Ponatinib, Lestaurtinib, and mTOR/PI3K Inhibitors Are Promising Repurposing Candidates against Entamoeba histolytica

ABSTRACT

Dysentery caused by Entamoeba histolytica affects millions of people annually. Current treatment regimens are based on metronidazole to treat invasive parasites combined with paromomycin for luminal parasites. Issues with treatment include significant side effects, inability to easily treat breastfeeding and pregnant women, the use of two sequential agents, and concern that all therapy is based on nitroimidazole agents, with no alternatives if clinical resistance emerges. Thus, the need for new drugs against amebiasis is urgent. To identify new therapeutic candidates, we screened 11,948 compounds assembled for the ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library against E. histolytica trophozoites. We identified 159 hits in the primary screen at 10 μM, and 46 compounds were confirmed in secondary assays. Overall, 26 were selected as priority molecules for further investigation, including 6 FDA approved, 5 orphan designations, and 15 that are currently in clinical trials (3 phase III, 7 phase II, and 5 phase I). We found that all 26 compounds are active against metronidazole-resistant E. histolytica, and 24 are able to block parasite recrudescence after drug removal. Additionally, 14 are able to inhibit encystation and 2 (lestaurtinib and LY-2874455) are active against mature cysts. Two classes of compounds are most interesting for further investigations: (i) the Bcr-Abl tyrosine kinase (TK) inhibitors, with ponatinib (50% effective concentration [EC₅₀], 0.39) as the most potent; and (ii) mTOR or phosphatidylinositol 3-kinase (PI3K) inhibitors, with 8 compounds in clinical development, of which 4 have nanomolar potency. Overall, these are promising candidates and represent a significant advance for development of drugs against E. histolytica.

INTRODUCTION

Entamoeba histolytica is an anaerobic protozoan and a waterborne pathogen generally spread in situations associated with contaminated food and water and lack of access to sanitation, as occurs in developing countries. The parasite is estimated to infect 500 million people annually (1, 2), with a prevalence of infection as high as 40% in some populations in regions of endemicity (3) and is estimated to cause 55,000 deaths each year (4). Amebiasis is also a major cause of traveler’s diarrhea (5). Among returning travelers reporting morbidity at GeoSentinel sites (mostly in Europe and the United States), the estimated incidence of E. histolytica infection is 12.5% of all microbiologically confirmed cases of infectious gastrointestinal diseases (6). Traveler’s diarrhea is also a problem among military personnel returning from regions of endemicity, with an incidence as high as 36.3 cases per 100 person-months (7). Sexual transmission and immigration are also among the reasons for amebiasis reemergence in developed countries (8).

Infection occurs when E. histolytica cysts are ingested in contaminated food or water and undergo excystation to produce the replicative and invasive trophozoite form. Cysts are resistant in the environment and responsible for spreading the disease to new patients. Disease begins with the adherence of the trophozoite form to colonic mucins and epithelial cells: the most common manifestation is amebic colitis, but infection may spread to other organs, particularly the liver, lung, and brain (9). Amebic colitis can be misdiagnosed as inflammatory bowel disease, which may lead to fulminant colitis after corticosteroid treatment (10).

Metronidazole (MNZ) is the most commonly prescribed treatment for invasive amebiasis. It is a low-cost nitroimidazole compound that is oral, rapidly absorbed, and highly effective against invasive trophozoites (9, 11). However, it has mild to severe side effects and is ineffective against luminal parasites and cysts: 40% to 60% of patients have parasites persist in the colonic lumen after treatment. Therefore, after metronidazole treatment, a second round of treatment with luminal agents such as paromomycin is required (12). The complexity of this regimen and the lack of symptoms during the second phase, when the second agent is needed, may reduce patient compliance and result in recurrent disease (13, 14). Other treatments for amebiasis are available; however, all rely on other nitroimidazole compounds with similar issues (11). Additionally, metronidazole resistance is easily induced under laboratory conditions (15). In Giardia and Trichomonas, other protozoan parasites that are treated with metronidazole, clinical metronidazole resistance is an increasing problem, indicating that similar problems may also arise with Entamoeba (16, 17). Thus, there is a need for development of new treatments for amebiasis, preferably with different modes of action to serve as optional therapy if parasite drug resistance emerges.

Drug development is costly and risky, with an average of $1.6 billion of investment for each drug launched (18). For this reason, diseases with lower predicted returns, including neglected diseases such as amebiasis, receive less attention from traditional routes of drug development. Repurposing drugs with known human safety profiles is an alternative that offers fewer risks, lower costs, and shorter timelines (19, 20). Auranofin is a successful example of repurposing, having been identified against amebiasis in a screening of ∼700 FDA-approved drugs (21); it has subsequently passed phase I clinical trials (22). Another recent screen of ∼3,400 unique compounds identified anisomycin, obatoclax, and prodigiosin as potent inhibitors of E. histolytica with activity against cysts and metronidazole-resistant parasites (23). Disulfiram, an antabuse FDA-approved drug, has also being highlighted as an interesting option for repurposing against amebiasis as its metabolite, zinc-ditiocarb complex, was shown to be efficient against E. histolytica in a mouse model (24, 25).

In this effort we screened the ReFRAME (Repurposing, Focused Rescue, and Accelerated Medchem) library, a set of ∼12,000 drugs that were assembled with a majority of compounds tested for clinical safety to accelerate discovery of drugs for new indications, including neglected and rare diseases (26). The ReFRAME library has been screened for many infectious disease indications, and the results are compiled in the a publicly available platform (https://reframedb.org). In our final analysis, we identified 26 priority compounds, including compounds with relevant features such as activity against metronidazole-resistant parasites, blockage of recrudescence, and inhibition of cysts. This effort demonstrates the value of screening the carefully assembled ReFRAME library against E. histolytica and represents a significant advance for drug development against amebiasis.

RESULTS

Screening the ReFRAME library identifies 86 hits against E. histolytica.

New treatments are needed for amebiasis, a neglected disease that is largely prevalent in areas of the developing world. To identify drug development candidates, we screened the ReFRAME library, a collection with 11,948 compounds assembled with the focus on accelerating drug development for neglected diseases (26). The workflow from screen to the list of final selected compounds is presented in Fig. 1. We identified 159 active compounds using a cutoff of 75% of viability inhibition (see Table S1 in the supplemental material). Primary screen hits were tested in 7-point dose-response curves (from 9 nM to 20 μM) in duplicate, using luciferase-expressing parasites. Setting a cutoff of 50% effective concentrations (EC₅₀s) lower than 20 μM, 86 hits were considered active (data available at the ReFRAME online portal at https://reframedb.org/assays/A00203 and in Table S2 in the supplemental material).

FIG 1.

Schematic workflow. The figure depicts the screening workflow of the ReFRAME library against E. histolytica trophozoites. The primary screen was performed with the whole library in a single point at 10 μM. With a cutoff of 75% of viability inhibition, 159 hits were selected. These drugs were tested in the secondary screen in a 7-point dose-response curve from 9 nM to 20 μM and with a cutoff EC₅₀ of 20 μM: 86 compounds were considered hits. A total of 51 of these compounds were selected based on potency, advanced clinical stage, and commercial availability to be purchased and tested in a 20-point dose response from 0.09 nM to 50 μM. A total of 20 derivatives or compounds with similar target specificity to the hits were added to this list. Of the 71 compounds, 46 were considered active, and 26 were selected for further assays based on potency and clinical stage.

Both primary and secondary screenings were performed using a transgenic cell line. Although G418 is removed from the medium for the assay, residues of the antibiotic could impact compound activity. This concern is resolved in subsequent confirmatory and phenotypic assays, in which we used the nontransgenic E. histolytica HM-1:IMSS strain. Other limitations should also be considered in drug discovery using a high-throughput library in vitro. For instance, in the case of therapies designed with prodrugs, the molecules we tested may not be the active form, which may lead to false-negative results. Moreover, in our screening we used a laboratory strain of E. histolytica. Under this condition, no host factor is considered, no pharmacology is addressed, and in the adaptation to the laboratory culture, the parasite may lose important aspects of its biology in vivo. Nevertheless, the significant findings emerging from high-throughput screenings with E. histolytica (21–23) are evidence that important drug discovery knowledge can be accessed with this methodology. Additionally, a number of hits which had been previously shown to inhibit E. histolytica viability were identified in this screen such as obatoclax, nithiamide, auranofin, and etofamine, further validating our approach (21, 23, 27).

Confirming compound activity.

With the list of 86 hits after the secondary screen, we selected 51 compounds based on potency, advanced clinical stages, and commercial availability for further testing. These compounds, plus an additional 20 related compounds, were purchased and tested in 20-point dose-response curves (0.09 nM to 50 μM). Assays were performed in triplicate, using E. histolytica HM-1:IMSS trophozoites in a fluorescent dye (fluorescein diacetate) viability assay to evaluate parasite survival. We chose to perform the confirmatory assay with a different readout to identify compounds that truly inhibit parasite viability and remove those that may have been identified in the first screen simply because they interfere with luciferase. All of the data obtained in this step, including EC₅₀, EC₉₀, R² values, and efficacy are shown in Table S3 in the supplemental material. After this assay, a total of 46 compounds were considered active with a confirmed EC₅₀ lower than 20 μM. We further selected compounds for ongoing testing using a variable threshold for potency depending on the clinical development stage of the compound. We applied the following criteria: EC₅₀ of <10 μM for drugs that received FDA approval (6 molecules) or orphan designation (5 molecules), EC₅₀ of <2 μM for drugs in phase III clinical trials (3 molecules), and EC₅₀ of <1 μM for drugs in phase II and I clinical trials (7 and 5 molecules). With these criteria, we selected 26 compounds that are more potent than metronidazole (EC₅₀, 11.85 μM), with 16 drugs presenting submicromolar potencies (Table 1). The targets in humans for each molecule are listed; a majority of kinases and some repeated antineoplastic targets such as mTOR/phosphatidylinositol 3-kinase (PI3K) and Bcr-Abl tyrosine kinase (TK) can be observed. Overall, these results identify a number of new druggable compounds that can be explored as therapeutic options against Entamoeba.

TABLE 1.

Activity of selected compounds against E. histolytica from screening the ReFRAME library^a

Highest phase	Compound name	EC50 (μM) for E. histolytica	Known target(s) in human cells
Positive control	Metronidazole	11.85
FDA approved	Plicamycin	0.26	DNA
	Bortezomib	0.61	Proteasome
	Ponatinib	0.39	Bcr-Abl TK
	Dasatinib	1.73	Bcr-Abl TK
	Bosutinib	2.44	Bcr-Abl TK
	Telotristat etiprate	6.70	TPH1
FDA orphan clinical trial
Phase III	Vosaroxin	2.85	DNA topoisomerase
	Lestaurtinib	4.77	FLK, TrK, JAK
	Bardoxolone methyl	6.31	Multiple
Phase II	Zotiraciclib (TG-02)	2.32	CDKs, JAK2, FLT3
	Milciclib maleate	7.60	CDK, TrKA
Clinical trial
Phase III	Beloranib	0.02	MetAP2
	Taselisib	1.39	PIK3
	Dactolisib	1.53	mTOR/PI3K
Phase II	CC-115	0.03	mTOR, DNA-PK
	NVP-BGT226	0.06	mTOR/PI3K
	TNP-470	0.07	MetAP2
	Bruceantin	0.11	Myc
	CUDC-907	0.44	HDAC, PI3K
	PF-04691502	0.47	mTOR
	Sapanisertib	0.93	mTOR
Phase I	Omipalisib	0.03	mTOR/PI3K
	LY-2874455	0.31	FGFR
	Latrunculin B	0.52	Actin
	R-547	0.53	CDK 1/2
	PF-03814735	0.82	Aurora kinase

^aThe table shows the EC₅₀s of the 26 selected compounds against E. histolytica trophozoites. The highest phase of clinical studies achieved with each compound and the target in humans of each compound are listed (30, 39, 60, 61).

Specific target inhibitors.

Among the 26 selected compounds in Table 1, we noted that 8 are mTOR or PI3K inhibitors. To understand if this activity against the parasite could be related to the potency of these compounds against the targeted enzymes, we expanded this panel by screening an additional 13 compounds, including 3 ReFRAME compounds that were active but not included on our priority compound list, and 10 new compounds selected from commercially available with specific activities that were organized in the following categories: selective to mTOR, selective to PI3K, selective to both mTOR and PI3K (dual inhibitors), and rapalogs, which contain rapamycin and derivatives.

Of the 13 new inhibitors included, 6 were active against E. histolytica, in addition to the 8 previously identified mTOR/PI3K inhibitors (Table 2). Further data for these compounds, such as EC₉₀, R² values, and efficacy, are shown in Table S3. Neither mTOR nor PI3K ATP binding pocket inhibitors seem to be preferred, since no relationship was found when the inhibition of E. histolytica viability and the target-specific inhibition potencies of the compounds tested were compared (Table 2). Interestingly, none of the rapalog compounds showed significant activity, which is notable since rapalogs have a different mechanism than PI3K or mTOR selective inhibitors. The former class binds to FKBP12, and the complex inhibits mTOR by blocking substrate access (28), while the latter class competes with ATP for the binding pocket of these kinases (29). Hence, the lack of activity of rapamycin and rapalogs compared to variable activity in the other three categories indicates that the inhibition in parasite viability may be related to the compounds’ affinity to the ATP binding site of mTOR and PI3K.

TABLE 2.

Activity of specific target inhibitors against E. histolytica^a

Selectivity	Compound name	Potency against:		Other target(s)	EC50 (μM) for E. histolytica	Highest clinical trial phase
		mTOR or Abl	PI3K or Bcr-Abl
		mTOR	PI3K
mTOR	Sapanisertib	++++	+		0.93	Phase II
	Gedatolisib*	++++	+		Not active	Phase II
	Vistusertib*	++++		P-Akt, PS6	Not active	Phase II
	CC-115	++	+	DNA-PK	0.03	Phase II
PI3K	Taselisib		+++++	C2β, hVps34	1.39	Phase III
	GDC-0084*	+	+++		19	Phase II
	NVP-BGT226		++		0.06	Phase I/II
	AZD-8835		+		5.71	Phase I
	Wortmannin*		+++	DNA-PK, ATM, MLCK	2.47	Preclinical
	GSK-2636771		+++		18.48	Phase I/II
	XL765*	+	++	DNA-PK	Not active	Phase II
	CUDC-907		++	HDAC	0.44	Phase II
Dual	Omipalisib	+++++	+++++		0.03	Phase I
	SB-2343	++++	+++		8.18	Phase I
	Dactolisib	+++	++	ATR	1.53	Phase III
	Bimiralisib*	++	+++		Not active	Phase II
	PF-04691502	++	++++	P-Akt	0.47	Phase II
	Apitolisib*	++	+++		15.53	Phase II
Rapalogs	Rapamycin*	++++		FKBP12	Not active	FDA approved
	Everolimus*	++++		FKBP12	Not active	FDA approved
	Temsirolimus*	++++		FKBP12	Not active	FDA approved
		Abl	Bcr-Abl
Bcr-bl	Ponatinib	+++++		PDGFRα, VEGFR2, FGFR1	0.3927	FDA approved
	Dasatinib	+++++		Src, c-Kit(D816V), c-Kit (WT)	1.728	FDA approved
	Bosutinib	+++++		STAT3, ERK, S6 kinase	2.44	FDA approved
	Rebastinib	++++		FLT3, KDR, Tie-2	13.4	FDA orphan (phase II)
	Imatinib	+		c-Kit, PDGFR	Not active	FDA approved
	Nilotinib		++		Not active	FDA approved
	Bafetinib	+++		Lyn	Not active	Phase II
	Danusertib	++		Aurora A, TrkA, RET	Not active	Phase II
	Tozasertib		++	Aurora A, Aurora C, Aurora B	Not active	Phase I

^aIn the top part of the table, inhibitors of mTOR and PI3K with distinct selectivity were tested in 8- or 16-point drug response curves against trophozoites of E. histolytica. The top part of the table is divided into categories of selectivity to mTOR, PI3K, or dual mTOR/PI3K inhibitors, as well as rapalogs. The potency of each molecule against the purified human protein is represented in the following scale: +++++, <1 nM; ++++, 1 nM to <5 nM; +++, 5 nM to <10 nM; ++, 10 nM to <50 nM; +, >50 nM (50, 62–82). In the bottom part of the table, inhibitors of Bcr-Abl TK are listed, and the potency of each molecule against the purified human protein is represented using the scale defined above (83–90). Activity is shown according to the EC₅₀ (μM). A compound name followed by an asterisk (*) indicates additional compounds not identified in the primary screen. All other molecules were identified from the ReFRAME library. The highest phase of clinical studies achieved with each compound and the targets in humans of each compound are listed.

ReFRAME screens against other intestinal parasites revealed less activity of mTOR/PI3K inhibitors than what we observed. SB-2343 is the only compound active against Giardia lamblia and NVP-BGT226, and sapanisertib, dactolisib, and gedatolisib are active against Cryptosporidium parvum (30). The activities of mTOR/PI3K inhibitors have been found enriched against amebic parasites that can infect the brain, including Naegleria, Balamuthia, and Acanthamoeba (31), suggesting this class of compounds may have greater activity against amebic parasites. Interfering with mTOR pathway in the host has also been studied as a possible therapeutic route for parasitic diseases, such as toxoplasmosis, leishmaniasis, and malaria (32).

Another target that is repeated among the selected compounds is Bcr-Abl TK, with ponatinib, dasatinib, and bosutinib as potent FDA-approved compounds against E. histolytica. We expanded this list by testing rebastinib, bafetinib, nilotinib, imatinib, danusertib, and tozasertib. We observed increasing activity against E. histolytica corresponding to increasing potency of these compounds against human ABL1 (Table 2). Although the parasite target has not been determined for these molecules, our results indicate that Bcr-Abl TK inhibitors may be an interesting class of compounds for further investigations of their activity against Entamoeba.

Activity against metronidazole-resistant parasites.

In order to identify compounds that are active against metronidazole-resistant parasites, we tested the 26 selected compounds against a metronidazole-resistant cell line that we previously generated (23). We performed parallel experiments in 6-point dose-response curves and in both wild-type (WT) and MNZ-resistant parasites.

The EC₅₀ calculated for metronidazole against the MNZ resistant parasites was almost 4× greater than that for the WT strain, validating the MNZ resistance of this cell line (Table 3). Of the 26 drugs tested against wild-type and metronidazole-resistant parasites, 24 had comparable EC₅₀s for WT and MNZ-resistant parasites, with a fold change lower than 2 indicating similar sensitivity to the drug of interest in the MNZ-resistant line. This may indicate that the drugs tested have mechanisms of action in E. histolytica that are different from the one of metronidazole, which will be useful in case of emergence of clinical resistance. A few compounds had higher EC₅₀s (fold change of >2) for the metronidazole-resistant parasites, such as bosutinib (fold change of 2.1) and NVP-BGT226 (fold change of 2.05). However, even for these drugs, the EC₅₀s against MNZ-resistant parasites were still low (5.2 and 0.13 μM, respectively). The slight increase in EC₅₀ for bosutinib is probably not related to its mechanism of action, since the effect was not observed for other Bcr-Abl TK inhibitors, ponatinib and dasatinib. Anisomycin and prodigiosin were previously identified as active against metronidazole-resistant parasites (23), and lestaurtinib and bardoxolone methyl have also been found active against Giardia (30), which makes these compounds promising for treating both intestinal parasitic diseases. The ReFRAME library has not been tested against Trichomonas vaginalis, but this represents another anaerobic pathogen typically treated with MNZ, but in which resistance has been demonstrated (17). These results show that all 26 compounds represent viable alternatives to nitroimidazole therapies as they are active against MNZ-resistant E. histolytica parasites, and some may also be interesting leads against other parasites that develop MNZ resistance. Of note, in this assay the absolute EC₅₀ value for metronidazole against the HM-1:IMSS strain is somewhat lower than the one found at the 20-point dose-response curve that we have previously demonstrated; this could be due to having lower resolution, with a 6-point dose-response curve versus a 20-point dose response curve, or to the parasite growth differences in 96-well plates versus the experiment’s 384-well plates.

TABLE 3.

Selected compounds are active against metronidazole-resistant parasites^a

Highest phase	Compound name	EC50 (μM) from 6-pt dose-response curves		Fold change
		WT (HM-1:iMSS)	MNZ resistant
Positive control	Metronidazole	4.93	18.77	3.8
FDA approved	Plicamycin	0.26	0.05	0.2
	Bortezomib	0.42	0.18	0.4
	Ponatinib	0.39	0.73	1.9
	Dasatinib	1.73	1.82	1.1
	Bosutinib	2.44	5.20	2.1
	Telotristat etiprate	6.70	5.15	0.8
FDA orphan clinical trial
Phase III	Vosaroxin	2.14	2.95	1.4
	Lestaurtinib	2.64	1.94	0.7
	Bardoxolone methyl	2.81	4.93	1.8
Phase II	Zotiraciclib (TG-02)	2.32	1.57	0.7
	Milciclib maleate	4.89	4.27	0.9
Clinical trial
Phase III	Beloranib	0.02	0.02	1.1
	Taselisib	0.83	1.39	1.7
	Dactolisib	0.17	0.04	0.3
Phase II	CC-115	0.03	0.04	1.4
	NVP-BGT226	0.06	0.13	2.1
	TNP-470	0.03	0.02	0.9
	Bruceantin	0.05	0.03	0.6
	CUDC-907	0.18	0.12	0.6
	PF-04691502	0.47	0.48	1.0
	Sapanisertib	0.41	0.30	0.7
Phase I	Omipalisib	0.01	0.02	1.4
	LY-2874455	0.28	0.48	1.7
	Latrunculin B	0.23	0.42	1.8
	R-547	0.55	0.50	0.9
	PF-03814735	0.57	0.66	1.2

^aThe 26 compounds were tested in 6-point dose-response curves against MNZ resistant in parallel with the wild-type parasite (HM1:IMSS) E. histolytica. Results are shown as EC₅₀ (μM) of both parasite lines, and the fold change (MNZ EC₅₀ versus WT EC₅₀) for each compound tested is shown in the last column.

Activity in preventing parasite recrudescence.

Recrudescence was evaluated by testing the parasite’s viability when treated with the compounds’ EC₉₀ before and after a 72-h period of drug removal. (Fig. 2; see Table S4 in the supplemental material). Metronidazole was included at its EC₉₀ for comparison with the standard of care and at 50 μM in attempt to promote complete inhibition of the parasite growth. Vosaroxin (33%) and bruceantin (61%) exhibited higher recrudescence than metronidazole (18%). All other 24 compounds were effective in preventing recrudescence, suggesting a cytotoxic effect, which is ideal for treating parasitic diseases and would help prevent recurrent disease by dormant parasites. These data are useful in helping to prioritize compounds for further development against Entamoeba. A similar approach in another amebic parasite, Balamuthia mandrillaris, identified plicamycin, zotiraciclib (TG-02), and auranofin as drugs that inhibit recrudescence (31).

FIG 2.

Activity of compounds in blocking recrudescence of E. histolytica. Selected compounds were tested at a concentration to inhibit 90% of parasite viability. After 72 h of treatment, parasites were removed from the drug and left to recover for another 72-h period. The graph depicts the percentage of fluorescence signal compared to DMSO treatment (100%). Asterisks (*) indicate results after 72-h drug removal that are above 10% viability and significantly higher than values with drug treatment. Shown are adjusted P values of <0.05.

Drug activity against encystation and mature cysts.

Current treatment of amebiasis involves metronidazole to treat trophozoites, followed by paromomycin to target luminal parasites’ trophozoites and cysts (11, 33). Compounds that target trophozoites and can also inhibit encystation or kill mature cysts can drastically reduce amebiasis transmission and could lead to a single-drug treatment for Entamoeba. To identify compounds with this feature, we tested the selected drugs against encystation and mature cysts. E. histolytica can be cultivated as trophozoites, but the encystation process has not yet been developed to high efficiency in the lab for the purposes of a drug screen. The currently accepted model for high-level, reliable in vitro encystation uses Entamoeba invadens, a species that infects reptiles (34). Thus, we defined the EC₅₀ for the 26 selected compounds in E. invadens (Table 4). R² values and efficacy are shown in Table S5 in the supplemental material. Of the 26 compounds, plicamycin is the only drug that did not have any effect on E. invadens viability. Bosutinib, telotristat etiprate, CUDC-907, and R-547 showed low efficacy (EC₅₀ higher than 20 μM) (Table S5). Drug susceptibility differences between E. histolytica and E. invadens have previously been noted, and so these results are not surprising (23).

TABLE 4.

Activity of selected compounds inhibiting encystation in E. invadens^a

Highest phase	Compound name	EC50 (μM) for E. invadens	% Encystation after treatment at 1× EC50	Significance vs DMSO
Positive control	A23187 (at 5 μM)	NA	26 ± 3.4	****
FDA approved	Plicamycin	Not calculated	Not tested
	Bortezomib	3.93	4 ± 0.9	****
	Ponatinib	6.97	76 ± 5.3	*
	Dasatinib	4.42	87 ± 8.8	NS
	Bosutinib	22.96	11 ± 1.6	****
	Telotristat etiprate	26.24	41 ± 7.5	****
FDA orphan clinical trial
Phase III	Vosaroxin	3.03	11 ± 2.2	****
	Lestaurtinib	1.27	5 ± 0.9	****
	Bardoxolone methyl	2.38	2 ± 0.3	****
Phase II	Zotiraciclib (TG-02)	15.62	4 ± 1.2	****
	Milciclib maleate	13.34	27 ± 4.2	****
Clinical trial
Phase III	Beloranib	0.15	11 ± 2.2	****
	Taselisib	1.43	55 ± 19.8	****
	Dactolisib	1.05	68 ± 9.0	**
Phase II	CC-115	0.31	68 ± 14.7	**
	NVP-BGT226	0.67	64 ± 11.4	**
	TNP-470	0.06	11 ± 2.6	****
	Bruceantin	0.06	79 ± 19.9	NS
	CUDC-907	27.96	3 ± 0.9	****
	PF-04691502	0.13	36 ± 8.4	****
	Sapanisertib	3.01	70 ± 14.3	**
Phase I	Omipalisib	0.01	113 ± 16.6	NS
	LY-2874455	1.40	9 ± 2.2	****
	Latrunculin B	0.10	24 ± 7.0	****
	R-547	5.01	14 ± 1.5	****
	PF-03814735	1.60	22 ± 9.7	****

^aTrophozoites of E. invadens were incubated in encystation medium for 48 h with the selected compounds at a concentration equal to the EC₅₀ obtained in E. invadens. Cysts were counted using high-content imaging. The results are presented as a percentage of the encystation value ± standard error. Treatment with DMSO is considered 100% encystation. NA, not applicable. Significance was calculated with a t test against DMSO treatment values. Asterisks: *, adjusted P values between 0.05 and 0.01; **, adjusted P values between 0.01 and 0.001; ****, adjusted P values of <0.0001. Nonsignificant differences are indicated (NS). Boldface indicates compounds that were considered active, reducing encystation efficiency (<30% encystation).

After defining the EC₅₀ in E. invadens trophozoites, the activity against encystation was tested (23). The results are expressed as a percentage of the control number of cysts after 48 h of treatment (Table 4). In this in vitro model, we have determined that the calcium ionophore A23187 (35) blocks encystation when added at the initiation of encystation (see Fig. S1 in the supplemental material). We adapted this compound for use as a positive control in our encystation assay and saw significant reduction of encystation. Treatment with dimethyl sulfoxide (DMSO) is considered to show 100% encystation efficiency, and the calcium ionophore A23187 is the positive control for blocking encystation. Metronidazole had been shown to be ineffective against encystation efficiency (23). Of the 25 molecules tested in this manner, 14 were considered active by using the criterion that treatment with 1× EC₅₀ resulted in >70% encystation inhibition. Encystation inhibitors have been previously identified from several repurposing drug libraries (23). These compounds may serve as a useful tool to block an important biological pathway of the parasite and would be of high priority as they may serve as a one-drug approach to treat both trophozoites and cysts.

The assay described above tests the encystation process, but additional inhibition of mature cysts is high priority as it is the shedding of mature cysts into the environment that propagates disease. We previously developed an assay to test this feature and have demonstrated that metronidazole does not inhibit mature cysts (23). We now evaluated the ability of 10 compounds that were efficient encystation inhibitors to target mature cysts. Data are expressed as percentage of inhibition compared to treatment with DMSO (Fig. 3). Obatoclax, previously identified as targeting mature cysts, was used as a positive control (23). In our assay, lestaurtinib was the most active compound, with 75% and 50% mature cyst inhibition at 20 and 10 μM, respectively, and no activity observed at 5 μM. LY-2874455 was also active at 20 μM, with 65% inhibition, and with a slight but not significant inhibition at 10 μM. Milciclib maleate, bardoxolone methyl, and vosaroxin presented slight inhibition in cyst viability, although without significant difference. R-547, PF-03814735, bortezomib, and zotiraciclib (TG-02) did not inhibit cyst viability. These results highlight lestaurtinib and LY-2874455 as interesting compounds for further development against amebic cysts; both are oral drugs (36, 37) and potent inhibitors of both E. histolytica life cycle stages, trophozoites and cysts.

FIG 3.

Lestaurtinib and LY-2874455 are active against E. invadens mature cysts. Ten compounds found active in blocking encystation were tested against mature cysts at 20 μM. Active cyst inhibitors were tested at lower concentrations, as indicated. Results are shown as a percentage of luminescence signal obtained from DMSO treatment. Obatoclax at 5 μM is the positive control (23). Asterisks indicate results that are significantly lower than DMSO control: **, adjusted P values between 0.01 and 0.001; ****, adjusted P values of <0.0001.

DISCUSSION

Screening of the ReFRAME library opened several drug development opportunities for amebiasis, with identification of 26 molecules in different stages of clinical trials and various target classes to be explored. Among this set of drugs, we identified compounds with advantages for clinical use, such as activity against MNZ-resistant parasites, blockage of parasite recrudescence, and efficacy against encystation or mature cysts. We compiled the results in a heat map to give an overview of the drugs, their regulatory status, and their features of interest in specific areas of parasite biology (Fig. 4).

FIG 4.

Heat map summary of the compounds. The potency against E. histolytica and results of three phenotypic assays are shown. Results obtained for E. histolytica EC₅₀, MNZ-resistant EC₅₀, the percentage of survival postrecovery, and encystation inhibition were normalized considering the highest value in the set as 100, represented as the darkest color (Worst activity) and 0 as the lightest gray color (Best activity). A white square with an X indicates a compound (plicamycin) that was not tested in that assay.

We identified six FDA-approved compounds highly active against E. histolytica, of which plicamycin (EC₅₀, 0.26 μM), ponatinib (EC₅₀, 0.39 μM), and bortezomib (EC₅₀, 0.61 μM) have submicromolar potency (Table 1). This highlights one advantage of the ReFRAME library, as it was assembled using compounds with significant clinical use. For a repurposing strategy, ponatinib is the most interesting among the three cited drugs as plicamycin has been discontinued and bortezomib is tolerated in mice in very low doses, which would make it challenging to test in animal models of amebiasis for in vivo efficacy (38, 39).

Ponatinib is a multikinase inhibitor with primary affinity to Bcr-Abl TK and with oral bioavailability (40). It is the most potent among the compounds in this class (EC₅₀, 0.39 μM, compared to dasatinib and bosutinib, with EC₅₀s of 1.73 and 2.44 μM, respectively) against E. histolytica and is 30× more potent than metronidazole. All three Bcr-Abl TK inhibitors are active against MNZ-resistant parasites and are able to block recrudescence, but are not able to inhibit encystation. These drugs, as well as other antineoplastic kinase inhibitors, were previously described to be active against E. histolytica with similar potencies (41). In addition, a cross talk was found between the Bcr-abl protein and the Trx system (42), a known target of amebiasis therapies with nitroimidazole molecules and auranofin (43, 44), highlighting the Bcr-Abl TK inhibitors as potential drug development candidates against the parasite.

All five drugs that have received FDA orphan designation were able to inhibit MNZ-resistant parasite viability: most blocked parasite recrudescence, were able to inhibit encystation, and, therefore, were tested against mature cysts. Lestaurtinib, a multitargeted tyrosine kinase inhibitor, was the most efficient compound, with 76% cyst inhibition at 20 μM and 50% at 10 μM. This molecule was found active against Naegleria (a free-living ameba) (31, 45), Trypanosoma cruzi, Leishmania major, Mycobacterium tuberculosis, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (30). Most interestingly, lestaurtinib was also found to inhibit G. lamblia, with similar potency found against E. histolytica (EC₅₀ of 4.25 μM) (https://reframedb.org/assays/A00229). Giardiasis is also an intestinal disease spread by infectious cysts that is treated with metronidazole, with significant clinical resistance (16). Thus, lestaurtinib should be considered for further studies as a treatment for multiple intestinal parasitic pathogens.

Beloranib and TNP470 are among the most potent compounds found in this study (Table 1). Both are MetAP2 inhibitors and chemically similar to fumagillin, previously shown be active against Entamoeba (46). Both are very potent against metronidazole-resistant parasites, block recrudescence at very low concentrations, and are also able to inhibit encystation. However, concerns about venous thromboembolism in beloranib-treated patients motivated the development of alternative MetAP2 inhibitors with better safety profiles (47).

The majority of the other compounds are mTOR/PI3K inhibitors; most are in clinical trials in phase II and III, with omipalisib in phase I. All mTOR/PI3K inhibitors among the selected compounds (dactolisib, CC-115, NVP-BGT226, CUDC-907, PF-04691502, sapanisertib, and omipalisib) are able to inhibit the metronidazole-resistant parasites and block recrudescence, but most are not efficient encystation inhibitors. These compounds are all orally bioavailable, and several have been reported in phase I clinical trials to be well tolerated (48–54). Even though no relationship was observed between enzymatic specificity or potency and amoebicidal activity (Table 2), the behaviors of mTOR and PI3K inhibitors were similar across the phenotypic assays. Considering the lack of activity of rapalogs, compounds with higher affinity to the mTOR and PI3K ATP binding sites are of interest for development of therapy against amebiasis and further target validation studies.

As a neglected disease endemic in communities with poor sanitation conditions, a good candidate for drug development against amebiasis is an inexpensive, orally bioavailable molecule. It should ideally be safe for children and elderly people, as well as pregnant and breastfeeding women, with no severe or intolerable side effects. Slow absorption would be an interesting feature to ensure good luminal concentration. In addition to good cytotoxic potency against trophozoites and cysts of E. histolytica, the preferred compounds would also have good activity against metronidazole-resistant parasites.

Given these considerations, ponatinib would be a good candidate for a repurposing strategy as an FDA-approved oral drug potent against E. histolytica. It has been noted to cause severe side effects (e.g., pancreatitis, arrhythmia, and hypertension), which is a concern; however, those events were identified over several months of treatment, and lower doses of the drug are associated with fewer side effects (55). Thus, lower drug doses and a shorter treatment of duration may be able to overcome these hurdles and make this a viable option for amebiasis therapy. Lestaurtinib, another orally bioavailable drug with orphan status, is generally well tolerated (36, 56), has activity against Entamoeba cysts and Giardia, and is thus a very interesting option for further development. Molecules that are in earlier stages of development, such as LY-2874455 and the mTOR/PI3K inhibitors, also have relevant activity against Entamoeba, are orally bioavailable, and should be considered for further studies as more robust data on clinical safety profile and pharmacology parameters become available.

In summary, we identified several molecules and potential targets with promising profiles for the development of therapies against amebiasis, a devastating disease that affects millions of people worldwide. All of our selected compounds are more potent than the current standard of care, metronidazole, and several performed better in our phenotypic assays, representing clinical advantages, such as the abilities to inhibit encystation or mature cysts, block recrudescence, and inhibit MNZ-resistant parasites. As we move to the next steps, it will be important to design animal studies considering the particularities of selected drugs, such as possible immunosuppression and other side effects, to obtain animal pharmacology profiles and to determine in vivo efficacy in an animal model of colitis.

MATERIALS AND METHODS

Parasite culture and strains.

Trophozoites of Entamoeba histolytica strain HM-1:IMSS were grown and maintained at 37°C in TYI medium under standard conditions (57). For primary and secondary screen assays, we used a stable transgenic heterogeneous cell line expressing luciferase (Eh-CS-luc) maintained in 6 μg/mL G418 (58). Trophozoites of Entamoeba invadens (strain IP-1) were grown and maintained at 25°C in LYI medium under standard conditions (34). For the cyst assay, a stable transgenic heterogeneous E. invadens cell line expressing luciferase (Ei-CK-luc) was established and maintained in 40 μg/mL G418 in LYI medium (59). Both E. histolytica and E. invadens parasites were maintained in glass tubes and expanded for 2 to 3 days before the assays into plastic T25 flasks.

Compound library and screening.

For the primary and secondary screen assays, the compounds from the ReFRAME library were pinned for a total of 0.1% of volume in white 384-well Greiner Bio-One plates, which were heat-sealed, shipped frozen, and kept at −20°C until the day before testing. Our screening was performed with the support of the Stanford High Throughput Biosciences Center (http://htbc.stanford.edu). The plates were thawed at room temperature overnight. E. histolytica cells expressing luciferase (Eh-CS-luc) grown in T25 flasks were iced for 10 min, centrifuged for 10 min at 230 relative centrifugal force (RCF), resuspended in fresh medium, and seeded into the library plates at 3,000 Eh-CS-luc cells/well in 100 μl of fresh TYI medium using a Multidrop DW (Thermo Fisher, USA). Plates were covered with a lid and incubated at 37°C for 48 h in an anaerobic chamber using a BD GasPak System with two CO₂ generator sachets (Becton Dickinson and Company, USA). BrightGlo (Promega, USA) reagent was prepared and kept at −80°C until the day before reading, when it was transferred to 4°C to thaw overnight. At 2 h before reading, the BrightGlo reagent and assay plates were placed on the bench to equilibrate at room temperature. After 40 min, plates were centrifuged for 10 min at 230 RCF, the liquid was aspirated with Bio-Tek ELx405 (BioTek, USA) to leave 10 μL per well, and then 20 μL per well of BrightGlo diluted in phosphate-buffered saline (PBS [1:1]) was added. Luminescence signal was read on the Tecan Infinite M1000 Pro (Molecular Devices, US). DMSO at 0.1% and metronidazole at 50 μM were used as negative and positive controls, respectively.

The primary screening was performed with the whole ReFRAME library at a single dose of 10 μM and a single replicate. Compounds that inhibited E. histolytica viability at least 75% compared to the DMSO control were selected as primary hits (Table S1). These were used for the secondary screen, in which compounds were pinned for 7-point dose-response curves (from 9 nM to 20 μM) in two biological replicates. Raw data were normalized based on percentage of blank control, and the EC₅₀ was calculated using Genedata software. The dose-response curves of the 86 compounds considered active were used to calculate EC₅₀, efficacy and R² values (Table S2). All data are available at https://reframedb.org/assays/A00203.

Confirmatory assays.

For the confirmatory assays, we purchased 71 molecules (51 identified from the ReFRAME library and 20 related compounds) from commercial sources and tested them in 20-point dose-response curves in Greiner-Bio Cellstar 384-well plates with black walls. For the assay, 10 μL of medium was added to all wells, and another 10 μL of medium containing 2 μL of DMSO stock solution of each compound at 5 mM was added to the well with the highest concentration of the dose-response curve. Serial dilution (1:2) was performed by transferring 10 μL of the diluted solution to the following 19 wells, to result in a curve ranging from 0.09 nM to 50 μM when completed to a final volume of 100 μL. Cells of wild-type E. histolytica strain HM-1:IMSS growing in T25 flasks were iced for 10 min, centrifuged for 10 min at 230 RCF, resuspended in fresh medium, and primed to the multidrop pipettor. Parasites were plated (3,000 cells per well in 90 μL), and plates were covered with a lid and incubated in anaerobic chamber at 37°C for 72 h. Two columns were left with medium only, to calculate the background value. Plates were then centrifuged for 10 min at 230 RCF, and the medium was aspirated with Bio-Tek ELx405 (BioTek, USA), leaving 10 μL. Then, 30 μL of fluorescein diacetate diluted in fresh medium at a concentration of 20 μg/mL was added to all wells. The plates were incubated for 20 min at 37°C. To wash the medium with fluorescein diacetate and avoid high fluorescence background, 70 μL of 1× PBS was added per well, the plates were centrifuged for 10 min at 230 RCF, the liquid was again aspirated with Bio-Tek ELx405 (BioTek, USA), leaving 10 μL, and another 90 μL of 1× PBS was added to each well. Fluorescence was read using the Tecan Infinite M1000 Pro. Metronidazole dose curves were used as a positive control in all biological replicates, and 1% DMSO was used as a negative control. EC₅₀s were calculated as described below. Each compound was tested in at least three biological replicates.

EC₅₀ calculation.

In order to calculate the EC₅₀, the percentage of inhibition relative to maximum and minimum reference signal controls was calculated using the formula % inhibition = [(mean of maximum signal reference control − experimental value)/(mean of maximum signal reference control − mean of minimum signal reference control)] × 100.

The relative dose-response data in triplicate were exported to GraphPad Prism software 8.0 for EC₅₀ calculations using 4PL sigmoidal nonlinear regression. R² values and efficacy were used for statistical analysis.

Evaluating E. histolytica recrudescence after drug treatment.

To investigate if any viable E. histolytica were remaining after compound treatment, we evaluated the ability of parasites to recover growth after treatment with the selected drugs for 72 h. For that, 200,000 parasites were treated in 8-mL glass tubes with each of the 26 compounds at a concentration able to inhibit 90% of E. histolytica growth in 72 h of treatment at 37°C (EC₉₀). After treatment, the tubes were iced and centrifuged for 6 min at 230 RCF. The pellets were resuspended in 8 mL of fresh medium, and 300 μL of the suspension was transferred to multiple wells of two 96-well black plates with clear bottoms. One of the plates was incubated with fluorescein diacetate dye immediately, and the viability was evaluated as described previously. The second plate was incubated in an anaerobic chamber at 37°C for 72 h to allow any remaining viable parasites to grow. After this 72-h recovery period, parasite viability was also evaluated with fluorescein diacetate dye. Treatment with metronidazole at 50 μM was used as a positive control and 0.5% DMSO as a negative control. Each compound was tested in three biological replicates. Percent values were exported to GraphPad Prism software 8.0. For statistical analysis, values obtained before and after the 72-h period of drug removal were compared using a t test. Recrudescence was considered positive when the value after drug removal was above 10% and significantly different (P < 0.05) from the value previously obtained for 72-h treatment with the same compound.

Drug effect on metronidazole-resistant E. histolytica.

Parasites resistant to metronidazole (MNZ) were generated by continuous growth of E. histolytica HM-1:IMSS in MNZ, with steadily increasing concentrations from 1 μM to 15 μM (23). Parasites growing well and able to be sequentially passaged multiple times at 15 μM MNZ were defined as MNZ resistant and used for further experiments. A total of 15,000 amebae from the MNZ-resistant strain were seeded into a 96-well plate with medium and dose-response curves or the DMSO control. The plates were covered with a lid and incubated in an anaerobic chamber for 72 h at 37°C. Dose-response curves were done with 6 points in a 1:3 dilution, and the maximum concentrations were 45 μM for drugs with an EC₅₀ of >1 μM, 5 μM for drugs with an EC₅₀ between 0.1 μM and 1 μM, and 1.6 μM for drugs with an EC₅₀ of <0.1 μM. Viability was assayed by fluorescein diacetate fluorescence, as detailed previously. Parallel dose-response curves were performed using the MNZ-sensitive E. histolytica HM-1:IMSS strain as a control, seeded at 6,000 parasites per well. Each compound was tested in three biological replicates. The compounds’ EC₅₀s against the WT (HM-1:IMSS) and MNZ resistance cell lines were calculated and analyzed as described above. Fold change (EC₅₀ in the MNZ-resistant strain versus EC₅₀ in the WT HM-1:IMSS strain) was used to evaluate if any change of activity is found when the parasite is resistant to metronidazole.

EC₅₀ definition in E. invadens.

E. invadens EC₅₀s were determined by seeding E. invadens IP-1 parasites in logarithmic growth in 96-well black plates with clear bottoms, which were covered with a lid in an anaerobic chamber at a density of 40,000 parasites per well, with the compounds in dose-response curves done with 9 points ranging from 7 nM to 45 μM. Potent drugs were further tested in dose-response curves ranging from 0.8 nM to 5 μM. After 72 h at 25°C, plates were centrifuged at 230 RCF, liquid was aspirated, 50 μL fluorescein diacetate dye was added at a concentration of 20 μg/mL, and the plates were incubated for 20 min. Once again, plates were centrifuged at 230 RCF, liquid was aspirated, and 300 μL of 1× PBS was added to each well. Fluorescence was read using the Tecan Infinite M1000 Pro. Each compound was tested in three biological replicates. Metronidazole dose curves were used as a positive control in all biological replicates, and 1% DMSO was used as a negative control. EC₅₀s were calculated as described above.

Evaluating drug effect on encystation efficiency.

Encystation assays were performed as previously described (23). Briefly, E. invadens IP-1 trophozoites were harvested in mid-log phase, washed once, resuspended in encystation medium (47% LG) (34), and plated in 96-well black plates with clear bottoms, at a density of 50,000 parasites per well. Drugs were added at a concentration that was 1× the E. invadens EC₅₀, as determined above. Plates were covered with a lid and incubated for 48 h at 25°C in an anaerobic chamber and then spun, and the medium was removed. Cysts were stained by addition of 50 μL of 50 μM calcofluor white (Sigma) and 4% paraformaldehyde (PFA) in PBS, incubated at room temperature for 20 min, and evaluated by high-content imaging in an ImageExpress Micro (Molecular Devices). The number of cysts per well was quantified using MetaExpress software. Each compound was tested in three biological replicates. Parasites treated with DMSO only were used as a positive control, and media without parasites were used to establish background signal. A23187 was used as a positive control. A23187 is a calcium ionophore that blocks encystation when incubated at 5 μM with trophozoites in encystation medium for 48 h, and this was evaluated by counting cysts after a 30-min incubation with 0.1% Sarkosyl (Fig. S1). We adapted this compound for use as a positive control in our encystation assay and saw significant reduction of encystation. Percent values were exported to GraphPad Prism software 8.0 for statistical analysis. The percentage of encystation was calculated as the mean ± standard error. Significance was calculated based on t test using percentage of encystation obtained with DMSO treatment as a reference for comparison.

Evaluating effect on mature cyst viability.

Compounds identified as encystation inhibitors were tested for the ability to reduce mature cyst viability, as previously described (23). E. invadens cells expressing luciferase (Ei-CK-luc) were induced to encyst by incubation in encystation medium (47% LG). After 72 h, cells were harvested, washed once in distilled water, resuspended in water, and incubated at 25°C for 5 h to lyse trophozoites. Purified cysts were pelleted, counted, resuspended in encystation medium, and distributed in 8-mL glass tubes to have 4 × 10⁵ cells per tube. Drug or DMSO was added, and tubes were incubated at 25°C for 72 h. On the day of the assay, cysts were pelleted, washed, and treated once more with distilled water (5 h) to lyse any trophozoites that had emerged during treatment. Purified cysts were then resuspended in 75 μL cell lysis buffer (Promega) and sonicated for 2 × 10 s to break the cyst wall. The luciferase assay was performed using the Promega luciferase assay kit according to the manufacturer's instructions, using 100 μL of luciferase substrate and 35 μL of cyst lysate, keeping equal volumes, and not normalizing by protein content. Obatoclax was used as a positive control (23), and 0.2% DMSO was used as a negative control. Each compound was tested in at least two biological replicates. The effect of the drug was calculated by comparison to the DMSO control, after subtraction of background signal. Percent values of replicates were exported to GraphPad Prism software 8.0 and calculated as the mean ± standard error. Significance was calculated based on t test using values obtained with DMSO treatment as a reference for comparison.