Identification of potent inhibitors of Leishmania donovani and Leishmania infantum chagasi, the causative agents of Old and New World visceral leishmaniasis

Abstract

The drug discovery pipeline for neglected kinetoplastid diseases remains sparse. In particular, the field of leishmaniasis drug discovery has had limited success in translating potential drug candidates into viable therapies. Here, we describe the development of two lead compounds, BZ-1 and BZ1-I, which have potent in vitro anti-leishmanial activity against Leishmania donovani DD8 intracellular amastigotes (0.59 ± 0.13 µM and 0.40 ± 0.38 µM) with corresponding selectivity ( > 33.89 and > 49.12) for differentiated THP-1 cells (Human monocytic cell line), respectively. Further characterization and biological profiling identified that in addition to the activity against L. donovani DD8 (Old World - Indian strain), compounds were active against intracellular parasites from other species and strains of the Old and New World, namely L. donovani (Old World - Sudanese strain) and L. infantum chagasi (New World-South American strain). In vivo evaluation using the hamster model illustrated that the activity observed in vitro was translated in vivo, with outstanding results. Our data suggests that these compounds represent a promising starting point for developing a novel lead series for future anti-leishmanial therapeutics.

Introduction

Kinetoplastid parasites of the genus Leishmania are the causative agent of the infectious disease, leishmaniasis¹, endemic in 98 countries, with more than one billion people at risk of acquiring the disease^2,3. The mode of transmission is via the bite of a sand fly, genus Phlebotomus (Old World) and Lutzomyia (New World)⁴. The life cycle of the Leishmania parasite exists between the sand fly (promastigote form) and the mammalian host (amastigote form)⁵. Current treatment regimens often have poor toxicity profiles and resistance has begun to emerge against the standard of care therapies^6,7. There also exists a significant imbalance in the drug discovery pipeline for leishmaniasis^8,9, with ongoing challenges including insufficient understanding of the pathogen biology, plus a lack of cellular and pathophysiological animal models that mimic the disease in humans^10,11,12. An additional hurdle for new chemical entities is the variation in activity often observed against different species, contributing to different clinical outcomes based on geographical location of the parasite^13,14,15,16. Many compounds in the past have exhibited efficacy against Leishmania species, including antibiotics, organo-metallics, nucleosides and sterol biosynthesis inhibitors^17,18,19,20. Most of these were not progressed due to host cell cytotoxicity, plus lack of efficacy and safety in in vivo models. Even the currently available treatments face similar issues. Pentavalent antimonials, the treatment of choice in South America, Africa, Nepal, Bangladesh, and India (except North Bihar)²¹, have demonstrated differences in in vitro sensitivity between the different species in the amastigote macrophage models. L. brasiliensis and L. donovani were shown to be three to five fold more sensitive to antimonials compared to L. tropica, L. major and L. mexicana^22,23. Amphotericin B deoxycholate is considered the second-line therapy in cases that do not respond to antimonials²⁴ as it was very effective against visceral leishmaniasis (>90% cure rate)²⁵. A study conducted by Escobar et al. compared the sensitivity of different Leishmania species to amphotericin B, observing that the rank order of species sensitivities measured in Median effective dose (ED₅₀), was L. mexicana > L. aethiopica > L. tropica > L. major > L. panamensis > L. donovani for the promastigote assay and L. mexicana > L. panamensis > L. aethiopica > L. tropica > L. major > L. donovani for the amastigote assay”²⁶. Variations in drug sensitivities of different species have also been demonstrated in vitro for miltefosine with L. tropica, L. aethiopica, L. mexicana, L. panamensis and L. donovani²⁷. The Half maximal effective concentration (EC₅₀) values obtained for miltefosine varied from 2.63 to 10.63 µM between these species, whereas the EC₅₀ for L. major was found to be significantly lower at 37.17 µM²⁶. Paromomycin has been approved by World Health Organization (WHO) for the treatment of cutaneous leishmaniasis in its topical form²⁸. In vitro testing suggests that sensitivity to paromomycin in Leishmania amastigotes in murine macrophages varies between species. This drug tends to be more effective against L. tropica and L. major compared to L. mexicana and L. braziliensis when compared in the same assay models¹⁹; and is moderately active against all strains of L. donovani (mean IC₅₀ in-between 6.1 and 43.8 µM), except L. donovani DD8 (mean IC₅₀ 158.1–165.7 µM)¹⁹. The basis for this difference has not yet been elucidated.

A target candidate profile (TCP) and a target product profile (TPP) were developed by Drugs for Neglected Diseases Initiative (DNDi) for visceral leishmaniasis to guide the progression of compounds in the drug discovery pipeline²⁹. These guidelines define the minimum criteria for developing a safe, effective, and orally administered anti-leishmanial drug that can be used in a short treatment regimen, aiming to replace current therapies²⁹.

As per the DNDi TCP, for a compound to be considered an active hit against visceral leishmaniasis, the molecule should exhibit activity against the intracellular amastigote stage of L. donovani or L. infantum with an IC₅₀ value below 10 µM. The compound must show at least a 10-fold selectivity over mammalian cell lines. Additionally, it should not possess any structural alerts that could negatively affect its metabolism, stability, or reactivity and demonstrate no toxicity in both in silico and in vivo models²⁹.

Historically, drug discovery for leishmaniasis has been slow and inefficient, primarily due to the limited commitment of the pharmaceutical industry for neglected tropical diseases (NTDs). Collectively, the lack of financial incentive, as well as the complexity of Leishmania biology, has resulted in few novel drugs entering the pipeline in recent decades. Nevertheless, recent advancements in drug discovery and greater engagement from pharmaceutical and philanthropic partners have begun to reshape this landscape, offering hope for more effective treatments^16,30

This study involved screening a structurally diverse synthetic library in an effort to discover new compounds active against Leishmania donovani (MHOM/IN/80/DD8). Hit molecules were defined as active against both promastigote and intracellular amastigote stages, with minimal cytotoxicity not only against the host cell THP-1 (human monocytic cell), but also a panel of mammalian cells comprised of various cell lines from human and mice representing different organ systems including HepG2 (human liver cancer cell), HEK-293 (human embryonic kidney cell), RAW 264.7 and J774.1 (both murine macrophages) cell lines. This panel served as a valuable tool to address compound cytotoxicity early in development. Two 1H-cyclopenta[b]quinolin-9-amine compounds (BZ1 and BZ1-I) that fulfilled the hit criteria with respect to both activity and selectivity were subjected to further in vitro and in vivo profiling.

To compare the difference of activity between the Old world and New world species, two species of Leishmania were selected, namely L. donovani causing visceral leishmaniasis in the Indian subcontinent WHO reference strain MHOM/IN/80/DD8 (Old world)³¹ and L. infantum chagasi (New world) which causes visceral leishmaniasis in Brazil, MHOM/BR/1972/BH46³². In addition, to compare the strain specific differences, L. donovani MHOM/SD/62/1S-CL2D, LdBOB-eGFP (Old world)³³, a genetically modified strain of a wild type isolated from the Sudanese population was also utilized. Histopathological and cytokine data were acquired to determine whether the compounds elicited an immunological response against the Leishmania infection.

Results

Discovery of compound BZ1

Compound N-(4-Ethoxyphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine, referred to herein as BZ1 (Fig. 1A) was discovered via a screening campaign. From 5560 compounds tested in the promastigote assay, 29 compounds fulfilled our criteria having >60% activity at 10 µM (hit rate: 0.52%). Of these, 22 compounds returned activity at >60% activity against the promastigotes when retested at 10 µM. Testing against the intracellular amastigote identified 40 compounds with >50% activity at 10 µM, which was the criteria defined for a hit molecule, and representing a hit rate of 0.71%. Following retest, 26 of these compounds reconfirmed activity at 10 µM.

Fig. 1: Structures and in vitro activities of compound BZ1 and BZ1-I.

A Structure of compound “N-(4-Ethoxyphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine” referred as BZ1. B In vitro activity of the compound BZ1 against L. donovani DD8 intracellular amastigote promastigotes. C Structure of compound “N-(4-phenoxyphenyl)-1H,2H,3H-cyclopenta[b]quinolin-9-amine” referred to as BZ1-I. D In vitro activity of the compound BZ1-I against L. donovani DD8 intracellular amastigotes and promastigotes. The data points are means and error bars represent standard deviations of three replicates of two independent experiments. Individual data points are shown as lighter-shaded markers in the corresponding colour of each group.

Of the compounds which confirmed upon retest, four demonstrated >50% activity against both the promastigote and the amastigote forms at 10 µM. The most promising hit molecule BZ1 had an IC₅₀ value of 0.59 ± 0.13 µM against the intracellular amastigotes and an IC₅₀ value of 2.37 ± 0.85 µM against the extracellular promastigotes (Fig. 1B). The selectivity index of BZ1 was estimated to be >33 for both HEK-293 and THP-1 cells.

A series of substructure searches, performed in Scifinder^® (CAS, American Chemical Society), ChemSpider® (Royal Society of Chemistry) and ChEMBL^® (European Bioinformatics Institute), in addition to Compounds Australia^® structural portal (CASPeR) were performed to retrieve analogues most relevant to structure–activity relationship (SAR) interpretation. Analogues with no structural alerts (metabolism/stability/reactivity) were chosen based on the following criteria: no more than 5 H-bond donors (total number of N–H and O–H bonds), no more than 10 H-bond acceptors (all N or O atoms), and a molecular mass < 500 g/mol and octanol–water partition coefficient (log P) < 5. Thirty compounds were selected (referred to as BZ1-A–BZ1-d), the structures of which are shown in Supplementary Fig. 1.

Of the 30 analogues tested against L. donovani DD8 promastigotes and amastigotes, compound BZ1-I (Fig. 1C), showed improved activity (IC₅₀: 0.60 ± 0.04 µM) against the promastigotes compared to the original hit compound, BZ1 (IC₅₀: 2.15 ± 0.14 µM). BZ1-I also demonstrated comparable activity (IC₅₀: 0.40 ± 0.38 µM) to BZ1 against the intracellular form of the parasite (amastigotes)(IC₅₀: 0.59 ± 0.13 µM) (Fig. 1D).

The activity exhibited by these analogues against both the Leishmania promastigotes and intracellular amastigotes has been presented in the form of a spider plot (Fig. 2A). Selectivity of these compounds for the Leishmania intracellular amastigotes, with respect to HEK-293 and THP-1 host cells, is shown in Fig. 2B. According to the target candidate profile (TCP) established by DNDi, we aimed to identify compounds with an IC₅₀ value < 10 µM against L. donovani intracellular amastigotes and 10-fold more selective for the parasites than mammalian cells. The DNDi guidelines provide the minimal requirements for the development of a safe, oral, effective anti-leishmanial drug, enabling short course treatment schedule with a bid to replace the existing current treatments available.

Fig. 2: Anti-parasitic activity exhibited by analogues against both Leishmania promastigotes and intracellular amastigotes, with associated selectivity for HEK-293 and THP-1 host cells.

A Spider plot of the IC₅₀ values of compounds for L. donovani DD8 promastigotes (blue) and L. donovani DD8 intracellular amastigotes (orange). B Selectivity of compounds active against L. donovani intracellular amastigotes in relation to HEK-293 (blue) and THP-1 cells (green).

The IC₅₀ values of the reference drugs, amphotericin B, miltefosine and the preclinical candidate, VL-2098, were determined for the intracellular amastigote assay; amphotericin B 0.08 ± 0.01 µM (Fig. 3A), miltefosine 1.97 ± 0.25 µM (Fig. 3B) and VL-2098 0.70 ± 0.13 µM (Fig. 3C). The negative control, 0.4% DMSO, was used to define 100% viability of the amastigotes in the host cells. Amphotericin B was used at 1 µM as the positive control, to define 0% viability of amastigotes in the host cells and no associated host cell cytotoxicity.

Fig. 3: In vitro activity of the reference drugs against L. donovani DD8 intracellular amastigotes.

A Amphotericin B, B miltefosine and C VL-2098. The percentage inhibition was determined from quadruplicate assay data points after normalization of the data with 1 µM amphotericin B (100% death, final assay) as the positive control and 0.4% DMSO (0% inhibition) as the negative control. Left-Y-axis: percentage inhibition of L. donovani DD8 (blue dots/triangles and curves); Right-Y-axis: percentage inhibition of THP-1 cells (red dots/triangles and curves); X-axis: Log of micromolar compounds concentration. Data points are means and error bars represent the standard deviations of two independent experiments (dots for experiment 1 and triangles for experiment 2). Individual data points are represented as lighter-shaded markers in the corresponding colour of each group.

Activity on different strains and species of Leishmania in the Old and New World

The intracellular amastigote activities of BZ1 and BZ1-I were compared between the different strains and species of Leishmania representative of Old and New World leishmaniasis (Table 1). It was observed that our novel compounds, BZ1 and BZ1-I, demonstrated sub-micromolar activity against L. donovani WHO reference strain MHOM/IN/80/DD8 which results in visceral leishmaniasis in the Indian subcontinent³⁴, with IC₅₀ values of 0.59 ± 0.13 and 0.40 ± 0.38 µM, respectively, in the intracellular amastigote assay (Fig. 4A, B). In contrast, the IC₅₀ values of both BZ1 and BZ1-I were found to be 4.40 ± 0.12 and 4.26 ± 0.24 µM against L. donovani MHOM/SD/62/1S-CL2D, LdBOB-eGFP intracellular amastigotes, a genetically modified strain, the wild type of which was isolated from the Sudanese population. L. donovani MHOM/SD/62/1S-CL2D, LdBOB-eGFP was chosen as it had previously been used for high-content screening³⁵. Relative to the variation in activity against the parasite strains, the selectivity for THP-1 cells shifted from >25.64 to >11.76 for BZ1 and from >44.44 to >11.49 for BZ1-1, respectively (Table 1—72 h incubation data). Amphotericin B was used as a reference compound exhibiting IC₅₀ values of 0.20 ± 0.02 µM for L. donovani MHOM/IN/80/DD8. The reported IC₅₀ values for miltefosine were 2.54 ± 0.57 µM for L. donovani MHOM/IN/80/DD8.

Fig. 4: In vitro activity and selectivity of the BZ1 and BZ1-I compounds against different species and strains causing VL.

A Concentration response curve (CRC) of the in vitro activity of the compound BZ1 generated against L. donovani DD8 and L. donovani LdBOB intracellular amastigotes for 72 h incubation time. B Concentration response curve of the In vitro activity of the compound BZ1-I generated against L. donovani DD8 and L. donovani LdBOB intracellular amastigotes. For A and B, Black line represents L. donovani DD8 and Red for L. donovani LdBOB. C In vitro cytotoxicity represented as CRC for compound BZ1 against a panel of mammalian cell lines for 72 h incubation time. D In vitro cytotoxicity represented as CRC of the compound BZ1-1 against a panel of mammalian cell lines for 72 h incubation time. For both figure C and D the green line represents HepG2 cells, red line represents Raw 264.1, blue line represents J774.1 cells, burgundy line represents HEK-293 cells and orange line represents THP-1 cells. For the activity against the L. donovani LdBOB (A, B), the data represents the mean and standard devitaion of three independent replicates (each replicate run as a single technical replicate). For C, D the data represents the mean and standard deviations of three replicates of two independent experiment (N = 2). Individual data points are indicated as lighter-shaded markers in the corresponding colour of each group.

Table 1 Tabulated form of in vitro activity of the BZ1 and BZ1-I against different Old and New World strains and species of Leishmania intracellular amastigotes with different incubation times

In the cytotoxicity assay, a stable plateau of activity was not observed for either BZ1 or BZ1-I at 80 µM (final assay concentration) which was the highest concentration tested due to compound solubility issues in 100% DMSO, hence accurate IC₅₀ values could not be determined (Fig. 4C, D). Arbitrary IC₅₀ values were therefore calculated for compounds against HepG2, Raw264.7, J774.1, HEK-293 and THP-1 cells (Supplementary Table 1).

The promastigote activities of BZ1 and BZ1-I were compared between the different strains and species of Leishmania representative of Old and New World leishmaniasis (Table 2).

Table 2 Tabulated form of in vitro activity of the BZ1 and BZ1-I against different Old and New World strains and species of Leishmania promastigotes

Determining cidal action of compounds (Promastigote assay)

Compound activity can either be cidal (direct killing) or static in nature, thus has the ability to hinder parasite division, resulting in a delayed response. The time and concentration dependent susceptibility of the BZ compounds was evaluated in comparison to known reference compounds/drugs. Measurement of the number of promastigotes following exposure to the compounds at various concentrations for 24, 48 and 72 h are shown in Fig. 5. To determine cidal activity, physical deformation and loss of motility allowed accurate assessment of live/dead parasite. No viable promastigotes were observed for amphotericin B, miltefosine, BZ1 and BZ1-I at 0.33, 6.66, 6.66, and 1.66 µM respectively after 24 h incubation (Fig. 5A), indicating that at these concentrations, the mode of action of these compounds was direct killing.

Fig. 5: Cidal/static mode of action and impact of time on efficacy of compounds BZ1 and BZ1-I.

A Cidal activity of amphotericin B, miltefosine, BZ1 and BZ1-I based on MIC at various time intervals 24, 48 and 72 h. Different concentrations are colour-coded ranging 66.66–0.03 µM as described in the figure legend. Each evaluation was performed in triplicate, for N = 2. B Concentration response curve for BZ1 against L. donovani DD8 promastigote following exposure from 24 to 96 h to ascertain speed of action. C Concentration response curve for BZ1-Iagainst L. donovani DD8 promastigote following exposure from 24 to 96 h to ascertain speed of action. D Concentration response curve for BZ1 against L. donovani DD8 intracellular amastigotes following exposure from 24 to 96 h to ascertain speed of action. E Concentration response curve for BZ1-I against L. donovani DD8 intracellular amastigotes following exposure from 24 to 96 h to ascertain speed of action. B–E Green represents 24 h, red 48 h, blue 72 h and brown 96 h incubation times. Each evaluation was performed in triplicate, for N = 2. Individual data points are indicated as lighter-shaded markers in the corresponding colour of each group.

Time to kill assay (promastigotes and intracellular amastigote assay)

Ascertaining the “time to kill” can serve as a determinant of anti-leishmanial compound activity relative to the time needed to kill the parasite, ideally as fast as possible. To determine the speed with, which the compounds exerted their effects, the IC₅₀ values of BZ1 and BZ1-I were determined for both promastigotes and amastigotes. BZ1 activity against promastigotes showed a plateau of activity from 24 h (4.31 ± 0.22 µM) onwards (considered as 2 concentrations or more at >90%), suggesting that BZ1 was able to achieve 100% inhibition of the parasite number reduction after 24 h (Fig. 5B). After 48 h the IC₅₀ value of BZ1 was consistent (2.37 ± 0.74 µM) remaining in the same range for 96 h. In contrast, whilst BZ1-I also demonstrated 100% kill at 24 h (3.08 ± 1.11 µM) there was a significant shift in IC₅₀ value at 48 h (0.69 ± 0.35 µM) (Fig. 5C) which then stabilized. This suggested BZ1-I was a faster acting compound than BZ1 against the promastigotes at 48 h time point. For the intracellular amastigote assay BZ1 did not elicit a stable plateau of activity (100% inhibition) until after 48 h incubation time (Fig. 5D). BZ1-I demonstrated activity within 24 h (4.84 ± 0.83 µM) however with significantly reduced efficacy than observed for the longer incubation periods (0.40 ± 0.38 µM) (Fig. 5E). Stable consistent activity was observed after 48 h. This delay in BZ1 and BZ1-I complete activity, or suboptimal effects, might be due to permeability issues, as the compounds must cross multiple barriers to reach the amastigotes which reside within the parasitophorous vacuoles. Alternatively, this may also reflect the nature of the mode of action of this compound, highlighting an accumulative effect following increased incubation times. Overall, this would warrant future investigations as we believe this is an interesting finding.

Generating resistant cell lines

Resistance was generated for both BZ1 and BZ1-I. In parallel, resistance was also generated for the reference drugs amphotericin B and miltefosine to enable assessment of BZ1 and BZ1-I against amphotericin B and miltefosine resistant parasites, thus informing whether the compounds acted via a similar mechanism of action to these drugs. After a duration of 428 days, clones of resistant strains were generated for amphotericin B (Fig. 6A), miltefosine (Fig. 6B) and BZ1 (Fig. 6C) that could withstand concentrations of these drugs/compounds of 420 nM, 40 and 11 µM, respectively. Resistant clones were also generated for BZ1-I taking 210 days to reach a concentration of 5.5 µM, (Fig. 6D). Continued exposure to increase resistance to BZ1-I is ongoing. When both compounds were compared, resistance generated with BZ1-I occurred more rapidly than BZ1, suggesting a different mechanism or uptake.

Fig. 6: Generation of resistance, confirmation and stability of resistance after freeze–thaw cycle in L. donovani DD8 promastigotes for both compounds BZ1 and BZ1-I.

A Generating resistance against amphotericin B. B Generating resistance against miltefosine. C Generating resistance against BZ1. D Generating resistance against BZ1-I, line 1 (blue), line 2 (red) and line 3 (green) are representative of 3 different clones. E Resistance confirmation against amphotericin B after freeze–thaw cycle in promastigote and intracellular amastigote assays. F Resistance confirmation against miltefosine after freeze–thaw cycle in promastigote and intracellular amastigote assays. G Resistance confirmation against BZ1after freeze–thaw cycle in promastigote and intracellular amastigote assays. H Resistance confirmation against BZ1-Iafter freeze–thaw cycle in promastigote and intracellular amastigote assays. I Resistance stability against amphotericin B after freeze–thaw cycle in promastigote and intracellular amastigote assays. J Resistance stability against miltefosine after freeze–thaw cycle in promastigote and intracellular amastigote assays. K Resistance stability against BZ1 after freeze–thaw cycle in promastigote and intracellular amastigote assays. L Resistance stability against BZ1-I after freeze–thaw cycle in promastigote and intracellular amastigote assays. Green and red represent promastigote assay N1 and N2, respectively. Blue and brown represent intracellular amastigote assay N1 and N2, respectively. Each evaluation was performed as duplicates for N = 2. Individual data points are represented as lighter-shaded markers in the corresponding colour of each group.

Resistance in these clonal populations for amphotericin B (Fig. 6E), miltefosine (Fig. 6F), BZ1 (Fig. 6G) and BZ1-I (Fig. 6H) were confirmed using both promastigote and intracellular amastigote assays. The results obtained co-align with previously reported data for amphotericin B and miltefosine, where a 14-fold increase in drug resistance was obtained for miltefosine³⁶ and a 20-fold resistance generated for amphotericin B³⁷.

Studies were also conducted to determine the stability of resistance after removing drug pressure for 10 passages for amphotericin B (Fig. 6I), miltefosine (Fig. 6J), BZ1 (Fig. 6K) and BZ1-I (Fig. 6L). Compounds BZ1 and BZ1-I resulted in IC₅₀ values of 5.35 ± 0.18, 4.94 ± 0.69, 14.52 ± 0.55, and 8.93 ± 0.67 µM for the intracellular amastigote and promastigote assays, respectively. The IC₅₀ values obtained were compared with a control non-resistant culture of the same passage number. Our results indicated that resistance was stable for all reference and test compounds after removal of drug pressure.

Following the confirmation of resistance generation and stability, the activity of BZ1 and BZ1-I was evaluated on L. donovani DD8 strains resistant to amphotericin B and miltefosine, using the promastigote and intracellular amastigote assays, respectively. The findings reveal no significant difference in the activity of BZ1 and BZ1-I against the resistant strains compared to the wild-type L. donovani DD8 strain (Fig. 7). These results suggest that both compounds exert their antiparasitic effects through a mechanism distinct from that of amphotericin B and miltefosine.

Fig. 7: In vitro activity of compounds BZ1 and BZ1-I on the amphotericin B and miltefosine resistant parasites utilizing the promastigote and the intracellular amastigote assays.

A In vitro activity of compounds BZ1 on amphotericin B resistant parasites. B In vitro activity of compounds BZ1-I on amphotericin B resistant parasites. C In vitro activity of compounds BZ1 on miltefosine resistant parasites. D In vitro activity of compounds BZ1-Ion miltefosine-resistant parasites. Each evaluation was performed as duplicates for N = 2. Individual data points are shown as lighter-shaded markers in the corresponding colour of each group.

Drug metabolism and pharmacokinetics studies

Effective anti-leishmanial drugs must exhibit favorable properties to ensure bioavailability, target specificity, and minimal off-target effects. Both compounds were analyzed for parameters critical to oral drug absorption and pharmacokinetic performance. The summary and interpretation of key metrics has been outlined in Tables 3 and 4.

Table 3 Physiochemical properties of compounds BZ1 and BZ1-I

Table 4 The pharmacokinetic properties of the compounds BZ1 and BZ1-I, based on human microsome studies, revealing their metabolic stability and clearance potential

In vivo efficacy

To fully appreciate the potential of BZ1 and BZ1-I for visceral leishmaniasis, the compounds were assessed in a chronic hamster model allowing parasite load evaluation in two target organs (liver and spleen), as for the human disease. The in vivo efficacy of BZ1 and BZ1-I against L. infantum chagasi (MHOM/BR/1972/BH46), was assessed in the L. infantum chagasi chronic phase hamster model (Fig. 8A). Post 60 days infection the animals were administered with 0.5% of carboxymethyl cellulose (vehicle) suspension orally (n = 5 animals), (untreated vehicle only group, or UTG); 50 mg/kg/day of GLU (Meglumine Antimoniate), by intraperitoneal injection (GLU group; n = 5); 10 mg/kg/day of BZ1 or BZ1-I, administered orally as suspensions in 0.5% of carboxymethyl cellulose (n = 6 each group) for 10 consecutive days, with repeated doses each day. Meglumine antimoniate is a pentavalent antimony (SbV) drug, used with a maximum dose of 20 mg/kg intramuscularly to treat all types of leishmaniasis and is recommended as the first line treatment option by WHO^38,39. Upon completion of the experiment, animals were sacrificed in a carbon dioxide (CO₂) chamber, and a sample of the spleen and the liver (~50 mg) removed, weighed, and used for total RNA extraction. Real-time PCR quantification of the amastigotes per gram was determined post treatment in spleen and liver samples to ascertain efficacy. The standard curve for DNA for L. infantum chagasi (MHOM/BR/1972/BH46) promastigotes presented optimal linearity and reproducibility (Fig. 8B), with R² = 0.9925, indicating a high correlation between the variables. Cycle thresholds (Ct) linearity describes the degree to which the cycle thresholds for the parasite number is precisely proportional to the promastigotes in the spleen and liver samples. The detection limit of the curve corresponds to Ct of 41.52. The Ct values obtained from the spleen and liver samples were inserted into the equation of the line and the number of parasites calculated per gram of organ.

Fig. 8: In vivo activity of compounds BZ-1 and BZ1-I in the L. infantum chagasi hamster model.

A Schematics of in vivo L. infantum chagasi (MHOM/BR/1972/BH46) Hamster model. B Standard curve for serial dilutions of DNA from promastigote forms from L. infantum chagasi (MHOM/BR/1972/BH46) culture to determine the absolute number of parasites present per gram of sample. C Real-time PCR quantification (amastigotes per gram in spleen and liver) to measure the in vivo efficacy of BZ1 and BZ1-I in the L. infantum chagasi (MHOM/BR/1972/BH46) Hamster model. GLU: meglumine antimoniate, UTG: untreated control.

As shown in Fig. 8C, the average number of parasites in the UTG, was 1.54 × 10⁶/g and 2.09 × 10⁵/g of tissue, in the spleen and liver, respectively, confirming an established infection in these animals (clinical symptoms such as ascites and alopecia). The GLU-treated group resulted in a parasite load reduction, with average values of 1.22 × 10⁴ (spleen) and 2.53 × 10³ (liver) reported. Treatment with BZ1 significantly reduced the number of amastigotes in the spleen to 2.54 × 10⁴/g and liver to 3.02 × 10³ when compared with the UTG (p < 0.005). These values were comparable to those obtained with GLU. Treatment with BZ1-I also reduced the number of amastigotes in the spleen and liver considerably, resulting in 1.62 × 10²/g and 1.20 × 10²/g respectively, when compared with the group UTG (p < 0.005); resulting in ~21-fold greater activity compared to the GLU treatment group.

Histopathological analysis of golden Hamster tissue fragments

Histopathological analysis of the spleen and liver was carried out blinded for all experimental groups with respect to immuno-inflammatory patterns associated with infection by L. chagasi (Fig. 9).

Fig. 9: Histopathological analysis of golden Hamster tissue fragments.

A Fragments of liver processed histologically and stained with Haematoxylin and Eosin in L. infantum chagasi (MHOM/BR/1972/BH46) hamster model. B Fragments of spleen processed histologically and stained with Haematoxylin and Eosin in L. infantum chagasi (MHOM/BR/1972/BH46) Hamster model. Normal: Healthy control, UTG: Untreated control. The scale bar (20 µm) is shown as a yellow line.

Histopathological changes observed in the livers of untreated animals were characterized by the presence of intense mononuclear inflammatory infiltrate in the portal space with the formation of nodules. Of note, intense inflammatory infiltrates of lymphocytes and macrophages forming nodules were observed in the hepatic parenchyma, in addition to evident Kupffer cells hyperplasia and hypertrophy. In the spleen, there was slight hyperplasia of lymphoid follicles in the white pulp and considerable increase of macrophages forming nodules in the red pulp.

The presence of inflammatory nodules in the hepatic parenchyma, lymph histiocytic inflammatory infiltrate in the portal space forming nodules of infected animals treated with BZ1 was observed in n = 6 mice, where BZ1 was administered orally as suspensions in 0.5% of carboxymethyl cellulose. An increase of macrophages in the red pulp forming nodular aggregates and granulomas was shown in the spleen. Infected animals treated with BZ1-I (n = 6) showed a mild inflammatory infiltrate in the portal space and nodules in the parenchyma with the presence of giant cells. In the spleen, an evident increase in macrophages in the red pulp with the formation of nodules was observed, in addition to white pulp hypoplasia.

Effect of BZ1 and BZ1-I on cytokine profile

Next, we analyzed the cytokine profile of liver and spleen cells isolated from the in vivo hamster animal model, which we classified as healthy (uninfected), untreated (UTG); treated with GLU; or treated with BZ1 or BZ1-I (Fig. 10). Alterations in both the proinflammatory cytokine INF-γ (Fig. 10A), TNF-α (Fig. 10B) and IL-17 (Fig. 10C) and anti-inflammatory cytokine IL-4 (Fig. 10D), IL-10 (Fig. 10E) and TGF-β (Fig. 10F) profiles representing both the Th1 and Th2 immune responses, were compared between the different groups. As expected, there was a significant difference in INF-γ levels between the untreated and GLU treatment for spleen samples. Compound BZ1 produced similar results as GLU treatment for the INF-γ levels. A significant downregulation of IL-17 was observed for BZ1 (P < 0.001) compared to GLU in liver samples. A significant downregulation of TNF-α (P < 0.01) was observed with BZ1 treatment compared to GLU and untreated liver and spleen samples. The anti-inflammatory cytokines, such as IL-4 and TGF-β, were also significantly downregulated following exposure to BZ1 (P < 0.01) compared to untreated spleen and liver samples.

Fig. 10: Cellular immune response (pro-inflammatory and anti-inflammatory) in liver and spleen cells of healthy (uninfected), untreated (UTG); treated with GLU; or treated with BZ1 or BZ1-I compounds.

INF-γ (A), TNF-α (B), IL-17 (C), IL-4 (D), IL-10 (E), and TGF-β (F) with the material obtained from the spleen and liver fragments of the L. infantum chagasi (MHOM/BR/1972/BH46) Hamster model. CTL: Healthy (uninfected) animals, GLU: meglumine antimoniate, UTG: untreated control.

Discussion

Much advancement has been made in VL treatment over the last 15–20 years, with the discovery of antimonial monotherapy and the development of newer treatments, such as liposomal formulations of amphotericin B, injectable paramomycin and the first orally available drug miltefosine^16,40. Nevertheless, these drugs still bear limitations such as poor safety profiles and the need for cold chain storage⁴¹. Not to mention the associated non-compliance to therapy because of the difficult route of administration and long durations of therapy^30,42. This calls for an innovative new therapy that is efficacious, safe, has pan-geographical efficacy against multiple species and strains responsible for the disease. Here we report the discovery of two compounds from phenotypic screening and the subsequent data that supports their selection for further development as candidates for VL.

Compound BZ1 (N-(4-Ethoxyphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine), exhibited activity against both the intracellular (amastigote) and extracellular (promastigote) forms of the Leishmania donovani DD8 (Old World- Indian strain) parasite, with IC₅₀ values 0.59 ± 0.13 and 2.37 ± 0.85 µM, respectively. BZ1 also displayed good selectivity (>33) for the mammalian host cells THP-1 and an independent cell line, HEK-293 cells. Thus, BZ1 fulfilled the Drugs for Neglected diseases initiative (DNDi) target candidate profile criteria for promising molecules based on this in vitro activity and selectivity profile²⁹. It also does not have any structural alerts and does not exhibit in vivo toxicity in the hamster VL model.

Liposomal amphotericin B (LAMB) is the treatment of choice in the Indian subcontinent and Mediterranean regions^43,44. Whilst both amphotericin B deoxycholate (Fungizone®) and liposomal amphotericin (LAMB) (Ambisome®) have shown potent activity⁴⁵ against the different species of Leishmania, both variants have significant adverse effects including nephrotoxicity, thrombophlebitis and hypokalemia⁴⁶. When compared with amphotericin B, which has an IC₅₀ 0.20 ± 0.02 µM in the L. donovani DD8 intracellular amastigote assay (96 h), BZ1 had ~3 fold lower in vitro activity (0.59 ± 0.13 µM) in the same assay. However, BZ1 had a greater selectivity index (SI) for the THP-1 host cells, with >33 compared to amphotericin B which is 10.24.

In comparison to the other reference drug, miltefosine, which is also used in combination with LAMB in the clinical settings⁴⁷, displayed an IC₅₀ of 2.54 ± 0.57 µM in the L. donovani DD8 intracellular amastigote assay (96 h), whereas BZ1 demonstrated 5-fold greater activity (0.59 ± 0.13 µM) in the same assay. BZ1 also demonstrated an improved selectivity profile (>33) than miltefosine, as the SI of miltefosine is only 15.66 and 7.87 for HEK-293 and THP-1 cells, respectively. Overall, BZ1 showed better in vitro selectivity profiles than the currently used drugs available.

Initial SAR studies, with the objective of illustrating biological proof of concept for this chemical class, included thirty compounds (BZ1-A–BZ1-d), resulting in a range of IC₅₀ values from 0.57 to 11.48 µM for the intracellular amastigote assay (Supplementary Table 2). Analogue BZ1-I demonstrated improved activity against the L. donovani DD8 intracellular amastigotes (IC₅₀ 0.40 ± 0.38 µM) and comparable activity to BZ1 against L. donovani DD8 intracellular amastigotes (IC₅₀ 0.59 ± 0.13 µM). The SI for BZ1-I was >49.12 and >24.11 for THP-1 host cells and HEK-293, respectively, when compared to the activity observed against the intracellular amastigote.

Our goal was to establish a strong foundation of efficacy and supportive pharmacological profile, to ascertain the therapeutic potential of the candidates, subsequently enabling more informed and targeted medicinal chemistry efforts.

Whilst in vitro anti-leishmanial activity has been reported for chemotypes from the class of Quinolines and 9-Anilinoacridines^48,49,50, of which the BZ compounds are members, there are no previous reports of BZ1 and BZ1-I anti-leishmanial activity.

It has been reported that therapeutic efficacy is dependent on the species of Leishmania, geographic region and clinical manifestations, therefore experimental and clinical data should not be generalized^23,30,51. The taxonomic classification for the genus Leishmania is based on the molecular and biochemical characterization of each species¹. These differences contribute to the variable sensitivity of compounds and may present important clinical implications^52,53. It has been reported in the past that a three to five-fold intrinsic variation in drug sensitivity exists among Leishmania species for antimonials, azoles, miltefosine and paromomycin^14,23,27.

A variation in activity was observed for compounds BZ1 and BZ1-I following the pattern of increased activity from L. donovani (MHOM/IN/80/DD8) > L. donovani (MHOM/SD/62/1S-CL2D, LdBOB). To minimize the possibility of methodology impacting the data¹⁶, all evaluations were undertaken using the same image-based approach⁵⁴.

The data suggested that the variation of the in vitro activity between the strains is substantial. This is most evident in the case of L. donovani (MHOM/IN/80/DD8) and L. donovani (MHOM/SD/62/1S-CL2D, LdBOB), with a 5-fold and 10-fold difference in activity for BZ1 and BZ1-1 in the intracellular amastigote 72 h assay, respectively. Possible reasons for the difference in in vitro activity between the two strains exist, such as media components used in the assay (media composition, fetal bovine serum); multiplicity of infection and biological make-up of the parasites themselves or the specific target of the molecule.

The physicochemical parameters (Table 3) of a compound significantly influence its bioavailability, permeability, and ability to interact with the target pathogen, whereas the pharmacokinetic properties (Table 4) of these compounds, reveal their metabolic stability and clearance potential. Both compounds show promising drug-likeness, with BZ1 exhibiting a better solubility and balanced lipophilicity, which are critical for achieving adequate systemic concentrations and minimizing off-target effects. On the other hand, BZ1-I, with higher lipophilicity, risks aggregation and lower solubility but may have stronger permeability.

Leishmaniasis is characterized by the parasite’s need for intracellular survival, making permeability, solubility, and metabolic stability critical for a compound’s effectiveness. We believe BZ1 is the more promising candidate for anti-leishmanial drug discovery due to its favorable solubility, balanced lipophilicity, and sufficient metabolic stability. Whereas BZ1-I requires optimization to reduce lipophilicity and improve solubility while retaining permeability and metabolic stability. Whilst reduced activity of BZ1 and BZ1-I against Leishmania donovani (MHOM/SD/62/1S-CL2D, LdBOB) compared to the L. donovani (MHOM/IN/80/DD8) intracellular amastigote was observed, both compounds fulfill the DNDi target candidate profile criteria for promising molecules based on this in vitro activity profile in terms of activity and selectivity. As a result, BZ1 and BZ1-I progressed for evaluation in a L. infantum chagasi (strain MHOM/BR/1972/BH46) chronic hamster model for visceral leishmaniasis^55,56.

The in vivo model used incorporates GLU (meglumine antimoniate), belonging to the class of antimonials, as the reference drug. Antimonials remain the treatment of choice in South America, Africa, Nepal, Bangladesh, and India (except North Bihar)²¹. GLU at a dose of 50 mg/kg/day via intraperitoneal route reduced the parasite burden to 99.21% and 98.79% in spleen and liver, respectively. The efficacy of GLU corresponds to the values reported previously for the same dosage regimen^56,57. Both BZ1 and BZ1-I showed excellent in vivo activity when administered orally (10 mg/kg/day), with BZ1 being comparable to GLU, reducing parasitemia to 98.56% and 98.36% in the spleen and liver, respectively. Of particular note, BZ1-I was found to be more active than GLU, with parasite load reduced to 99.95% in spleen and 99.99% in liver. The efficacy of both BZ1 and BZ1-I illustrates that these are highly effective molecules fulfilling the criteria for a lead molecule (DNDi’s target product profile) for visceral leishmaniasis²⁹.

In the experimental visceral leishmaniasis hamster model, the main organs affected are the liver and spleen, where the disease presents a specific organ immune response⁵⁸. In our study, the main histopathological changes observed in the liver were the presence of periportal infiltrate and nodules. The development of granulomas in the liver is closely associated with an attempt from the immune system to control Leishmania infection⁵⁹. Furthermore, the main changes observed in the spleen included increased macrophages forming nodular aggregates and granulomas, as well white pulp hypoplasia as previously described by Corbett⁶⁰, when animals reach 60 days of infection. The reduction in granulomas observed in both BZ1 and BZ1-I treated groups demonstrated that the compounds were effective at controlling the infection. Mangoud et al. has demonstrated that an increase in the number of granulomas and subsequent formation of necrosis and fibrosis occurs with the progression of infection caused by L. infantum chagasi⁶¹, which was observed in the UTG controls.

In the present study, we investigated cellular immune responses by evaluating the mRNA expression of the proinflammatory (TNF-α, IFN-γ and IL-17) and anti-inflammatory (IL-4, IL-10 and TGF-β) cytokines in the spleen and liver samples collected from different groups as previously described⁶². The GLU treated animals were found to have an increased INF-γ (Th1) expression, which signifies a decrease in the parasitic load. The same effect has been seen in miltefosine treated VL-hamsters’ models in which the animals exhibit a resistance to infection^63,64. A similar trend with compound BZ1 was shown, where it shifted the profiles more towards the healthy controls compared with the untreated control (UTG) showing a decrease in parasitic load. Earlier studies have also indicated that a high expression of IL-10 and TGF-β suggests parasite proliferation and establishment of an active infection^64,65. In the present study, this was observed in the UTG group compared to the CTL (healthy, uninfected) group. Interestingly, both BZ1 and BZ1-I have decreased expression of TGF-β when compared to UTG in spleen samples. This correlates with the reduction of the granulomas observed with both BZ1 and BZ1-I, and better spleen architecture and preserved periportal spaces of the liver when compared to the untreated controls signifying susceptibility to infection.

Collectively, the in vitro and in vivo data provides strong evidence of selective anti-leishmanial activity and efficacy against both new and old-world strains of Leishmania. Current efforts, including whole genome sequencing, are focused on ascertaining the biological target or mode of action to enable more extensive interrogation for new further optimized lead molecules. Focused medicinal chemistry optimization, exploring the structure activity relationships of this chemical class and incorporating more comprehensive pharmacological profiling, is thus warranted.

Conclusion

Here we have discussed the identification and development of lead compounds which fulfill the DNDi target candidate profile with promising activity, being selective for the parasite with minimal effect on host cells. Of note, these compounds demonstrated activity across multiple species and strains of Leishmania parasite representative of both the Old and New World. The data generated to date provides evidence to support further investigation and development of the lead series, with the potential to develop a new anti-leishmanial drug candidate.

Methods

Library specifications, reference compound and assay plate preparation

A subset of 5560 compounds representative of a larger diverse synthetic scaffold library housed within Compounds Australia^®, a compound management facility based at Griffith University, Australia, were screened. The scaffold library is comprised of >33,000 pure compounds, acquired from various commercial vendors including Enamine^® and ChemDiv^®, ~30 compounds per scaffold (1200 scaffolds in total). The 5560 compounds collection was handpicked as a representative set of the total 1200 scaffolds within the library.

For the initial screen, compounds were provided by Compounds Australia^® in master plates in single point 5 mM concentrations in 100% DMSO. These compounds were diluted in RPMI without heat inactivated fetal bovine serum (HIFBS) and screened at 10 (for promastigotes) and 20 µM (for intracellular amastigotes) as 0.4% dimethyl sulfoxide (DMSO) for the promastigote and intracellular amastigote assays, respectively. The data was normalized with in-plate controls based on 100% inhibition of parasites with 1 µM amphotericin B, 0% parasite inhibition in the presence of 0.4% DMSO, and the percentage inhibition of each test molecule was calculated for each concentration. Retest of the active hits (with 60% activity in the promastigote assay and 50% activity in the intracellular amastigote assay) was performed using the following concentration range, 20–0.2 µM (0.4% DMSO vehicle) based on a 7-point concentration response scale. New stocks of the active hits were ordered, and their activity was assessed. Amphotericin B, miltefosine and VL-2098⁶⁶ were used as reference drugs and compounds.

In vitro anti-leishmanial assays

L. donovani DD8 promastigote viability assay

An initial parasite density of 1 × 10⁵ parasites/mL was inoculated into a 75 cm² flask (in a total of 30 mL of M199 medium + 10% (v/v) HIFBS) and incubated at 27 °C. After 96 h, the parasites reached the mid-log phase and were seeded in the 384-well Greiner™ black/clear bottom plates at a concentration of 5 × 10⁵ parasites/mL in a volume of 55 µL using an Agilent BRAVO™ automated liquid handling platform. The stocks were diluted 1:25 in M199 media without HIFBS using a dilution plate. The compounds were dispensed in 5 µL using an Agilent BRAVO™ and plates incubated for an additional 68 h at 27 °C at normal atmospheric conditions. Resazurin was then added (0.142 mM final assay concentration) to the plates in a volume of 10 µL/well using a Multidrop™ 384 Reagent Dispenser (Thermo Scientific^®, Newington, NH) and incubated at 27 °C at normal atmospheric conditions. The plates were read after 4 h using an EnVision™ Multilabel plate reader (PerkinElmer^®) using fluorometry settings with excitation of 530 nm and emission 590 nm. The IC₅₀ value for each compound was calculated by normalizing the data based on 1 μM amphotericin B (100% inhibition of parasites) serving as the positive control and 0% inhibition = 0.4% DMSO as the negative control against log of concentration in PRISM™ 10 software (GraphPad Software Inc., San Diego, CA) (denoted IC₅₀ by PRISM™). Each compound concentration was screened in duplicate.

Leishmania infantum chagasi and Leishmania amazonensis promastigote viability assay

Promastigotes of the species Leishmania infantum chagasi (MHOM/BR/72/BH46) were cultured in Schneider’s medium, supplemented with 10.0% (v/v) heat-inactivated fetal bovine serum (FBS) and 1.0% penicillin (10,000 UI mL⁻¹)/streptomycin (10.0 mg mL⁻¹) (Sigma, USA) and Leishmania amazonensis (MHOM/BR/71973/M2269) were grown in LIT medium, supplemented with 10.0% (v/v) HIFBS and 1.0% penicillin (10,000 UI mL⁻¹)/streptomycin (10.0 mg mL⁻¹) (Sigma, USA).

Log-phase growth promastigotes were added to 96-well cell culture plates (tissue culture treated) at a concentration of 1 × 10⁶ cells per well with compounds BZ1 and BZ1-I in triplicate (concentration range: 100–0.781 μg/mL dilution 1:2 in DMSO 0.6% v/v). Amphotericin B, 9.24 µg/mL was used as positive control. Cultures were maintained in 5% CO₂ at 25 °C in a 72 h incubation, then washed with PBS and centrifuged three times at 3000 rpm for 10 min at 4 °C for the removal of the compounds. The promastigote viability analysis was performed using 100 μL/well of a sterile solution of a 4 μg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate buffered saline (PBS), incubated for 4 h, followed by the addition of 50 μL DMSO. The reading was undertaken using wavelength 560 nm. Using GraphPad Prism, version 5.0 the dose response curves were generated and the IC₅₀ estimated. The second experiment was performed using a compound concentration range from 5 to 0.039 μg/mL.

Murine peritoneal macrophages for cytotoxicity studies were obtained from the peritoneal lavage of Swiss mice, performed in accordance with the Guide for the Care and Use of Laboratory Animals, and approved by the Research Ethics Commission of the Universidade Federal de Alfenas. A suspension of 1 × 10⁶/mL murine peritoneal macrophages, in RPMI 1640 medium, supplemented with 10.0% HIFBS, and 1% penicillin (10,000 UI/mL)/streptomycin (10 mg/mL) was added to each well in 96-well plates. The plates were incubated in a 5% CO₂ air mixture at 37  °C to allow for adhesion of the cells. After 24 h, non-adherent cells were removed by washing with the RPMI 1640 medium. Then, compounds and reference drugs, at concentrations ranging from 500 to 3.9 µg mL⁻¹ in DMSO at the final concentration of 0.6% (v/v), were added to the wells containing the cells and the plates incubated for 48 h. Non-adherent cells were removed by washing with the RPMI 1640 medium, and MTT) added to RPMI 1640 medium. The absorbance of each individual well was calculated at 570 nm. Each experiment was performed in triplicate, and the percentage of viable cells was calculated using GraphPad Prism, version 5.0. The second experiment also was performed at concentrations ranging from 5 to 0.039 μg/mL.

L. donovani MHOM/IN/80/DD8 intracellular amastigote assay

The L. donovani MHOM/IN/80/DD8 intracellular amastigote assay⁵⁴ was used to determine the IC₅₀ values of the compounds against L. donovani DD8 intracellular amastigotes described briefly as follows:

Cell culture: L. donovani MHOM/IN/80/DD8 (ATCC 50212) promastigote parasites were maintained in modified M199 Hanks salt medium (pH 6.8) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 27 °C. Parasites were subcultured every 7 days at a concentration of 10⁵ cells/mL. THP-1 (ATCC TIB202) cells were maintained in RPMI medium and 10% FBS at 37 °C/5% CO₂. The cells were subcultured every 2–3 days to maintain a cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Assay protocol: THP-1 cells were seeded in 384-well cell carrier imaging plates (PerkinElmer, Waltham, MA) using a Bravo automated liquid handling platform (Agilent Technologies, Santa Clara, CA, USA) at a concentration of 12,500 cells per well in RMPI plus 10% HIFBS medium containing 25 ng of phorbol 12-myristate 13-acetate (PMA)/mL in order to induce differentiation of the THP-1 cells. Assay plates were incubated at room temperature for 30 min to allow cells to adhere before being incubator at 37 °C and 5% CO₂ for 24 h. After incubation, the PMA was removed by discarding the medium within wells and washing the assay plates three times in phosphate-buffered saline (PBS) on an HydroSpeed plate washer (Tecan, Melbourne, VIC, Australia). After washing, 40 μL of fresh RMPI and 10% FBS medium were added to the assay plates, followed by incubation for 48 h at 37 °C and 5% CO₂.

The number of metacyclic promastigotes present in a 7-day-old L. donovani DD8 promastigote culture was subsequently determined, and parasites were added to the assay plates containing the transformed THP-1 cells (72 h after initial cell seeding) at a multiplicity of infection of 1:2.5 (ratio of host cells to parasites). Assay plates were incubated at room temperature for 30 min, followed by 24 h incubation at 37 °C and 5% CO₂. Non-internalized parasites were subsequently removed by aspirating the medium within wells and washing the assay plates three times in PBS on a HydroSpeed plate washer before the addition of 45 μL of fresh RMPI (10% FBS and 25 ng/mL PMA). Controls consisted of positive wells containing a final assay concentration of 1 μM amphotericin B, and negative wells containing 0.4% DMSO were used as in-plate controls for all experiments. Then, 1 μL of each compound, prepared by Compounds Australia, was diluted by the addition of 24 μL of RPMI medium containing no FCS. Next, 5-μL portions of this dilution were dispensed via a Bravo liquid handler to assay plates to give final assay concentrations of 80 μM (at 0.4% DMSO). For IC₅₀ confirmation, the final assay concentrations ranged from 80 to 0.004 μM. Assay plates were incubated for 96 h at 37 °C and 5% CO₂ before being fixed with 4% paraformaldehyde and stained with SYBR green and CellMask Deep Red (ThermoFisher Scientific, Wlatham, MA, USA).

Images were acquired on an Opera high-content imaging system (Perkin-Elmer). Healthy host (THP-1) cells were identified based on the CellMask Deep Red cytoplasmic and SYBR green nuclear area and intensities. Segmentation of nuclear and cell boundaries were used to identify the region of host cell cytoplasm. Intracellular parasites were then identified within this region based on spot detection algorithms of the SYBR green staining (with size and intensity measurements used to define parasite nucleus of kinetoplast) to determine the number of parasites present within THP-1 host cells. An infected cell was defined as a host cell containing >3 parasites within the cytoplasm boundary. The compound activity was determined based on the number of infected cells normalized to the positive (1 μM Amphotericin B) and negative (0.4% DMSO) controls. Non-linear sigmoidal dose–response curves with no constraints were plotted, and IC₅₀ values were calculated using GraphPad Prism 6 from two independent experiments.

L. donovani MHOM/SD/62/1S-CL2D, LdBOB intracellular amastigote assay

The L. donovani MHOM/SD/62/1S-CL2D, LdBOB intracellular amastigote assay was performed as previously described³³, with minor modifications. In brief, a 96 h incubation period was utilized to replace the 72 h incubation period with compound treated infected cells. The selectivity of the compounds was assessed using THP-1 host cells.

Determination of the cidal action of compounds

To determine the cidal action of the selected compounds, the promastigotes from two different cultures in two separate M199 media (N = 2) were seeded at a concentration of 5 × 10⁵ parasites/mL and volume of 55 µL/well in Greiner 384-well plates using a Bravo liquid handler. A master compound plate was created with the compounds dissolved in 100% DMSO diluted in M199 + 10% HIFBS media using a dilution plate with the final assay concentration of 66.660, 33.330, 16.660, 6.660, 3.330, 1.660, 0.660, 0.330, 0.160, 0.06, 0.033, 0.016, 0.006 and 0.003 µM. The compound exposure was for 24, 48 and 72 h. The compounds were dispensed from the master plate to the assay plate in a volume of 5 µL into 55 µL of parasite culture/well. Cell numbers were determined using the method described previously⁶⁷. If no cells were identified at the above concentrations, compounds were considered to have been effectively cidal at that time point.

Time to kill assay (promastigote viability assay)

To calculate the time to kill for compounds in the promastigote assay the IC₅₀ values were determined for compounds BZ1, BZ1-I and reference compounds (amphotericin B and miltefosine) following exposure of the promastigotes to compound and reference drugs for 24, 48 and 72 h. The starting concentration was 40 µM and the IC₅₀ values were determined from a 14-point concentration–response curve. The compound exposure was for 24, 48, 72 and 96 h.

Time to kill assay (Intracellular amastigote assay)

To calculate the time to kill for compounds in the intracellular amastigote assay the IC₅₀ values were determined for compounds BZ1, BZ1-I and reference drugs (amphotericin B and miltefosine) following incubation for 24, 48 and 72 h. The starting concentration was 40 µM and the IC₅₀ values were determined from a 14-point concentration-response curve. The assay conditions were the same as previously described for the intracellular amastigote assay.

Host cell and compound pre-incubation studies

A study was performed to assess the percentage infectivity in the host cells after pre-incubation with BZ1, BZ1-I and reference drugs (amphotericin B and miltefosine) at various time points (12, 24, 48 and 72 h). THP-1 cells were plated as described previously⁵⁴, after 24 h, cells were washed 3 times with PBS and 45 µL RMPI + 10% HIFBS (v/v) added to the assay plates. The reference compounds and BZ1 and BZ1-I were diluted 1:25 in RPMI with no HIFBS with a final assay concentration in a 14-point concentration response ranging from 80 to 0.004 µM. Five µL were dispensed into assay plates using a Bravo liquid handler (Agilent). The assay plates were incubated for 24, 48 and 72 h at 37 °C/5% CO₂. After 24, 48 and 72 h the plates were washed 3 times with PBS. The addition of parasites, washing the extracellular parasites away and staining the plates were subsequently performed as mentioned previously.

Cytotoxicity assays

The HepG2, RAW-264.7, J774.1 and macrophage (Induced THP-1) resazurin viability assays were undertaken as a modification of the HEK-293 resazurin viability assay previously described⁵⁴.

HEK-293 resazurin viability assay

HEK-293 human embryonic kidney cell line obtained from the ATCC®, USA (HEK-293 CRL-1573™) was maintained in DMEM supplemented with 10% (v/v) HIFBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The cells were passaged as they reached 80% confluence. The assay was undertaken as previously described⁵⁴.

HepG2 resazurin viability assay

HepG2 cells, a human liver cancer cell line obtained from the ATCC®, USA (Hep G2-ATCC^® HB-8065™), were maintained in DMEM supplemented with 10% (v/v) HIFBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. HepG2 cells at 5 × 10⁴ cells/mL in DMEM + 10% HIFBS (v/v), were added in a total volume of 55 µL to Greiner™ black 384-well plates using the Multidrop™ liquid handling system. The compounds prepared at 20 mM in 100% DMSO were diluted in DMEM without HIFBS in a ratio 1:25 and 5 µL of the compounds were added to give a final concentration of 0.4% DMSO and desired compound final concentration. The plates were incubated for 48 h at 37 °C with 5% CO₂. Resazurin was diluted in DMEM + 10% HIFBS to give a concentration of 0.49 mM. Ten microliters of this dilution were added to assay plates, which were further incubated for 5 h at 37 °C/5% CO₂, then left at room temperature for 19 h. The plates were read on the EnVision™ Multilabel plate reader using fluorometry settings with an excitation of 530 nm and an emission of 590 nm. DMSO (0.4%) was used as the negative control and 5 µM of puromycin was used as the positive control.

Macrophage (Induced THP-1) cytotoxicity assay

THP-1 cells, a human monocytic cell line obtained from the ATCC®, USA (THP-1 TIB-202™), were maintained in RPMI 1640 medium GlutaMAX™ supplemented with 0.05 mM mercaptoethanol and 10% (v/v) HIFBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The THP-1 cells were suspended at a concentration of 2.5 × 10⁵ cells/mL in RMPI medium + 10% (v/v) HIFBS in a 150 mL sterile clear polystyrene bottle (Corning®). One mg/mL of PMA stock solution was diluted 1:20 in DMSO and then the working solution diluted 1:2000 in cell suspension to give a final concentration of 25 ng/mL. 50 µL of cell suspension at a density of 2.5 × 10⁵ cells/mL were added to Greiner™ black/clear bottomed 384-well plates using a BRAVO™, which was also used for all subsequent additions unless otherwise stated. After 24 h incubation at 37 °C/5% CO₂ the plates were washed 3X with PBS and incubated for an additional 48 h at 37 °C/5% CO₂ in accordance with the intracellular amastigote assay conditions. The reference compounds, amphotericin B, miltefosine were prepared in 100% DMSO, whereas puromycin was prepared in MilliQ water. The reference compounds were serially diluted in 100% DMSO to give 14 concentrations (40 µm–0.002 µM). An intermediate dilution of the reference plate was prepared in water, PBS or RMPI medium and 5 µL of these dilutions subsequently dispensed into the THP-1 assay plates. The plates were incubated for 64 h at 37 °C/5% CO₂. Cell viability was assessed using resazurin at a concentration of 1.5 mM (0.3 mM final assay concentration) in a volume of 10 µL using a Multidrop™ 384 Reagent Dispenser for an incubation period of 8 h. The plates were read on an EnVision™ Multilabel plate reader using fluorometry settings with excitation of 530 nm and emission 590 nm.

RAW-264.7 resazurin viability assay

RAW-264.7 murine macrophage cell line derived from Abelson’s leukaemia obtained from the ATCC^®, USA (RAW 264.7-ATCC® TIB-71™) was maintained in DMEM supplemented with 10% (v/v) HIFBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The protocol used was as described for the THP-1 cytotoxicity assay with a slight modification in the number of cells initially seeded per 384-well plate: reduced from 2.5 × 10⁵ to 2.0 × 10⁴ cells/mL.

J774.1 resazurin viability assay

J774.1 cell line, a murine macrophage cell line from reticulum cell sarcoma obtained from the ATCC^®, USA (J774A.1-ATCC® TIB-67™), was maintained in DMEM supplemented with 10% (v/v) HIFBS at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. The protocol used was similar to THP-1 cytotoxicity assay with a slight modification in the number of cells initially seeded per 384-well plate: from 2.5 × 10⁵ to 4 × 10⁴ cells/mL.

Statistics and reproducibility

To accurately assess the activity of compounds in inhibiting Leishmania parasite survival, the data was normalized based on the number of infected cells in comparison to control samples. The negative control, which contains 0.4% DMSO, represents the baseline or no-compound condition, while reference compound (amphotericin B) serves as positive controls for known activity. By normalizing the data in this manner, the calculated activity of each compound was directly comparable, ensuring a reliable evaluation of their anti-parasitic effects.

The normalization formula used is as follows:

$${{\rm{Normalization}}}= \; 100-[100\times ({{\rm{sample}}}-{{\rm{average}}}\,{{\rm{of}}}\,{{\rm{minimum}}}\,{{\rm{signal}}})/ \\ ({{\rm{average}}}\,{{\rm{of}}}\,{{\rm{maximum}}}\,{{\rm{signal}}}-{{\rm{average}}}\,{{\rm{of}}}\,{{\rm{minimum}}}\,{{\rm{signal}}})]$$

The Z-factor, a key statistical parameter used to assess the quality of an assay was calculated for all assays. The Z-factor was calculated as follows:

$${{Z}}^{\prime} \,=\, 1{-}\left[\right.3 \times \left({{\rm{SD}}}\; {{\rm{of}}}\; {{\rm{wells}}}\; {{\rm{with}}}\; {{\rm{negative}}}\; {{\rm{control}}}\right)+3 \\ \left. \times \left({{\rm{SD}}}\; {{\rm{of}}}\; {{\rm{wells}}}\; {{\rm{with}}}\; {{\rm{positive}}}\; {{\rm{control}}}\right)\right]/ \\ \left[{{\rm{Mean}}}\; {{\rm{of}}}\; {{\rm{negative}}}\; {{\rm{control}}}-{{\rm{Mean}}}\; {{\rm{of}}}\; {{\rm{positive}}}\; {{\rm{control}}}\right]$$

A Z-factor value close to 1 indicates excellent assay quality, whilst between 0.5 and 1 is typically considered acceptable for cell-based assays.

Drug metabolism and pharmacokinetics studies

Physicochemical parameters using ChemAxon JChem software

A range of physicochemical properties evaluating drug-likeness and likely oral absorption characteristics were calculated using the ChemAxon chemistry cartridge via JChem for Excel software (version 16.4.11). A brief description of each parameter is provided below, along with a suggested ideal range based on research reported in the Absorption, distribution, metabolism, and excretion (ADME) literature from key industry and academic sources.

MW (<500): Molecular weight.

PSApH 7.4 (<140 Ų): Polar surface area also inversely correlates with membrane permeability.

HBD (<5) and HBA (<10): Number of hydrogen bond donors and acceptors gives an indication of the hydrogen bonding capacity, which is inversely related to membrane permeability.

FRB (≤10): Number of freely rotating bonds represents the flexibility of a molecule’s conformation.

Aromatic rings (<4): Total number of aromatic and heteroaromatic rings is also related to molecular flexibility.

Fsp³ (>0.3): Fraction of sp³ carbons to total carbons indicates the complexity of a molecule’s 3D structure.

cpK_a: Ionization constants impact solubility and permeability. Only physiologically relevant predicted values are provided here (i.e. 0 < pK_a < 12).

cLogP/cLogDpH (<5): Partition coefficients reflect the lipophilic character of the neutral structure, while distribution coefficients reflect the partitioning properties of the ionized molecule at a specific pH.

Kinetic solubility estimation using nephelometry (SolpH)

Compound in DMSO was spiked into either pH 6.5 phosphate buffer or 0.01 M HCl (pH ~2.0) with the final DMSO concentration being 1%. After 30 min had elapsed, samples were then analyzed via Nephelometry to determine a solubility range. See Bevan and Lloyd (2000) Anal Chem, 72:1781-1787.

Distribution coefficient estimation using chromatography (gLogDpH)

Partition coefficient values (LogD) of the test compounds were estimated at pH 7.4 by correlation of their chromatographic retention properties against the characteristics of a series of standard compounds with known partition coefficient values. The method employed is a gradient HPLC based derivation of the method developed by Lombardo (see Lombardo et al. (2001) J. Med. Chem. 44:2490–2497).

In vitro metabolic stability

Incubation

The metabolic stability assay was performed by incubating each test compound in liver microsomes at 37 °C and a protein concentration of 0.4 mg/mL. The metabolic reaction was initiated by the addition of an NADPH-regenerating system and quenched at various time points over a 60-min incubation period by the addition of acetonitrile containing diazepam as internal standard. Control samples (containing no NADPH) were included (and quenched at 2, 30 and 60 min) to monitor for potential degradation in the absence of cofactor. The human liver microsomes used in this experiment were supplied by XenoTech, lot #1410230. Microsomal incubations were performed at a substrate concentration of 0.5 μM.

Data analysis

Species scaling factors from Ring et al.⁶⁸ were used to convert the in vitro Clint (μL/min/mg) to an in vivo CLint (mL/min/kg). Hepatic blood clearance and the corresponding hepatic extraction ratio (EH) were calculated using the well stirred model of hepatic extraction in each species, according to the “in vitro T1/2” approach⁶⁹. The EH was used to classify compounds as low (<0.3), intermediate (0.3–0.7), high (0.7–0.95) or very high (>0.95) extraction compounds. Predicted in vivo clearance values have not been corrected for microsomal or plasma protein binding. Species scaling calculations are based on two assumptions: (1) NADPH-dependent oxidative metabolism predominates over other metabolic routes (i.e. direct conjugative metabolism, reduction, hydrolysis, etc.) and (2) rates of metabolism and enzyme activities in vitro are truly reflective of those that exist in vivo. If significant non-NADPH-mediated degradation is observed in microsome control samples, then assumption (1) is invalid and predicted clearance parameters are therefore not reported.

In vivo anti-leishmanial assay

Ethics statement

All experimental procedures involving animals were approved by the Research Ethics Commission of the Federal University of Alfenas, Brazil (project number 394/2012), and were performed according to the Guide for the Care and Use of Laboratory Animals⁷⁰. We have complied with all relevant ethical regulations for animal use.

Experimental animals

Male golden hamsters (Mesocricetus auratus) recently weaned (~120 g) were housed inside boxes with sterile absorbent material, with food and water ad libitum, on ventilated shelves with controlled local temperature and in natural light/dark cycle. Hamsters were infected intraperitoneally with 1 × 10⁷ promastigotes of L. (L.) infantum chagasi (MHOM/BR/1972/BH46) and maintained in sterile absorbent material boxes, with water and food ad libitum.

In vivo model for Leishmaniasis

The L. chagasi in vivo evaluation was performed as previously described by Colombo et al.⁵⁷. During the chronic phase of infection (~60 days post infection), animals were divided into 4 groups, and subjected, to one of the following treatments for 10 consecutive days; vehicle 0.5% of carboxymethyl cellulose (vehicle) suspension (n = 5 animals), administered orally (untreated group, or UTG); 50 mg/kg/day of GLU, by intraperitoneal injection (GLU group; n = 5); 10 mg/kg/day of compounds BZ1 and BZ1-I, administered orally as suspensions in 0.5% of carboxymethyl cellulose (n = 6 each group). After 10 days of treatment, animals were sacrificed in a CO₂ chamber, and a sample of the spleen and the liver (~50 mg) removed, weighed and used for total RNA extraction.

DNA extraction for standard curve

Standard curves of parasite DNA for use in quantitative real-time PCR (qPCR) experiments were produced as described previously⁷¹. Briefly, promastigotes from stationary phase cultures were harvested by centrifugation at 1000×g for 10 min, washed twice in PBS (pH 7.2), and counted in a hemocytometer. Then, parasites were re-suspended in PBS (pH 7.2) to a concentration of 1 × 10⁸ cells/mL, and serially diluted (1:10) up to a concentration of 1 × 10² cells/mL (in triplicates). Lysis buffer (10 mM Tris–HCl, pH 8.0, containing 10 mM EDTA, 0.5% SDS, 0.01% N-Lauroylsarcosine sodium salt, 100 µg/mL Proteinase K) was then added to parasite suspensions, at a ratio of 1:4 (v/v), samples were mixed by vortexing and incubated at 56 °C, for 40 min. DNA was extracted from lysed samples using the QIAamp DNA extraction Mini Kit (Qiagen), according to the manufacturer’s instructions.

RNA extraction and cDNA synthesis

Fragments of liver and spleen (~50 mg; weighed using sterile and disposable surgical material) removed from treated mice were placed in sterile microfuge tubes and frozen immediately at −80 °C in storage buffer (RNAlater, Thermofisher). RNA extraction was performed 24 h after fragment removal, using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. RNA samples were frozen immediately after extraction. For reverse-transcription into cDNA, 1 μl of dNTPs mix (10 mM) and 1 μl of random primers (3 μg/μL) were added to 11 μl of RNA sample, and samples were incubated in a thermal cycler for ~5 min, at 65 °C. Then, tubes were placed on ice for 20 s, and 2 μl of DTT (100 mM) and 4 μl 5x buffer (Tris–HCl 250 mM, pH 8.3, containing 375 mM KCl, 15 mM MgCl₂) were added, and samples incubated again in the thermal cycler for 20 s, at 37 °C. Finally, 1 μl (200 U/μL) M-MLV RT enzyme was added and samples incubated for 50 min, for cDNA synthesis. The purity of the cDNA sample was confirmed by measuring the absorbance at 260/280 in a NanoDrop ND2000, and sample integrity was verified by agarose gel electrophoresis and PCR. Samples were frozen at −20 °C for subsequent use in qPCR⁵⁷.

Parasite load estimation by LINJ31 quantitative PCR (qPCR)

Quantitative real time PCR (qPCR) was performed using the TaqMan® probe 5’CCT CCT TGG ACT TTG C3’ (double-labeled with FAM at the 5’-end and a non-fluorescent quencher at the 3’-end), and the primers LINJ31F (5’CCG CGT GCC TGT CG3’) and LINJ31R (5’CCC ACA CAA GGA GCG ACT3’), which amplify L. (L.) infantum hypothetical protein (partial mRNA; GeneBank accession number LinJ31.1310). Reactions were performed in a StepOne Real Time PCR System (Applied Biosystems), and reaction mixtures contained 2 μL of DNA or cDNA samples, 10 μL of 2X TaqMan Universal PCR Master Mix, 1 μL of a mixture of forward (LINJ31F) and reverse (LINJ31R) primers (at a concentration of 18 μM), and 5 μM of the labeled TaqMan® probe, in a final volume of 20 μL. For negative and positive controls, water or DNA extract from L. (L.) infantum chagasi (MHOM/BR/1972/BH46 were added, respectively. The following PCR conditions were used: one step of 50 °C for 2 min, followed by one step of 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The number of parasites per gram of spleen or liver tissue was calculated based on the linear regression data from the standard curve performed with promastigote DNA. Statistical analysis was performed by quantitative Student’s t-test with Mann–Whitney (unpaired, two-tailed) for the significance test (p < 0.05)⁷².

Histopathological assays

The L. chagasi histopathological assay was performed as previously described by Silva et al.⁶². Male golden hamsters (Mesocricetus auratus) were housed; infected and treated as described above.

Fragments of spleen and liver were collected, processed histologically, and stained with Haematoxylin and Eosin (HE). The histopathological analyses for the different experimental groups were performed blinded. The spleen slides were analyzed to evaluate the white pulp and red pulp ratio, using Image-Pro Plus software to measure the areas relative to these pulps. The liver slides were analyzed to measure the intensity of the periportal infiltrate and to count of the number of granulomas. Analysis of the liver involved identifying the presence of inflammatory infiltrates, nodules and inflammatory cells (mononuclear and polymorphonuclear cells) in the periportal region. In hepatic parenchyma, the presence of lobular infiltrate, nodules, Kupffer cells hyperplasia and hypertrophy, necrosis and inflammatory cells (mononuclear and polymorphonuclear cells). The inflammatory infiltrate near periportal region and parenchyma were quantified according to intensity (mild, moderate or intense). In the spleen, the presence of hyperplasia or hypoplasia in white pulp were evaluated, in addition to the presence of nodules and inflammatory cells (macrophages, plasmocytes, neutrophils) in red pulp.

Evaluation of the relative expression of cytokines by qPCR

The relative expression of cytokines was measured as previously described by Silva et al.⁶². The assay to evaluate the gene expression of the cytokines IL-10, IL-4, TGFβ, IFN-γ, IL-17 and TNFα was performed with the material obtained from the spleen and liver fragments of the animals. RNA was extracted from the fragments and cDNA synthesized using real-time PCR. The Taqman-type hydrolysis probes used to quantify the relative expression of the cytokines were normalized against hamster microgobulin obtained from the Genebank gene sequence.

The primers were designed using Software Primer Blast and recently published⁶². The nucleotide sequences for the genes evaluated in this study were obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). The primers that were produced by Integrated DNA Technologies (IDT) and their sequences are shown in Table 2.

Primers sequences

IL-10 Primer 1: CCA GCT GGA CAA CAT ACT ACT C

Primer 2: CTG GAT CAT TTC TGA TAA GGT TTG G

Probe: /56-FAM/TG CAG GAC T/Zen/T TAA GGG TTA CTT GGG T/3IABkFQ/

IL-4 Primer 1: GAA GAA CTC CAC GGA GAA AGAC Primer 2: GGG TCA CCT CAT GTT GGA AAT A Probe:/56-FAM/CT TCC CAG G/Zen/T GCT TCG CAA GTT T/3IABkFQ

TGF-β Primer 1: GGC AGC TGT ACA TCG ACT TT Primer 2: GAC AGA AGT TGG CGT GGT AG Probe: /56-FAM/TG GAA GTG G/Zen/A TTC ACG AGC CCA AG/3IABkFQ/

IFN-γ Primer 1: GAG GAG CAT AGA CAC CAT CAA G Primer 2: CCT GAA GGT CAT TTA CCG GAA T Probe: /56-FAM/TC TTC AAC A/Zen/G CAG CAT GGA GAA ACT CA/3IABkFQ/

IL-17 Primer 1: CTC CAG CAG CAA CTC TTC TT Primer 2: TCT TCT GTT GCT GGT CTC TTG Probe: /56-FAM/CC AGC CAG G/Zen/G TTC TTC TCA AGC TC/3IABkFQ/

TNF-α Primer 1: GGT TTA CTC CCA GGT TCT CTT C Primer 2: GGA CAG GAG GTT GAC GTT AT Probe: /56-FAM/TC AGC CGC A/Zen/T TGC TGT GTC CTA /3IABkFQ/

MG (Microglobulin, hamster constitutive gene) Primer 1: GGT CTT TCT ATC TCT TGG CTC A Primer 2: CTT GGG CTC CTT CAG AGT TAT G Probe: /HEX/ACT GCG ACT/ Zen/ G ATA AAT ACG CCT GCA/3IABkFQ/

The reactions were carried out in a Step One Real Time PCR System (Applied Biosystems), in a final volume of 10 μL per reaction. 0.5 μL of a mixture including the forward, reverse, and the TaqMan probe, labeled with Fluorescein amidites (FAM) for the cytokines and Hexachloro-fluoresceine (HEX) for the constituent gene, were added using non-fluorescent quencher (NFQ) on the plate. Then a mix containing 5 μL of 2X TaqMan Universal PCR Master Mix and 3.5 μL of DNAse and RNAse free water and 1 μL of cDNA from the samples were prepared. Homogenization was performed for all markers, to standardize with the same final concentration of cDNA, and then added to the plate. A negative control of DNAse and RNAse free water was used for each marker. Amplifications occurred with an initial cycle of 50 °C for 2 min; then a cycle at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and a final 60 °C step for 1 min. The results were expressed by the ΔCt (Cycle Threshold) method, which consists of a relative quantification, where normalization of expression of each target is performed by subtracting the Ct value found for each cytokine, by the Ct value of the constitutive gene.

Selection of resistant parasites

Amphotericin B, miltefosine and compounds BZ1 and BZ1-I were used to select resistant parasites to assess any differences in THP-1 cell infectivity and determine whether resistance could be maintained in the intracellular amastigote assay. The promastigotes were cultured in the presence of gradually increasing concentrations of amphotericin B, miltefosine, and either compound BZ1 or BZ1-I beginning at starting concentrations of 0.000135 µM (0.125 µg/mL), 0.03330 µM (13.572 µg/mL), 0.0033 µM (1.004 µg/mL) and 0.0033 µM (1.004 µg/mL) respectively, (a sublethal concentration) to generate drug-resistant strains. Throughout the process the concentrations for amphotericin B, miltefosine, BZ1 and BZ1-I were increased gradually and only once the cells displayed growth and mobility characteristics after 96 h incubation comparable to the control cultures grown in the absence of compounds.

Three independent experiments were performed (the promastigotes from three different cultures in three separate M199 media were seeded in 25 cm² flasks at a concentration of 5 × 10⁵ promastigotes/mL in a volume of 10 mL) with amphotericin B, miltefosine, compounds BZ1 and BZ1-I at sublethal concentrations. These cultures were sub-cultured every 96 h with the addition of amphotericin B, miltefosine, compounds BZ1 and BZ1-I.

Confirmation of resistant cell lines

For confirming resistance, clonal populations from the resistant parasites were obtained via limiting dilution in a 384-well plate and the confirmation of resistant studies performed on isolated clones.

Resistant clones were evaluated using a promastigote viability assay as described previously. Following confirmation that resistance in the promastigotes was achieved, these cultures were used to infect the THP-1 cells and the infectivity of the cells was assessed. Resistance to amphotericin B, miltefosine, compound BZ1 and BZ1-I was also assessed with the intracellular amastigote assay.

Stability of compound resistance assessed

Following generation of resistant cultures, the sensitivity to the reference drugs (amphotericin B and miltefosine), plus test compounds BZ1 and BZ1-I, were evaluated. In addition, the stability of drug resistance post drug pressure removal throughout time in culture, was assessed. The resistant parasite cultures were grown in the absence of amphotericin B, miltefosine, compounds BZ1 and BZ1-I for 10 passages before the sensitivities of the cultures to these compounds and drugs were retested in the promastigote viability assay and intracellular amastigote assays. This experiment provided an indication as to whether the generated resistance was reversible or not.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.