Fig 1.
EC50 determination of antineoplastic kinase inhibitors against E. histolytica trophozoites.
Dose response curve plotting percentage inhibition of E. histolytica trophozoites compared to drug concentrations of antineoplastic kinase inhibitors. Trophozoites were assayed for cell viability following treatment with each drug for a period of 48 hours. The three drugs with the lowest EC50 values (ibrutinib, dasatinib, and bosutinib) are plotted in red, purple, and blue. All drugs with EC50 values > 2 μM are plotted in gray. Each data point represents mean values of percentage inhibition. Error bars represent standard deviation. Complete list of drugs can be found in Table 1.
Table 1.
Results of primary screen of AKI drugs against E. histolytica trophozoites.
Color scale indicates drug potency: darker blue = more potent, lighter blue = less potent. Anti-amoebic activity classified based on EC50 value as: Very high (0.001–0.999 μM), High (1.000–4.999 μM), Moderate (5.000–9.999 μM), Low (10.000–19.999 μM), Very low (20.000–99.999 μM), or None (EC50 > 100.000 μM).
Fig 2.
Generation of human target profile for inhibitors of E. histolytica.
Human kinase proteins scored and ranked based on targeting activity data for active drugs versus inactive drugs from the screen. Heatmap represents the calculated activity values (pAct, see Materials and Methods) of individual drugs against individual human protein targets. Darker colors indicate stronger drug activity against the protein. Dashed line represents the cutoff pAct value for proteins to be included in the target profile for the purpose of identifying additional E. histolytica drug candidates.
Fig 3.
Graphical screening workflow of antineoplastic kinase inhibitors against E. histolytica.
Chemical structures represent the three drugs found to possess the lowest EC50 values in each screen (ibrutinib, dasatinib, bosutinib, and ponatinib, neratinib, olmutinib respectively).
Fig 4.
In silico screen based on human target profile to determine new potent amoebicidal drug candidates.
Antineoplastic kinase inhibitor drugs scored and ranked based on activity data regarding all 15 proteins in the amoebicidal drug target profile shown in Fig 3. Score shown in the second column is calculated from the weighted sum of pAct values (see Materials and Methods.) Shown are all drugs meeting the cutoff score of 15 for further screening. Heatmap displays the calculated pAct of individual drugs against individual protein targets. Darker colors indicate stronger drug activity against the protein. Purple highlight indicates drugs included in the initial in vitro screen. Green highlight indicates new candidate drugs.
Table 2.
Analysis of drugs based on activity towards individual proteins in the active drug target profile.
Drugs are ranked based on the number of proteins from the active drug target profile towards which they possess an activity score greater than a threshold value. Darker color indicates a greater number of proteins. Drugs possessing the desired level of activity towards more than two target profile proteins were considered for further screening, shown above red line.
Fig 5.
Network map of active drug profile proteins with orthologous E. histolytica ORFs.
Orange ovals represent human sequences. Blue ovals represent E. histolytica sequences. Lines represent alignment relationships possessing a calculated pP greater than the cutoff value of 10. Line color represents pP value, with darker lines denoting higher pP. Blue ovals shown in the center with the highest number of connecting lines represent the most likely E. histolytica orthologs of the human target proteins.
Fig 6.
Extended screen of antineoplastic kinase inhibitors against E. histolytica trophozoites.
Dose response curves plotting percentage inhibition of E. histolytica trophozoites at different drug concentrations. Trophozoites were assayed for cell viability following treatment with each drug for a period of 48 hours. The three drugs with the lowest EC50 values (ponatinib, neratinib, and olmutinib) are plotted in red, purple, and blue. All drugs with EC50 values > 2 μM are plotted in gray. Each data point represents mean values. Error bars represent standard deviation. Complete list of drugs tested found in Table 3.
Table 3.
Results of extended screen of AKI drugs against E. histolytica trophozoites.
Color scale indicates drug potency: darker blue = more potent, lighter blue = less potent. Anti amoebic activity classified based on EC50 value as: Very high (0.001–0.999 μM), High (1.000–4.999 μM), Moderate (5.000–9.999 μM), Low (10.000–19.999 μM), Very low (20.000–99.999 μM), or None (EC50 > 100.000 μM).
Fig 7.
Amoebicidal effects of antineoplastic kinase inhibitor drugs.
Live cell number calculated in comparison to aliquots of known amounts of cells. All drugs tested at 10μM, vehicle = 0.5% DMSO. Dotted line represents the 5000 cells originally seeded into all wells for each treatment group. Error bars represent standard deviation.
Fig 8.
Timing of drug action against E. histolytica trophozoites.
(A—D) Dose-response curves measured at 12, 24, 36, and 48hr for ponatinib, neratinib, olmutinib, and metronidazole. (E) Plot of EC50 values calculated from the data shown in (A-D) graphed over time. (F) Plot of E. histolytica trophozoite inhibition by AKI drugs after varying exposure times. Trophozoites were treated with ponatinib, neratinib, olmutinib, or metronidazole at 5μM for 2, 6, 12, 24, 36, or 48hr, followed by drug washout and continued incubation until 48hr. Percent inhibition was then determined. Points represent % inhibition for each drug and exposure time scaled to the 48hr % inhibition value for the same drug. Statistical difference between groups was determined by 1-way ANOVA for each exposure time. (** = p < 0.01) (* = p < 0.05).
Fig 9.
Activity of antineoplastic kinase inhibitors against Entamoeba cysts.
Cyst survival measured using luminescence values of luciferase-expressing E. invadens cysts after drug treatments, compared with DMSO-treated controls. Cysticidal effect corresponds to cyst survival values below 100%. Metronidazole tested at 20μM. All other drugs tested at 10μM. Data points represent biological replicates. Asterisk indicates (p < 0.05).