Structure-activity relationship of anticancer and antiplasmodial gold bis(dithiolene) complexes.

Monoanionic gold bis(dithiolene) complexes were recently shown to display activity against ovarian cancer cells, Gram-positive bacteria, Candida strains and the rodent malaria parasite, P. berghei. To date, only monoanionic gold(III) bis(dithiolene) complexes with a thiazoline backbone substituted with small alkyl chains have been evaluated for biomedical applications. We now analyzed the influence of the length and the hydrophobicity vs. hydrophilicity of these complexes' alkyl chain on their anticancer and antiplasmodial properties. Isomer analogues of these monoanionic gold(III) bis(dithiolene) complexes, this time with a thiazole backbone, were also investigated in order to assess the influence of the nature of the heterocyclic ligand on their overall chemical and biological properties. In this report we present the total synthesis of four novel monoanionic gold(III) bis(dithiolene) complexes with a long alkyl chain and a polyoxygenated (PEG) chain aiming to improve their solubility and biological properties. Our results showed that the complexes with a PEG chain showed promising anticancer and antiplasmodial activities beside improved solubility, a key parameter in drug discovery and development.

Synthesis, design, and optimization of a potent and selective series of pyridylpiperazines as promising antimalarial agents.

An optimization of the pyridylpiperazine series against Plasmodium falciparum has been performed, exploring a structure-activity relationship carried out on the toluyl fragment of hit 1, a compound with low micromolar activity against Plasmodium falciparum discovered by high-throughput screening. After confirming the crucial role played by this aryl fragment in the antiplasmodial activity, the replacement of the ortho-methyl substituent of 1 by halogenated ones led to an improvement for four analogs, either in terms of potency, expected pharmacokinetics profile, or both. Further introduction of endocyclic nitrogens in this fragment identified two more optimized compounds, 20 and 23, which are expected to be much more metabolically stable than 1. Additional assessment of the cytotoxicity, Ligand Lipophilic Efficiency, potency against the chloroquine-resistant Dd2 strain and in silico ADMET predictions revealed a satisfactory profile for most compounds, ultimately identifying the four optimized compounds 7, 9, 20 and 23 as promising compounds for further lead optimization of this series against Plasmodium falciparum.

Antimalarial activity of cecropin antimicrobial peptides derived from Anopheles mosquitoes.

The emergence of clinically drug-resistant malaria parasites requires the urgent development of new drugs. Mosquitoes are vectors of multiple pathogens and have developed resistance mechanisms against them, which often involve antimicrobial peptides (AMPs). An-cecB is an AMP of the malaria-transmitting mosquito genus Anopheles, and we herein report its antimalarial activity against Plasmodium falciparum 3D7, the artemisinin-resistant strain 803, and the chloroquine-resistant strain Dd2 in vitro. We also demonstrate its anti-parasite activity in vivo, using the rodent malaria parasite Plasmodium berghei (ANKA). We show that An-cecB displays potent antimalarial activity and that its mechanism of action may occur through direct killing of the parasite or through interaction with infected red blood cell membranes. Unfortunately, An-cecB was found to be cytotoxic to mammalian cells and had poor antimalarial activity in vivo. However, its truncated peptide An-cecB-1 retained most of its antimalarial activity and avoided its cytotoxicity in vitro. An-cecB-1 also showed better antimalarial activity in vivo. Mosquito-derived AMPs may provide new ideas for the development of antimalarial drugs against drug-resistant parasites, and An-cecB has potential use as a template for antimalarial peptides.

Characterization of antimalarial activity of artemisinin-based hybrid drugs.

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed, and their activity profile was characterized against drug-sensitive and drug-resistant Plasmodium falciparum parasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide, inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

Ganopyrone A, a highly rearranged lanostane triterpenoid with antimalarial activity from artificially cultivated fruiting bodies of Ganoderma colossus.

Three previously undescribed highly modified lanostane triterpenoids, ganopyrone A, ganocolossusin I, and ganodermalactone Y, were isolated from the artificially cultivated fruiting bodies of the basidiomycete Ganoderma colossus TBRC-BCC 17711. Ganopyrone A possesses an unprecedented polycyclic carbon skeleton with an α-pyrone ring and C-18/C-23 bond. It showed antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain) with an IC50 value of 7.8 µM (positive control: dihydroartemisinin, IC50 1.4 nM), while its cytotoxicity (Vero cells) was much weaker (IC50 103 µM).

Optimization of diastereomeric dihydropyridines as antimalarials.

The increase in research funding for the development of antimalarials since 2000 has led to a surge of new chemotypes with potent antimalarial activity. High-throughput screens have delivered several thousand new active compounds in several hundred series, including the 4,7-diphenyl-1,4,5,6,7,8-hexahydroquinolines, hereafter termed dihydropyridines (DHPs). We optimized the DHPs for antimalarial activity. Structure-activity relationship studies focusing on the 2-, 3-, 4-, 6-, and 7-positions of the DHP core led to the identification of compounds potent (EC50 < 10 nM) against all strains of P. falciparum tested, including the drug-resistant parasite strains K1, W2, and TM90-C2B. Evaluation of efficacy of several compounds in vivo identified two compounds that reduced parasitemia by >75 % in mice 6 days post-exposure following a single 50 mg/kg oral dose. Resistance acquisition experiments with a selected dihydropyridine led to the identification of a single mutation conveying resistance in the gene encoding for Plasmodium falciparum multi-drug resistance protein 1 (PfMDR1). The same dihydropyridine possessed transmission blocking activity. The DHPs have the potential for the development of novel antimalarial drug candidates.

Novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives and their antitrypanosomal activities against T.brucei.

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the <1 µM range. We have demonstrated that substituted 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidines present promising antitrypanosomal hit molecules with potential for further preclinical development.

Multifunctional Organometallic Compounds Active against Infective Trypanosomes: Ru(II) Ferrocenyl Derivatives with Two Different Bioactive Ligands.

Human African trypanosomiasis (HAT, sleeping sickness) and American trypanosomiasis (Chagas disease) are endemic zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Trypanosoma cruzi, respectively). Just a few old drugs are available for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. Only fexinidazole has been recently incorporated into the arsenal for the treatment of HAT. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these pathogens by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: pyridine-2-thiolato-1-oxide ligand (mpo) and polypyridyl ligands (NN). Three [Ru(mpo)(dppf)(NN)](PF6) compounds and their derivatives with chloride as a counterion were synthesized and fully characterized in solid state and solution. They showed in vitro activity on bloodstream T. brucei (EC50 = 31-160 nM) and on T. cruzi trypomastigotes (EC50 = 190-410 nM). Compounds showed the lowest EC50 values on T. brucei when compared to the whole set of metal-based compounds previously developed by us. In addition, several of the Ru compounds showed good selectivity toward the parasites, particularly against the highly proliferative bloodstream form of T. brucei. Interaction with DNA and generation of reactive oxygen species (ROS) were ruled out as potential targets and modes of action of the Ru compounds. Biochemical assays and in silico analysis led to the insight that they are able to inhibit the NADH-dependent fumarate reductase from T. cruzi. One representative hit induced a mild oxidation of low molecular weight thiols in T. brucei. The compounds were stable for at least 72 h in two different media and more lipophilic than both bioactive ligands, mpo and NN. An initial assessment of the therapeutic efficacy of one of the most potent and selective candidates, [Ru(mpo)(dppf)(bipy)]Cl, was performed using a murine infection model of acute African trypanosomiasis. This hit compound lacks acute toxicity when applied to animals in the dose/regimen described, but was unable to control parasite proliferation in vivo, probably because of its rapid clearance or low biodistribution in the extracellular fluids. Future studies should investigate the pharmacokinetics of this compound in vivo and involve further research to gain deeper insight into the mechanism of action of the compounds.

Exploring antimalarial potential: Conjugating organometallic moieties with organic fragments for enhanced efficacy.

In the search for novel ligands with efficacy against various diseases, particularly parasitic diseases, molecular hybridization of organometallic units into biologically active scaffolds has been hailed as an appealing strategy in medicinal chemistry. The conjugation to organometallic fragments can be achieved by an appropriate linker or by directly coordinating the existing drugs to a metal. The success of Ferroquine (FQ, SR97193), an effective chloroquine-ferrocene conjugate currently undergoing the patient-exploratory phase as a combination therapy with the novel triaminopyrimidine ZY-19489 for malaria, has sparked intense interest in organometallic compound drug discovery. We present the evolution of organometallic antimalarial agents over the last decade, focusing on the parent moiety's class and the type of organometallics involved. Four main organometallic antimalarial compounds have been chosen based on conjugated organic moieties: existing antimalarial drugs, other clinical drugs, hybrid drugs, and promising scaffolds of thiosemicarbazones, benzimidazoles, and chalcones, in particular. The presented insights contribute to the ongoing discourse on organometallic compound drug development for malaria diseases.

Stereochemical insights into enantioselective antiplasmodial lignanamides from the twigs and leaves of Solanum erianthum.

Stereochemical investigations on the twigs and leaves of Solanum erianthum afforded five pairs of lignanamide enantiomers and a previously undescribed phenolic amide (3). Particularly, two pairs of previously undescribed lignanamide racemates (1a/1b-2a/2b) represent the first case of natural products that feature an unreported 5/5-fused N/O-biheterocyclic core. Their structures, including the absolute configurations, were determined unambiguously by using spectroscopic analyses and electronic circular dichroism calculations. A speculative biogenetic pathway for 1-3 was proposed. Interestingly, these lignanamides exhibited enantioselective antiplasmodial activities against drug-sensitive Plasmodium falciparum 3D7 strain and chloroquine-resistant Plasmodium falciparum Dd2 strain, pointing out that chirality plays an important role in drug development.

Synthesis and biological evaluation of orally active anti-Trypanosoma agents.

In previous studies, we developed anti-trypanosome tubulin inhibitors with promising in vitro selectivity and activity against Human African Trypanosomiasis (HAT). However, for such agents, oral activity is crucial. This study focused on further optimizing these compounds to enhance their ligand efficiency, aiming to reduce bulkiness and hydrophobicity, which should improve solubility and, consequently, oral bioavailability. Using Trypanosoma brucei brucei cells as the parasite model and human normal kidney cells and mouse macrophage cells as the host model, we evaluated 30 new analogs synthesized through combinatorial chemistry. These analogs have fewer aromatic moieties and lower molecular weights than their predecessors. Several new analogs demonstrated IC50s in the low micromolar range, effectively inhibiting trypanosome cell growth without harming mammalian cells at the same concentration. We conducted a detailed structure-activity relationship (SAR) analysis and a docking study to assess the compounds' binding affinity to trypanosome tubulin homolog. The results revealed a correlation between binding energy and anti-Trypanosoma activity. Importantly, compound 7 displayed significant oral activity, effectively inhibiting trypanosome cell proliferation in mice.

Novel saturated 1,2,4-trioxanes and their antimalarial activity against multidrug resistant Plasmodium yoelii nigeriensis in Swiss mice model via oral route.

Novel saturated 6-(4'-aryloxy phenyl) vinyl 1,2,4-trioxanes 12a(1-3)-12d(1-3) and 13a(1-3)-13d(1-3) have been designed and synthesized, in one single step from diimide reduction of 11a(1-3)-11d(1-3). All the newly synthesized trioxanes were evaluated for their antimalarial activity against multi-drug resistant Plasmodium yoelii nigeriensis via oral route. Cyclopentane-based trioxanes 12b1, 12c1 and 12d1, provided 100 % protection to the infected mice at 24 mg/kg × 4 days. The most active compound of the series, trioxane 12b1, provided 100 % protection even at 12 mg/kg × 4 days and 60 % protection at 6 mg/kg × 4 days. The currently used drug, ß-arteether provides only 20 % protection at 24 mg/kg × 4 days.

In Vitro Evaluation of Anti-Parasitic Activities of Quinolone-Coumarin Hybrids Derived from Fluoroquinolones and Novobiocin Against Toxoplasma gondii.

PURPOSE: Toxoplasmosis is caused by the parasite Toxoplasma gondii (T. gondii). In immunocompetent individuals, the infection is often asymptomatic; however, in expectant mothers and those with immune system deficiencies, complications may arise. Consequently, there is a need for new drugs that cause minimal damage to host cells. The purpose of this study was to investigate the in vitro antiparasitic efficacy of quinolone-coumarin hybrids QC1-QC12, derived from quinolone antibacterials and novobiocin, against T. gondii. METHODS: The derivatives were compared with novobiocin and ciprofloxacin during testing, with pyrimethamine used as a positive control. We conducted the MTT assay to examine the anti-toxoplasmic effects of the test compounds and novobiocin. Evaluation included the infection and proliferation indices, as well as the size and number of plaques, based on the viability of both healthy and infected cells. RESULTS: The in vitro assays revealed that QC1, QC3, QC6, and novobiocin, with selectivity indices (SIs) of 7.27, 13.43, and 8.23, respectively, had the least toxic effect on healthy cells and the highest effect on infected cells compared to pyrimethamine (SI = 3.05). Compared to pyrimethamine, QC1, QC3, QC6, and novobiocin Without having a significant effect on cell viability, demonstrated a significant effect on reducing in both infection index and proliferation index, in addition to reducing the quantity and dimensions of plaques ( P < 0.05). CONCLUSION: Based on our results, QC1, QC3, QC6, and novobiocin due to their significant therapeutic effects could be considered as potential new leads in the development of novel anti-Toxoplasma agents.

Towards development of new antimalarial compounds through in silico and in vitro assays.

Malaria is one of most widespread infectious disease in world. The antimalarial therapy presents a series of limitations, such as toxicity and the emergence of resistance, which makes the search for new drugs urgent. Thus, it becomes necessary to explore essential and exclusive therapeutic targets of the parasite to achieve selective inhibition. Enoyl-ACP reductase is an enzyme of the type II fatty acid biosynthetic pathway and is responsible for the rate-limiting step in the fatty acid elongation cycle. In this work, we use hierarchical virtual screening and drug repositioning strategies to prioritize compounds for phenotypic assays and molecular dynamics studies. The molecules were tested against chloroquine-resistant W2 strain of Plasmodium falciparum (EC50 between 330.05 and 13.92 µM). Nitrofurantoin was the best antimalarial activity at low micromolar range (EC50 = 13.92 µM). However, a hit compound against malaria must have a biological activity value below 1 µM. A large number of molecules present problems with permeability in biological membranes and reaching an effective concentration in their target's microenvironment. Nitrofurantoin derivatives with inclusions of groups which confer increased lipid solubility (methyl groups, halogens and substituted and unsubstituted aromatic rings) have been proposed. These derivatives were pulled through the lipid bilayer in molecular dynamics simulations. Molecules 14, 18 and 21 presented lower free energy values than nitrofurantoin when crossing the lipid bilayer.

Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate.

In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.

Antimony susceptible Leishmania donovani: evidence from in vitro drug susceptibility of parasites isolated from patients of post-kala-azar dermal leishmaniasis in pre- and post-miltefosine era.

Post-kala-azar dermal leishmaniasis (PKDL) patients are a key source of Leishmania donovani parasites, hindering the goal of eliminating visceral leishmaniasis (VL). Monitoring treatment response and parasite susceptibility is essential due to increasing drug resistance. We assessed the drug susceptibility of PKDL isolates (n = 18) from pre-miltefosine (MIL) era (1997-2004) with isolates (n = 16) from the post-miltefosine era (2010-2019) and post-miltefosine treatment relapse isolates (n = 5) towards miltefosine and amphotericin B (AmB) at promastigote stage and towards sodium antimony gluconate (SAG) at amastigote stage. PKDL isolates were examined for mutation in gene-encoding AQP1 transporter, C26882T mutation on chromosome 24, and miltefosine-transporter (MT). PKDL isolates from the post-miltefosine era were significantly more susceptible to SAG than SAG-resistant isolates from the pre-miltefosine era (P = 0.0002). There was no significant difference in the susceptibility of parasites to miltefosine between pre- and post-miltefosine era isolates. The susceptibility of PKDL isolates towards AmB remained unchanged between the pre- and post-miltefosine era. However, the post-miltefosine era isolates had a higher IC50 value towards AmB compared with PKDL relapse isolates. We did not find any association between AQP1 gene sequence variation and susceptibility to SAG, or between miltefosine susceptibility and single nucleotide polymorphisms (SNPs in the MT gene. This study demonstrates that recent isolates of Leishmania have resumed susceptibility to antimonials in vitro. The study also offers significant insights into the intrinsic drug susceptibility of Leishmania parasites over the past two decades, covering the period before the introduction of miltefosine and after its extensive use. IMPORTANCE: Post-kala-azar dermal leishmaniasis (PKDL) patients, a key source of Leishmania donovani parasites, hinder eliminating visceral-leishmaniasis. Assessment of the susceptibility of PKDL isolates to antimony, miltefosine (MIL), and amphotericin-B indicated that recent isolates remain susceptible to antimony, enabling its use with other drugs for treating PKDL.

Incomplete Plasmodium falciparum growth inhibition following piperaquine treatment translates into increased parasite viability in the in vitro parasite reduction ratio assay.

Antimalarial resistance to the first-line partner drug piperaquine (PPQ) threatens the effectiveness of artemisinin-based combination therapy. In vitro piperaquine resistance is characterized by incomplete growth inhibition, i.e. increased parasite growth at higher drug concentrations. However, the 50% inhibitory concentrations (IC50) remain relatively stable across parasite lines. Measuring parasite viability of a drug-resistant Cambodian Plasmodium falciparum isolate in a parasite reduction ratio (PRR) assay helped to better understand the resistance phenotype towards PPQ. In this parasite isolate, incomplete growth inhibition translated to only a 2.5-fold increase in IC50 but a dramatic decrease of parasite killing in the PRR assay. Hence, this pilot study reveals the potential of in vitro parasite viability assays as an important, additional tool when it comes to guiding decision-making in preclinical drug development and post approval. To the best of our knowledge, this is the first time that a compound was tested against a drug-resistant parasite in the in vitro PRR assay.

In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

Design, synthesis and identification of novel molecular hybrids based on naphthoquinone aromatic hydrazides as potential trypanocide and leishmanicidal agents.

In pursuit of potential agents to treat Chagas disease and leishmaniasis, we report the design, synthesis, and identification novel naphthoquinone hydrazide-based molecular hybrids. The compounds were subjected to in vitro trypanocide and leishmanicidal activities. N'-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)-3,5-dimethoxybenzohydrazide (13) showed the best performance against Trypanosoma cruzi (IC50 1.83 µM) and Leishmania amazonensis (IC50 9.65 µM). 4-Bromo-N'-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzohydrazide (16) exhibited leishmanicidal activity (IC50 12.16 µM). Regarding trypanocide activity, compound 13 was low cytotoxic to LLC-MK2 cells (SI = 95.28). Furthermore, through molecular modeling studies, the cysteine proteases cruzain, rhodesain and CPB2.8 were identified as the potential biological targets.

Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites.

BACKGROUND: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.