INTRODUCTION
Neglected diseases have a significant global impact, primarily afflicting the world's poorest and most vulnerable populations. Leishmaniasis is a group of infectious diseases caused by the intracellular parasite Leishmania spp., transmitted through the bites of infected female sandflie mosquitoes. It encompasses cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL), with a focus on VL due to its severity and high fatality rates (DNDi, 2023).
Leishmaniasis currently lacks vaccines for human prevention, and treatment relies on therapeutic drugs, some of which have severe side effects. Commonly used treatments include antimony compounds, Amphotericin B, Miltefosin, and Paromomycin. However, their use is associated with side effects and the development of drug resistance over the years (DNDi, 2023).
As a result, there is an urgent need for new drugs to combat leishmaniasis. Morita-Baylis-Hillman reaction-derived adducts (MBH) have gained attention for their diverse biological activities, including their potential as anti-Leishmania agents (Lima-Junior; Vasconcellos, 2012). This study aims to explore the potential of Morita-Baylis-Hillman adducts as a promising source of new drugs for parasitic diseases like leishmaniasis.
AIMS
Main Aim
To Assess the Anti-Leishmania Activity in L. infantum of Homodimeric 3-Hydroxyindolin-2-one Morita-Baylis-Hillman Adducts and their Biological Effects.
Specific Aims:
1. Evaluate the anti-Leishmania activity of Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adducts against L. infantum promastigote forms to determine the 50% growth inhibition concentration (IC50).
2. Assess the anti-Leishmania activity of Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adducts on axenic amastigote forms of L. infantum to obtain the effective concentration against 50% of amastigotes (EC50).
3. Investigate the cytotoxic effect of the compounds on peripheral blood mononuclear cells (CC50) and erythrocytes (HC50) from healthy individuals.
4. Determine the selectivity index of the compounds concerning human cells.
5. Perform an in silico analysis of the interaction nature of Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adducts with host proteins.
METHODS
3.1. ETHICAL CONSIDERATIONS
The present study obtained approval from the Ethics Committee for Research Involving Human Subjects at the University Hospital Lauro Wanderley/UFPB (CAAE: 17813013.8.0000.5183). All research participants volunteered to participate in the study, and they were duly informed about its significance and provided their informed consent by signing the Informed Consent Form (ICF).
3.2. CULTURE AND MAINTENANCE OF L. INFANTUM
The promastigote forms of Leishmania (Leishmania) infantum [IOC/L0579 (MHOM/BR/1974/PP75)] were cultured in Schneider's medium (24.084 g/L Schneider powder + 0.4 g/L Sodium Bicarbonate (NaHCO3) + 0.6 g/L anhydrous calcium chloride - CaCl2), pH 7, supplemented with 20% heat-inactivated fetal bovine serum (FBS), 1% male human urine, 100 U/mL of penicillin, and 100 mg/L of streptomycin, and the parasites were maintained at 26 ± 1 °C. Passages were performed weekly, not exceeding the twentieth. The procedure to obtain the extracellular axenic amastigote form of L. infantum was based on methods previously described (Debrabant et al., 2004), using a Schneider medium readjusted to pH 5.5 at 37ºC. Temperature treatment differentiated the promastigote forms in the stationary growth phase into axenic amastigotes (37 °C). Parasites were maintained in culture for no more than 20 passages.
3.3. COMPOUND
The Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adducts, namely HNA, HCM, HIB, HDM, HDB, and HCA, were obtained from the Laboratory of Organic and Medicinal Synthesis (LASOM)/UFPB. Amphotericin B (Amphotericin B®; UNIANF®; União Química Farmacêutica Nacional S.A, São Paulo, Brazil), a second-line drug for leishmaniasis treatment in Brazil, was used as a positive control. A stock solution of Morita-Baylis-Hillman adducts (16 mg/mL) and Amphotericin B (10 mg/mL) were prepared in DMSO.
The compounds underwent an initial screening for biological activity, and those showing strong anti-promastigote activity were chosen for further studies.
3.4. IN VITRO ANTI-LEISHMANIA ACTIVITY
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay assessed the promastigote growth inhibition assay. Promastigotes were harvested during the exponential growth phase. They were incubated (approximately 1 x 106) in the presence and absence of several concentrations of Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adducts (608.33 to 4.75 µM) and AmB (10 to 0.078 µM) as the positive control. The plate was incubated at 26 °C for 72 h in a biological oxygen demand (B.O.D.) incubator using a Schneider’s medium (Schneider’s Insect Medium 24.5g/L; L-glutamine 1.8g/L; glucose 2g/L and sodium bicarbonate 0.4g/L; Sigma-Aldrish – St. Louis - USA) supplemented. According to the manufacturer's protocol, the growth inhibition was evaluated using an MTT assay kit (Amresco, Ohio, USA). After 4 h of incubation, the plates were centrifuged for 10 min at 400 x g, the supernatants were removed, and cells were resuspended with 100 ?L of DMSO to dissolve the formazan crystals. The absorbance (540 nm) was measured using a plate reader (Biosystems model ELx800; Curitiba, PR, Brazil). The same procedure was followed to assess inhibition of the axenic amastigote form using these compounds with the following modifications: the treatment time was shortened to 24 h, and the test temperature was raised to 37 °C at a pH of 5.5. Three independent experiments were performed in triplicate.
3.5. RED BLOOD CELL LYSIS ASSAY
The hemolytic activities of Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adducts were measured using human red blood cells from healthy adults (n = 3) according to methods previously described (Almeida et al., 2023) by making some modifications. Briefly, 80 µL of a 5% erythrocyte/phosphate-buffered saline (PBS) suspension was mixed with 20 µL of HDM, HDB, HCA (608.33 to 4.75 µM) and AmB (100 to 0.78 µM). After incubation at 37 °C for 1 h, 100 µL of PBS (1.5 mM KH2PO4, 8.1 mM Na2HPO4, 136.9 mM NaCl, and 2.6 mM KCl, pH 7.2) was added to stop the hemolysis process, and the samples were centrifuged for 10 min at 1,000 ×g. The supernatants were collected, and the extent of hemolysis was measured spectrophotometrically at 540 nm. The hemolysis percentage was determined as [(Abssam ? Abscon)/ (Abstot ? Abscon) × 100], where Abssam is the absorbance of the sample, Abscon is the absorbance of the blank control (without drugs), and Abstot is the absorbance of total hemolysis (replacing the sample solution with an equal volume of ultrapure water by Direct-Q UV®, France).
3.6. IN VITRO CYTOTOXICITY IN PERIPHERAL BLOOD MONONUCLEAR CELLS
In vitro, cytotoxicity was evaluated using peripheral blood mononuclear cells (PBMCs) collected from three healthy volunteers using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. PBMC were separated by density gradient using Ficoll Paque PLUS (GE Healthcare, USA), pelleted by centrifugation (400 g for 40 minutes), and resuspended in RPMI 1640 medium, supplemented with 10% fetal bovine serum and 1% antibiotic solution (penicillin/streptomycin 5000 ?g/mL). The cells were stained with trypan blue and counted using a hemocytometer. Next, the cells were seeded in 96-well plates, where 2.5 x 105 cells/well were cultured in RPMI medium. Several concentrations of HCA, HDM, HDB (608.33 to 4.75 µM), and AmB (10 to 0.078 µM) were added. DMSO 0.5% as vehicle control and cells with media as a negative control. After preparation, the plates were incubated in a 5% CO2 incubator at 37°C for 24 hours of treatment.
After the treatment, the plates were centrifuged (1500 rpm for 10 minutes at 4°C), and 100 µL of the supernatants were carefully aspirated from each well. Finally, MTT was added to the plates, and they were incubated once again following the cell viability protocol described earlier in section 3.4 for the promastigote and axenic amastigote forms of L. infantum.
3.7. EVALUATION OF THE ENZYMATIC INHIBITORY POTENTIAL OF MORITA-BAYLIS-HILLMAN HOMODIMERIC 3-HYDROXYINDOLIN-2-ONE ADDUCT THROUGH MOLECULAR DOCKING
The in silico analysis was conducted using Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adduct HCA. The compound was obtained in .mol format and subsequently converted to .pdb format using the Open Babel software, enabling the retrieval of spatial distribution information for each atom (O'boyle et al., 2011). To identify suitable targets, a protein with a known crystallographic structure from the PDB database was selected (Bernstein et al., 1977). The target was Human DNA Topoisomerase I (Human Reconstituted DNA Topoisomerase I in Covalent Complex with a 22 base pair DNA Duplex - PDB code: 1A31). The goal was to determine if the cytotoxic activities in PBMCs could be associated with the inhibition of this protein, which is exclusively present in nucleated cells.
Both the target protein and ligand were prepared for docking using AutoDock Tools 1.5.6 (Morris et al., 2009). This preparation involved adding polar hydrogen atoms and/or hydrogen bonds, assigning charges, and removing heteroatoms and water molecules if present. The molecular docking process was carried out using the three-dimensional protein and ligand structures in .pdbqt format through AutoDock Vina (Trott; Olson, 2010), with the exhaustiveness parameter set to 50. The affinity of the receptor-ligand complexes was evaluated in kcal/mol, utilizing the Gibbs free energy (Myint et al., 2021). A more negative affinity value indicates a more spontaneous interaction between the compound and the target protein.
The resulting complexes underwent visual inspection using the PyMOL Molecular Graphics System (Delano, 2002). Additionally, the YRB.py script was employed to highlight hydrophobicity and charge distributions on protein surfaces (Hagemans et al., 2015), aiding in generating complex images. The Discovery Studio Visualizer (DSV) v. 20.1.0.19295 (Biovia, 2017) was used to visualize protein-ligand interactions.
3.8. STATISTICAL ANALYSES
The values of average inhibitory concentration (IC50), average effective concentration (EC50) and cytotoxicity (CC50) were calculated using GraphPad Prism® software (version 6.0; San Diego, CA, USA). Statistical analysis was performed using nonlinear regression (curve fitting). The assays were performed in technical triplicates and three independent experiments, except for the hemolytic assay, which was evaluated in technical duplicates and biological triplicates. FlowJo 10.0.7 was used for flow cytometry data analysis. Statistical differences between treatments were evaluated by analysis of variance (One-Way ANOVA) with Tukey's post hoc test (p ? 0.05). The data are presented as mean ± standard deviation (SD).
RESULTS
This study evaluated the biological activity of five Morita-Baylis-Hillman homodimeric 3-hydroxyindolin-2-one adduct derivatives against L. infantum promastigote forms. The compounds underwent initial screening for biological activity, focusing on AMBH compounds HCA, HDM, and HDB, which exhibited promising activity. HCA, HDB, and HDM effectively inhibited the growth of L. infantum promastigotes, with IC50 values of 17.36 ± 0.39 ?M for HCA, 8.95 ± 0.29 ?M for HDM, and 10.55 ± 0.31 ?M for HDB. In comparison, Amphotericin B had a potent inhibitory effect with an IC50 value of about 0.13 ± 0.02 ?M.
The cytotoxicity of HCA, HDB, and HDM on human erythrocytes was assessed. HCA and HDB showed minimal hemolytic activity, while HDM exhibited hemolytic activity at 649 ?M. HDM's HC50 was 380.8 ± 0.07, whereas Amphotericin B displayed significant cytotoxicity with an HC50 of 58.9 ± 0.14 for leishmaniasis treatment. The AMBH compounds, including HCA, HDM, and HDB, demonstrated efficacy in anti-amastigote activity against L. infantum, with EC50 values of 8.77 ± 0.05 ?M for HCA, 5.48 ± 0.05 ?M for HDM, and 5.70 ± 0.04 ?M for HDB. Amphotericin B exhibited strong cytotoxicity against amastigotes with an EC50 of 0.15 ± 0.02 ?M.
Cytotoxicity assessment on peripheral blood mononuclear cells (PBMCs) showed that HCA, HDB, and HDM had high cytotoxic potential against immune system cells, with CC50 values lower than Amphotericin B (53.88 ± 0.09 µM). CC50 values were 10.91 ± 0.03 µM for HCA, 2.54 ± 0.04 µM for HDB, and 8.34 ± 0.03 µM for HDM. The selectivity index (CC50/CE50) was calculated to assess selectivity. HDM, HCA, and HDB did not exhibit selectivity for L. infantum amastigotes compared to leukocytes, with selectivity indices less than 2. In contrast, Amphotericin B displayed a selectivity index of 359.20, indicating greater selectivity against amastigotes.
Considering the cytotoxicity against PBMCs, the study conducted molecular docking of the least cytotoxic compound, HCA, against Human DNA Topoisomerase I. The docking showed a binding affinity of -8.4 kcal/mol, with HCA anchoring to double-helical DNA at the enzyme's active site. Interactions included alkyl-? bonds, carbon-hydrogen bonds, hydrogen-donor bonds, and hydrogen bridges between HCA and nucleotide bases. This study assessed the effectiveness and cytotoxicity of AMBH compounds HCA, HDM, and HDB against L. infantum promastigotes and amastigotes. While they demonstrated promising anti-parasitic activity, they exhibited cytotoxicity to human cells, highlighting the need for further research and refinement.
DISCUSSION
The AMBH compounds, sharing a common indole core but differing in substituent groups, were selected based on the presence of halogen groups (-Br, -Cl, and/or -O) in their structure, which seemed to influence their anti-promastigote activity. The Craig plot used to analyze the correlation between electronic effects (?) and lipophilicity (?) of substituent groups (Giraud et al., 2019), indicated that halogens had positive electronic and lipophilic effects, while heterocyclic aromatic rings showed positive lipophilic effects, suggesting the importance of lipophilicity in their activity. However, further studies are required to understand their mechanism of action fully.
These AMBH compounds, due to their diverse structures, have limited studies explaining their mechanism of action (Lima-Junior; Vasconcellos, 2012). This study hypothesizes that they may cross the parasite's plasma membrane, potentially creating pores and inhibiting essential enzymes for the parasite's metabolism. The absence of toxicity in erythrocytes (except for HDM) is promising. Still, the observed hemolytic activity in HDM may be related to the strong reactivity of halogenated groups, inducing pores in the red blood cell membrane. Disruption of the mitochondrial environment might also be involved (Nemkov et al., 2020).
The AMBH compounds were effective against amastigote forms of L. infantum and it could be due to their affinity for the parasite's membrane and inhibition of common enzymes in both forms, such as endonucleases and DNA topoisomerases (Khan et al.Keywords
Comissão Organizadora
Francisco Mendonça Junior
Pascal Marchand
Teresinha Gonçalves da Silva
Isabelle Orliac-Garnier
Gerd Bruno da Rocha
Comissão Científica
Ricardo Olimpio de Moura
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