cryptosporidiosis are highlighted in Figure 2B.

6. Conclusions

In recent times, intestinal protozoan diseases, which were traditionally considered as tropical problems, are not restricted to tropical countries anymore. Although the understanding of genomes and the complex and diverse life cycles of these pathogens has considerably improved, no significant progress is achieved in finding new drugs for the treatment. The currently available drugs are inadequate due to limited numbers and limited chemical class diversity. Moreover, the available treatment options are not completely effective, and drug resistance is emerging. Very few drugs are currently in clinical trials. These lacunae highlight stringent need to pay renewed attention towords the drug discovery to combat these neglected pathogens. In light of modern “omics” based technologies, fundamental research and development efforts must be encouraged in order to have a deeper understanding of the molecular pathways orchestrating the myriad of novel functions within these pathogens. Lastly, the host-pathogen interactions can be another area which can be explored further to strenghten drug discovery efforts.

Acknowledgments

Sarika Thakur acknowledges the Department of Biotechnology (DBT) for financial assistance. Alka Sharma and Reena Negi acknowledge the University Non-NET fellowship from the Central University of Haryana, Mahendragarh – We thank Dr. Namrata Dhaka from Central University of Haryana for the valuable comments and editing..

Conflict of interest

The authors declare no conflict of interest.