Application of Virtual Screening Methods for the Identification of Novel Antileishmanial Therapies: Rational Discovery of Synthetic Inhibitors Targeting Triosephosphate Isomerase

Abstract

Leishmaniasis is a neglected tropical disease of global relevance, and the emergence of parasite resistance highlights the urgent need for new therapeutic strategies. This study aimed to identify compounds with potential antileishmanial activity through virtual screening and in vitro validation. Seventy-seven Leishmania proteins were evaluated, leading to the identification of triosephosphate isomerase (TIM, PDB ID: 2VXN) as a promising therapeutic target. TIM was screened against more than 6,500 bioactive compounds, resulting in two potential inhibitors: ZINC9829539 and ZINC4270223. Molecular dynamics simulations revealed that both compounds established stable interactions with TIM, although with lower binding affinity compared to the crystallographic ligand phosphoglycolohydroxamic acid (PGH). In vitro assays were performed using THP-1-derived macrophages infected with intracellular amastigotes of Leishmania infantum, L. braziliensis, and L. amazonensis. The compounds exhibited cytotoxic concentrations between 15 and 30 µM, comparable to amphotericin B. Inhibitory concentrations ranged from 10.4-28 µM for ZINC4270223 and 22.1-26.6 µM for ZINC9829539. Selectivity indices ranged from 0.57-0.59 and 0.98-2.66, respectively, indicating limited parasite selectivity. Despite moderate efficacy, this study demonstrates the utility of virtual screening in identifying novel antileishmanial candidates. Further structural optimization may enhance the selectivity and potency of these compounds, making them more viable for therapeutic development against leishmaniasis.

Keywords:
antileishmanial therapy; virtual screening; in vitro assays

Introduction

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania, transmitted to humans through the bite of infected phlebotomine sandflies. More than 20 pathogenic species of Leishmania are known to infect humans, with Leishmania amazonensis and Leishmania braziliensis being the most prevalent and responsible for cutaneous leishmaniasis (CL), while Leishmania infantum causes visceral leishmaniasis (VL), also known as kala-azar.¹-³ CL is characterized by skin lesions that may or may not progress to ulcers, whereas VL affects internal organs such as the liver, spleen, and bone marrow, and can be fatal if left untreated.⁴

In the human host, Leishmania parasites survive and replicate within cells of the mononuclear phagocyte system, particularly macrophages, residing inside a structure known as the phagolysosome. This intracellular compartment is typically a hostile environment for pathogens, characterized by acidic pH and lysosomal enzymes responsible for degradation. However, Leishmania parasites have evolved adaptive mechanisms that allow them to survive and proliferate in this adverse environment, thereby reducing drug efficacy and contributing to treatment resistance.⁵ Currently, leishmaniasis is recognized as an expanding zoonosis, with an estimated 12 million people infected by different Leishmania species worldwide.²,⁶ This scenario underscores the urgent need for the development of more effective treatments.⁷

The current therapeutic options for leishmaniasis include pentavalent antimonials (SbV), such as meglumine antimoniate (glucantime), as well as amphotericin B and miltefosine. However, these drugs present significant limitations, including high toxicity, prolonged treatment regimens, species-dependent efficacy, and the emergence of resistant strains. For instance, the efficacy of SbV compounds, used since the 1940s, has declined in regions of high endemicity due to the selection of resistant parasites.⁸ Amphotericin B, while effective, is associated with severe nephrotoxicity. Miltefosine, the only available oral treatment, has also shown increasing reports of resistance.⁹

Given these limitations, the search for new drugs that combine greater efficacy with reduced toxicity is essential. In this context, drug repositioning has emerged as a promising strategy. This approach involves identifying new therapeutic applications for existing, approved drugs, aiming to expand their clinical indications more rapidly and at lower cost compared to the development of entirely new molecules.¹⁰ Drug repositioning can be guided by both virtual and experimental screening techniques.¹¹ The traditional drug development process-from discovery to market approval is lengthy, complex, and costly, often requiring years of research. In contrast, repositioning offers a significant advantage by accelerating this process, as many pharmacological and toxicological profiles of the repurposed compounds are already well established. This strategy is particularly relevant for neglected tropical diseases, especially in developing countries where resources for research and innovation are limited.¹²,¹³

Several successful cases of drug repositioning have been reported in the treatment of VL. Pentamidine and amphotericin B, for instance, were originally developed for the treatment of fungal infections but are now employed as therapeutic options against VL.⁸ Another notable example is miltefosine, which was initially designed as an antitumor agent and later became the first oral drug approved for the treatment of leishmaniasis.¹⁴

Virtual screening (VS) is a key tool in the rational design of drugs, employed to identify bioactive compounds from large chemical libraries.¹⁵ This computational approach enables the prediction of binding affinity between molecules and their biological targets, providing estimates of ligand interactions with the active sites of target proteins.¹⁶ VS can be applied using two main strategies: ligand-based virtual screening (LBVS) and structure-based virtual screening (SBVS).¹⁷ In LBVS, biological targets are not yet identified, and the selection of compounds is guided by shared structural features among known bioactive ligands. In contrast, SBVS involves docking compounds directly into the active site of known target proteins, using structural databases to predict molecular interactions.¹⁸

Comprehensive databases such as the Protein Data Bank (PDB), ChEMBL, and the ZINC database offer a wide range of bioactive compounds and structural information on proteins, which are essential for conducting effective virtual screenings.¹⁹-²¹ These platforms have been proven highly effective in identifying promising molecules, particularly for neglected tropical diseases such as leishmaniasis. For example, the PDB is a primary source of three-dimensional structures of biological macromolecules and their complexes with ligands, and is widely used in molecular modeling and structure-based virtual screening.¹⁹ The ZINC database offers free access to over 100 million commercially available compounds, enabling structure-based screening based on ligand properties and their potential for interaction with molecular targets.²¹

Another critical element in the search for new antileishmanial compounds is the application of molecular dynamics (MD) simulations. This computational technique enables the analysis of molecular systems at the atomic level over time, offering dynamic insights into ligand-target interactions. MD simulations allow for the assessment of complex stability, conformational flexibility of the interacting molecules, and persistence of intermolecular interactions, thereby contributing to the refinement and validation of virtual screening results.

Therefore, the present study aims to identify and validate new potential molecular targets in Leishmania through molecular docking studies, and to explore other compounds with high binding affinity to these targets. The synthetic compounds ZINC9829539 and ZINC4270223 were evaluated as potential drug candidates with antileishmanial activity, using medicinal chemistry approaches such as molecular docking and dynamics simulations. In addition, the in vitro antileishmanial activity of these compounds was assessed against intracellular amastigote forms of Leishmania braziliensis, L. amazonensis, and L. infantum.

Experimental

Selection and validation of molecular targets

Three-dimensional models of the molecular targets were selected from the PDB using the keyword “Leishmania.” To ensure specificity and quality, filters were applied to prioritize eukaryotic protein structures, with an emphasis on crystallographic resolution and the presence of ligands in the binding site of the protein. When multiple structures were available for the same target, the model with the highest resolution and presence of a co-crystallized ligand was selected.²²

To properly prepare the proteins for simulation, loop regions of the structures were reconstructed using the SWISS-MODEL platform, widely employed for homology modeling.²³ The protonation state of each protein was adjusted to reflect physiological pH, based on predicted subcellular localization using PSORT II and the optimal pH values retrieved from the BRENDA database.²⁴,²⁵ Protonation refinement and the addition of missing hydrogen atoms were performed using the H++ software (Virginia Polytechnic Institute and State University, Virginia, USA), which estimates pK_a values and adjusts protein structures to the intended experimental conditions.²⁶

Target validation was carried out through molecular redocking, followed by evaluation of the receiver operating characteristic (ROC) curve and the area under the curve (AUC)-metrics widely used to assess the predictive accuracy of docking models. These analyses were performed using AutoDock Vina (Forli Lab, The Scripps Research Institute, CA, USA), a program known for its high precision and speed in protein-ligand redocking.²⁷,²⁸

In the redocking process, crystallographic ligands were first removed from their binding sites and then re-docked using the same docking protocol. This step assesses the accuracy of the method by calculating the root mean square deviation (RMSD) between the heavy atoms of the re-docked ligand and its original crystallographic conformation. RMSD values below 2.0 Å are considered indicative of good reproduction of the native pose.²⁹

To ensure the sensitivity and specificity of the docking protocol, ROC and AUC analyses were performed. These metrics evaluate the ability of the method to distinguish between bioactive compounds and decoys-molecules with similar structures but lacking activity.³⁰,³¹ Active compounds were obtained from the ChEMBL database, while decoys were generated using the DUDE-E platform. Both datasets were docked into the active sites of target proteins using the MolAr platform developed by the research group.³²

A total of 77 crystallographic structures of Leishmania were selected from the PDB for molecular docking studies. Protein structures were prepared by reconstructing loop regions through sequence alignment with homologous proteins and adjusting their protonation states. Ions and water molecules were removed from the system, and hydrogen atoms were added to the three-dimensional structures. Molecular redocking was conducted using AutoDock Vina, based on the x, y, and z coordinates of the ligand binding sites.²⁷ The redocking procedure was validated using RMSD values.

Virtual screening and compound selection

VS was conducted based on the pharmacophoric properties of the crystallographic ligand. Structures from four different databases available on the Pharmit platform-totaling 6,589 compounds-were filtered using ADME (absorption, distribution, metabolism, and excretion) properties with the software DataWarrior v4.7.2 (Actelion Pharmaceuticals, Switzerland). These databases consist of compound structures that have been approved for commercial use. The binding energy of the compounds, following molecular docking, was analyzed and compared to that of the crystallographic ligand. Docking simulations were performed using AutoDock Vina, targeting the defined binding site of the triosephosphate isomerase (TIM) protein.

Starting from the TIM target protein and its crystallographic ligand PGH (phosphoglycolohydroxamic acid), molecular docking was performed to determine the reference binding energy between the target and the ligand, which was found to be -5.9 kcal mol^-1. The Pharmit database was used to search for compounds with pharmacophoric features similar to those of the PGH ligand, which served as a reference.³³ A key pharmacophoric feature of PGH is the presence of a nitrogen atom bonded to a hydroxyl group, which directly interacts with the Glu167 residue of the TIM active site.³⁴

Based on this information, the search parameters were refined, and screening was conducted in databases such as ZINC Purchasable, Aldrich, and DrugBank Approved. For the selected compounds, molecular docking was carried out within the binding site of the Leishmania TIM target using AutoDock Vina. The docking grid was carefully adjusted, and the exhaustiveness parameter was set to 24 to enhance the robustness of the docking procedure.²⁷,³⁵ RMSD analysis of the resulting binding poses was performed using Discovery Studio Visualizer, ensuring that the docked conformations were consistent with the crystallographic ligand.³⁶

The molecular docking simulations aimed to predict the interaction of ZINC database compounds with the active site of TIM (structure ID: 2VXN). The process involved preparing the ligand and target protein structures, followed by the definition of the search grid centered on the previously identified active site. Docking was executed using standard parameters, and the best conformations were selected based on the lowest predicted binding free energy. To validate the docking protocol, a redocking of PGH into the TIM active site was performed, yielding an RMSD value below 2 Å, which confirmed the accuracy of the employed docking model.

Molecular dynamics simulations

To evaluate the stability of protein-ligand complexes over time, molecular dynamics (MD) simulations were performed using GROMACS software version 2016.4 (University of Groningen, The Netherlands).³⁷ Simulations were conducted using the CHARMM36 force field and a TIP3P water box,³⁸ with the addition of neutralizing ions to simulate physiological conditions. Temperature was maintained at 310 K using the V-rescale thermostat,³⁹ and pressure was kept constant at 1 bar using the Parrinello-Rahman barostat.⁴⁰,⁴¹ The simulated ligands were modeled in their most probable protonation states at physiological pH. Further information on the distribution of protonation microspecies is provided in the Supplementary Information section. Each protein-ligand complex was simulated for 100 ns. Resulting trajectories were analyzed to determine the number of hydrogen bonds, ligand-binding site distances, interaction energies, and the stability of the ligand within its binding site. Occupancy-based volumetric maps were employed to evaluate the regions of the protein surface most frequently contacted by the ligands. These maps were generated using VMD,⁴² with occupancy values accumulated over the entire 100 ns trajectory, corresponding to 25,000 sampled conformations. The resulting frame-averaged fractional occupancies were subsequently standardized through z-score normalization. The z-score was defined in equation 1.

(1) $$z\text{-score}=\frac{(X-\mu )}{\sigma }$$

where X represents the fractional occupancy at a given grid point, μ denotes the mean occupancy across all grid points, and σ corresponds to the grid-wide standard deviation. Following visual inspection of a range of cutoff values to ensure both clarity and consistency in the volumetric representations across all ligands, a z-score threshold of 20 was selected and consistently applied in the analyses presented herein.

Cytotoxicity in THP-1-derived macrophages

To evaluate the cytotoxicity of compounds ZINC4270223 and ZINC9829539, human monocytic THP-1 cells (ATCC® TIB-202™) were cultured in T75 flasks containing Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U mL^-1 penicillin, and 50 µg mL^-1 streptomycin. Cultures were maintained in a 5% CO₂ incubator at 37 °C. Cells were seeded in 96-well plates (1 × 10⁶ cells mL^-1) and differentiated into macrophage-like cells by incubation with 50 ng mL^-1 phorbol 12-myristate 13-acetate (PMA) for 72 h. After differentiation, cells were treated with various concentrations of the test compounds for 72 h.

Cytotoxicity was assessed using the MTT assay,⁴³ which measures the reduction of yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) by mitochondrial enzymes into purple formazan crystals. The formazan was solubilized with 100 μL of dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a spectrophotometer (SpectraMax M5, Molecular Devices). Amphotericin B was used as a positive control. Data analysis was performed using GraphPad Prism 10,⁴⁴ applying nonlinear regression analysis to determine the compound sensitivity profile and calculate the CC₅₀, defined as the concentration that reduces cell viability by 50%.

Evaluation of antileishmanial activity

The antileishmanial activity of the compounds was evaluated against intracellular amastigote forms of Leishmania braziliensis, L. amazonensis, and L. infantum species circulating in Brazil and associated with various clinical manifestations of both cutaneous and visceral leishmaniasis. The intracellular amastigote form, which is clinically relevant and found within vertebrate hosts, was selected for the assay.

THP-1 cells, after PMA-induced differentiation, were infected with luciferase-expressing Leishmania promastigotes at a parasite-to-macrophage ratio of 10:1 for 3 h. Post-infection, cells were washed with HEPES/NaCl buffer to remove non-internalized parasites and treated with different concentrations of the test compounds for 72 h. Antiamastigote activity was measured by luminescence using the One-Glo™ Luciferase Assay System (Promega), and luminescence was read with a luminometer (SpectraMax M5). Antiparasitic activity was expressed as IC₅₀, the concentration required to reduce luminescence by 50% compared to untreated controls.

Selectivity index

The selectivity index (SI) was calculated to assess the selective antileishmanial activity of the compounds against parasites relative to host cells. SI is defined as the ratio of CC₅₀ to IC₅₀. A higher SI indicates greater selectivity, meaning the compound is more active against the parasite and less toxic to host cells. Elevated SI values suggest that the compound can be used at higher concentrations with reduced cytotoxicity to host cells.⁴⁵,⁴⁶

Results and Discussion

Virtual screening

Initially, the 77 selected structures were analyzed for the presence of a crystallographic ligand in the binding site, druggability score, and other parameters established by our research group. Following this preliminary assessment, molecular docking was performed on the five top-ranked protein structures. The docking protocol was validated through redocking of the crystallographic ligands, followed by evaluation of RMSD and AUC. Among the targets analyzed, two proteins were particularly notable: TIM (PDB ID: 2VXN) and a putative serine/threonine kinase (PDB ID: 1Y63) (Table 1). Both exhibited RMSD values below 2.0 Å and high AUC scores, indicating accurate reproduction of ligand binding conformations and strong predictive performance of the docking methodology. Among the previously crystallized proteins of Leishmania spp., the enzyme TIM was selected as the molecular target of choice (Figure 1), due to its direct involvement in the glycolytic pathway.

Table 1
RMSD and AUC values for the validation of molecular redocking of the selected structures

Protein (ID - PDB)	Protein name	RMSD / Å	AUC
1E7W	dihydroorotate dehydrogenase	2.02	0.47
1EVY	glutathione reductase	0.71	0.06
2VXN	triosephosphate isomerase (TIM)	1.50	0.75
3TJX	dihydrofolate reductase	1.90	0.25
1Y63	putative serine/threonine kinase	0.44	0.80

Figure 1
Structure of triosephosphate isomerase 2VXN (from PDB).⁴⁸

Leishmania TIM catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P). This reaction is a component of the glycolytic pathway and is essential for the survival of the parasite.³⁴ A total of 6,589 compounds were retrieved from the Pharmit platform based on the crystallized ligand of structure 2VXN (PGH). Of these, 4,633 compounds were excluded from the virtual screening due to violations of Lipinski’s rule of five, a widely used guideline for predicting oral bioavailability in drug candidates.⁴⁷

The crystallographic ligand PGH was used as a reference model for the virtual screening process. In a preliminary redocking analysis, PGH demonstrated a binding energy of -5.9 kcal mol^-1. This value was subsequently used as a reference threshold for the binding energies of the compounds identified through the virtual screening. The search for potential inhibitors on the Pharmit platform was based on the pharmacophoric features of PGH, including a negatively charged nitrogen atom bonded to a hydroxyl group, which is responsible for the interaction with the Glu167 residue of the molecular target. The screening was performed across four compound libraries available on Pharmit: DrugBankApproved-2019, AldrichCPR_1, AldrichCPR_2, and ZINC Purchasable.

To ensure a rigorous and effective selection of bioactive candidates, a binding energy cut-off of ≤ -8.0 kcal mol^-1 was applied. Based on this criterion, four compounds were selected: ZINC1884569 and ZINC4270223, both exhibiting a binding energy of -8.0 kcal mol^-1 and commercial prices of US$ 26 and US$ 58 per milligram, respectively; ZINC9829539, with a binding energy of -8.1 kcal mol^-1 and a cost of US$ 58 per milligram; and ZINC3818418, which demonstrated the most favorable binding energy of -8.5 kcal mol^-1, albeit at a significantly higher price of US$ 697 per milligram (Table 2). Commercial pricing information was obtained from the Mcule platform.⁴⁹

Table 2
Selected compounds with binding energy and commercial value

ID - ZINC	Binding energy / (kcal mol-1)	Value / (US$ per mg)
ZINC1884569	-8.0	26
ZINC4270223	-8.0	58
ZINC9829539	-8.1	58
ZINC3818418	-8.5	697

Following the screening of the synthetic compounds, only two were deemed eligible for continuation in the present study. The compound ZINC1884569 was excluded due to discontinued synthesis by its commercial manufacturers, and ZINC3818418 was excluded due to its prohibitively high cost (US$ 697 mg^-1), rendering it unsuitable for application in the context of a neglected tropical disease.

Thus, the synthetic compounds selected for further analysis were ZINC9829539 (SMILES: COc1ccc(cc1)C(=O)NCc2nnc(n2Cc3ccccc3)SCC(=O)NO), ZINC4270223 (SMILES: c1ccc(c(c1)C(=O)Nc2nnc(s2)SCC(=O)NO)F) and the crystallographic ligand (PGH are shown in Figure 2.

Figure 2
Compounds ZINC9829539 (a), ZINC4270223 (b) and phosphoglycolohydroxamic acid - PGH (c) (PubChem).⁵⁰

Molecular dynamics simulation

MD simulations performed for the compounds ZINC9829539 and ZINC4270223, in comparison with the crystallographic ligand PGH, revealed critical insights into their molecular interactions with the target protein 2VXN. The analysis focused on parameters such as the number of hydrogen bonds, ligand-binding site distance, interaction energy, and ligand stability within the binding site, providing a detailed evaluation of binding affinity and interaction stability at the atomic level (Table 3).

Table 3
Molecular interaction parameters of compounds ZINC9829539, ZINC4270223, and the crystallographic ligand PGH with the target protein 2VXN

Number of hydrogen bonds
	Minimum	Medium ± s.d.	Maximum
ZINC4270223	0	2 ± 1	5
ZINC9829539	0	1 ± 1	8
PGH	1	7 ± 1	10
Ligand-binding site distance / Å
	Minimum	Medium ± s.d.	Maximum
ZINC4270223	1.36	1.72 ± 0.20	3.09
ZINC9829539	1.44	1.97 ± 0.19	2.68
PGH	1.36	1.60 ± 0.07	1.96
Interaction energy / (kJ mol-1)
	Minimum	Medium ± s.d.	Maximum
ZINC4270223	-337.43	-195.09 ± 52.64	-30.76
ZINC9829539	-351.51	-207.55 ± 39.81	-61.98
PGH	-578.96	-455.52 ± 37.05	-292.72

The in silico results highlighted notable features regarding the interactions of ZINC9829539 and ZINC4270223 with the target protein, compared to the crystallographic ligand PGH. First, the analysis of hydrogen bonding revealed that PGH maintained an average of seven hydrogen bonds-significantly higher than the synthetic compounds. According to Sliwoski et al.,⁵¹ this suggests stronger and more stable binding, a finding consistent with previous studies⁵²,⁵³ demonstrating that a greater number of hydrogen bonds can enhance ligand affinity and specificity. Throughout the entire simulation time, PGH consistently maintained at least one hydrogen bond with the target, whereas the synthetic compounds displayed time points with no hydrogen bonding. Moreover, at the point of maximum interaction, PGH formed two more hydrogen bonds than ZINC9829539 and five more than ZINC4270223, reinforcing the stronger interaction profile of PGHs.

Between the two synthetic compounds, the average number of hydrogen bonds was similar. However, at the peak interaction moment, ZINC9829539 formed three additional hydrogen bonds compared to ZINC4270223. It is important to note the structural differences between PGH and the synthetic compounds, as ZINC9829539 and ZINC4270223 exhibit substantially greater conformational freedom. This flexibility may influence the number of bonds formed, ligand mobility within the binding pocket, and structural variability during the simulation.

Ligand-binding site distance is also a critical parameter. On average, all compounds maintained intermolecular distances within interaction thresholds.⁵¹ Comparative studies⁵⁴ support the relevance of shorter intermolecular distances for enhancing binding efficiency and, consequently, biological activity. This was particularly evident with ZINC4270223, which demonstrated a shorter average interaction distance than ZINC9829539, though it also exhibited greater variability and maximum distances, as indicated by the higher deviations observed in Figure 3. Nevertheless, higher deviations do not necessarily imply a reduced interaction capacity.

Figure 3
Ligand-binding site distance relative to the TIM target. The black graph represents the compound ZINC4270223, the red represents ZINC9829539, and the green graph corresponds to the crystallographic ligand PGH.

The interaction energy analysis revealed that PGH displayed the most favorable average interaction energy (-455.52 kJ mol^-1), at least twice as negative as the values observed for the synthetic compounds, suggesting a more stable and stronger binding interaction with the target.⁵³ Both ZINC9829539 and ZINC4270223 exhibited less negative interaction energies compared to PGH, potentially reflecting weaker binding stability (Figure 4). Previous studies⁵⁴,⁵⁵ have demonstrated that interaction energy is a reliable indicator of drug potency, with more negative values generally correlating with higher therapeutic efficacy. Among the synthetic ligands, both demonstrated comparable average interaction energies with the target protein. However, at the peak interaction moment, ZINC9829539 displayed an interaction energy twice as favorable as that of ZINC4270223, reinforcing its more interactive character observed previously.

Figure 4
Interaction energy whit the TIM target. The black graph represents the compound ZINC4270223, the red represents ZINC9829539, and the green graph corresponds to the crystallographic ligand PGH.

It is important to emphasize that the binding sites for each compound differed. Thus, despite ZINC4270223 presenting a less favorable interaction energy compared to ZINC9829539, both compounds achieved structural equilibrium during the simulations and may exhibit experimental results that align with these theoretical predictions.

Finally, the volumetric map illustrates the mobility of the ligands throughout the MD simulations by highlighting the regions of highest compound density. These regions, depicted in orange in Figure 5, correspond to the areas where the compounds remained most frequently during the simulation period. Among the simulated compounds, PGH displayed the smallest volumetric region, indicating the lowest spatial mobility throughout the simulation, followed by compound ZINC9829539, and then ZINC4270223. This observation aligns with the analyses of interaction energy, ligand-binding site distance, and number of hydrogen bonds, suggesting that stronger intermolecular interactions confer increased structural stability between interacting partners. Moreover, this supports the notion of differential binding affinities of the compounds for the active site of the protein. These findings are consistent with previous studies⁵⁶,⁵⁷ that emphasize the importance of low interaction energies and favorable volumetric distributions in the effectiveness of enzyme inhibitors.

Figure 5
Volumetric map displaying the spatial distribution of atomic probability densities for the synthetic compounds and PGH around the TIM protein, highlighting regions of highest interaction.

The MD simulations suggest that, based on the evaluated interaction parameters, the synthetic ligands exhibit relevant interaction profiles with the target protein, albeit not as pronounced as the crystallographic ligand. Among the two synthetic compounds, ZINC9829539 demonstrated the more favorable interaction profile. Overall, the results reveal promising characteristics that warrant further investigation into the development of novel antileishmanial therapies.

In vitro assays: toxicity in THP-1 macrophages and antileishmanial activity against intracellular amastigote forms

The results of the antileishmanial activity assays for the synthetic compounds ZINC9829539 and ZINC4270223 are presented in Table 4. The CC₅₀ represents the concentration required to induce cytotoxicity in 50% of THP-1 MΦ cells (macrophage-differentiated THP-1 cells). Both compounds exhibited relatively higher CC₅₀ values compared to amphotericin B, indicating lower cytotoxicity than the commercial drug. However, IC₅₀ values revealed that antiparasitic activity required elevated concentrations of the compounds, reflecting moderate or low potency. The SI ranged between 0.57 and 2.66 µM, indicating a narrow margin between the concentration required for antiparasitic activity and that which induces host cell cytotoxicity. For ZINC9829539, an SI below 1 suggests greater toxicity to macrophages than efficacy against the parasite. These data suggest that although ZINC9829539 and ZINC4270223 exhibit moderate antileishmanial activity, they possess concerning toxicity profiles, especially when compared to amphotericin B, which shows SI values ranging from 200 to 600-indicating high parasite selectivity and low host cell toxicity.

Table 4
Cytotoxicity (CC₅₀), antileishmanial activity against intracellular amastigotes (IC₅₀), and selectivity index (SI) of the tested synthetic compounds

Compound	CC50 / µM	IC50 amastigotes (IC 95%) / µM				SI
	THP-1 MΦ	L. braziliensis	L. amazonensis	L. infantum		L. braziliensis	L. amazonensis	L. infantum
ZINC9829539	15.42 (ND)	26.6 (20.3-36.4)	22.1 (16.8-29.6)	25.05 (18.6-35.0)		0.57	0.69	0.61
ZINC4270223	27.74 (ND)	10.4 (7.5-14.4)	12.1 (9.2-15.9)	28 (24.8-31.9)		2.66	2.28	0.98
Amphotericin B	13.25 (12.1-14.5)	0.06 (0.05-0.06)	0.075 (0.06-0.08)	0.02 (0.02-0.26)		220	176	662

A comparison with the study by Al-Qahtani et al.⁵⁸ reveals that both works investigated 1,3,4-thiadiazole derivatives in Leishmania models, albeit under different experimental conditions. While Al-Qahtani et al.⁵⁸ reported an IC₅₀ of 0.5 µM for a compound tested against L. donovani promastigotes, the current study found IC₅₀ values between 10.4 and 28 µM for ZINC9829539 and ZINC4270223 against intracellular amastigotes of L. braziliensis, L. amazonensis, and L. infantum. This comparison must be interpreted cautiously, considering differences in parasite species, evolutionary stages (promastigotes vs. amastigotes), and experimental protocols, all of which significantly influence antiparasitic parameters.⁵⁹,⁶⁰ Nevertheless, the efficacy of compounds with similar pharmacophores in different contexts reinforces the potential of this chemical class as a promising starting point for new antileishmanial drug candidates. The structural and functional diversity of thiadiazoles supports rational modulation of their bioactive properties, justifying continued investigation into improving their selectivity and therapeutic efficacy.⁶¹

Various mechanisms have been proposed for the antileishmanial activity of 1,3,4-thiadiazole analogs. Al-Qahtani et al.⁵⁸ suggested that these compounds may permeate the cellular plasma membrane, causing damage to nucleic acids and/or intracellular proteins. Furthermore, they highlighted the ability of these compounds to generate free radicals, particularly those containing dihydroxyphenyl groups. Variations in pharmacological activity between different Leishmania species are widely reported and may significantly affect the development of effective treatments.⁶² These differences may necessitate varying compound concentrations to achieve effective antiparasitic activity, partly due to species-specific susceptibility and potential acquired resistance mechanisms that render certain compounds ineffective at even high concentrations, complicating treatment.⁶³

Guedes et al.⁶⁴ demonstrated that the compound PK11195 exhibited antileishmanial activity against several Leishmania species, including L. amazonensis. The authors noted that effective concentrations were higher for L. amazonensis compared to other species. This observation aligns with findings in the present study, where ZINC9829539 and ZINC4270223 displayed higher IC₅₀ values against L. amazonensis than against L. braziliensis, suggesting lower sensitivity of the former. Such pharmacological potency differences may be due to factors such as membrane permeability, resistance mechanisms (e.g., efflux transporter overexpression, detoxification enzymes), and the genetic diversity among Leishmania species.⁶⁵ Given the association of L. amazonensis with more treatment-resistant clinical forms, such as diffuse and anergic cutaneous leishmaniasis, individualized therapeutic strategies are crucial. Species-specific diagnosis and evaluation of compound efficacy across different parasite isolates are essential tools for developing novel therapies.

Understanding structure-activity relationships (SAR) is fundamental for the development of new antileishmanial drug candidates. Although both ZINC9829539 and ZINC4270223 belong to the 1,3,4-thiadiazole class, they exhibited notable differences in cytotoxicity and antiparasitic activity, suggesting that subtle structural variations may significantly affect pharmacological profiles. ZINC4270223 demonstrated greater antileishmanial activity (lower IC₅₀ values against L. braziliensis and L. infantum) and lower cytotoxicity in THP-1 macrophages (higher CC₅₀), resulting in a more favorable selectivity index compared to ZINC9829539. Structural analysis revealed that ZINC4270223 contains two sulfur atoms in distinct positions and a fluorine atom on the side chain, potentially contributing to its improved performance. Halogens such as fluorine are frequently employed to enhance lipophilicity and metabolic stability, thereby facilitating cellular uptake and target interaction.⁶⁶ Moreover, molecules with multiple heteroatoms, such as sulfur and nitrogen, are known for their ability to form specific interactions with enzymatic targets in parasites, enhancing selectivity.⁶¹

Conversely, ZINC9829539, despite structural similarity, demonstrated higher cytotoxicity and lower selectivity, possibly due to non-specific interactions with host cell components or lack of functional groups that promote selectivity for parasitic targets. This difference highlights the importance of rational modulation of chemical substituents in the 1,3,4-thiadiazole core to optimize antiparasitic potency and host safety. These results support the thiadiazole class as a promising structural scaffold for new antileishmanial agents, guiding further structural and pharmacological optimization. Continued studies, particularly employing rational design strategies and pharmacokinetic property predictions, may significantly advance the development of safe and effective leishmaniasis treatments.

Understanding molecular mechanisms of action is crucial in the design of new compounds with selective antiparasitic activity. In this study, ZINC4270223 and ZINC9829539 were selected based on their predicted binding affinity for the active site of TIM, an enzyme essential for Leishmania glycolytic metabolism, catalyzing the interconversion of DHAP and G3P-a key glycolysis step.³⁴,⁶⁷ Despite their high predicted in silico affinity for TIM, the compounds demonstrated moderate in vitro IC₅₀ values and limited selectivity.

This discrepancy may result from various factors, including cellular permeability, intracellular stability, and the challenge of achieving effective concentrations within the phagolysosome, the intracellular compartment hosting amastigotes.⁵,⁶⁸ Evaluating antiparasitic activity in amastigotes is critical for validating compound efficacy, as this form is responsible for infection in vertebrate hosts. To act effectively, antileishmanial agents must overcome multiple barriers, including the host cell membrane and intracellular compartments.

Structurally, ZINC4270223, which contains a thiadiazole unit with two sulfur atoms, exhibited lower cytotoxicity and better selectivity than ZINC9829539, which features a denser aromatic core and potentially more reactive functional groups. The presence of electronegative atoms, such as fluorine in ZINC9829539, may contribute to its higher cytotoxicity due to increased non-specific host interactions, a phenomenon noted for fluorinated bioactive compounds.⁶⁹ Moreover, molecules with sulfur atoms may form more stable complexes with cysteine residues in the TIM active site, potentially explaining ZINC4270223’s higher binding affinity.⁷⁰

Nonetheless, the absence of significant in vitro activity may indicate that compound binding to TIM does not necessarily result in functional inhibition or parasite death, emphasizing the need for complementary functional assays to validate TIM as a drug target.⁷¹ The modest activity may also be due to metabolic pathway redundancy or functional compensation in Leishmania TIMs, as reported in other parasites.⁷¹,⁷² Therefore, structural refinements are necessary to improve specificity for TIM and enhance cellular penetration, combined with direct enzymatic inhibition evaluation.

These findings support further structural modifications based on specific interactions with catalytic residues in Leishmania TIM, such as Glu167 and His95, which are essential for catalysis.⁷³ Optimizing interactions with these residues could significantly improve compound potency and selectivity. In conclusion, the results reinforce the need to integrate virtual screening with biochemical and SAR studies to rationally advance the discovery of antileishmanial drug candidates.

Rodrigues et al.⁷⁴ highlighted the potency of mesoionic compounds, particularly 4-phenyl-5-(4’-methoxyphenyl)-1,3,4-thiadiazol-2-phenylamine, which exhibited notably low IC₅₀ values (2.7 and 2.8 μM) against L. amazonensis amastigotes, mirroring results in promastigote models and demonstrating consistent antiparasitic efficacy. In contrast, the synthetic compounds ZINC9829539 and ZINC4270223 demonstrated moderate activity against amastigotes (IC₅₀ = 10.4-28 μM), highlighting the greater complexity of targeting intracellular amastigotes compared to extracellular promastigotes, given the need to reach effective concentrations within the phagolysosome.

Therefore, antileishmanial compounds must exhibit significantly enhanced absorption and bioavailability to ensure efficacy against amastigotes. This challenge is supported by studies⁷⁵ emphasizing the structural modification of antileishmanial agents to improve host cell penetration and intracellular parasite access. A recent work⁷⁶ also highlights the importance of drug formulation in enhancing bioavailability and therapeutic efficacy against amastigotes.

SI analysis is critical for evaluating the relative toxicity of synthetic compounds toward host cells versus parasites. SI values below 1 indicate higher toxicity to mammalian cells than to intracellular parasites,⁷⁷ suggesting the compound may impair macrophage viability before exerting any leishmanicidal effect. Conversely, SI values above 10 denote greater specificity for the parasite, allowing administration at higher concentrations without significant host toxicity.⁷⁸ Rodrigues et al.⁷⁴ reported high SI values for mesoionic compounds that significantly reduced L. amazonensis infection rates and macrophage infection percentages, indicating therapeutic potential.

However, ZINC9829539 and ZINC4270223 exhibited limited selectivity in the present study, with SI values ranging from 0.57 to 2.66. Notably, ZINC9829539’s SI < 1 indicates a negative selectivity margin, i.e., greater host cell toxicity than antiparasitic efficacy. This represents a major barrier to their therapeutic application and emphasizes the need for structural optimizations to enhance antiparasitic specificity. Rational modification of functional groups-such as halogen substitution, sulfur atom variation, or lipophilicity adjustments-could significantly influence target binding and improve safety and selectivity. Continued research based on this rationale could yield more effective derivatives with improved toxicological profiles for treating leishmaniasis.

Based on the cytotoxic and antileishmanial activity data obtained for the synthetic compounds in this study, and supported by previous literature, several strategies can be proposed to optimize their development. Chemical and structural modifications to ZINC4270223 and ZINC9829539 may yield new derivatives with reduced cytotoxicity or enhanced antileishmanial activity.⁷⁹ Combination therapies may also potentiate therapeutic effects.⁸⁰ Furthermore, evaluating the bioavailability and pharmacokinetics of these compounds in animal models is crucial to guide effective therapeutic development.⁵ These preclinical studies are essential for realistically assessing therapeutic potential and providing valuable insights for the development of new treatments for leishmaniasis.

Conclusions

This study highlights the effectiveness of virtual screening methodologies in the identification of novel antileishmanial compounds targeting triosephosphate isomerase. Despite the moderate selectivity observed in vitro, the identified molecules represent promising starting points for chemical optimization. The integration of computational and experimental approaches reinforces the potential of structure-based drug discovery in the development of new therapies against leishmaniasis.

Supplementary Information

Supplementary information (protonation microspecies distribution) is available free of charge at http://jbcs.sbq.org.br as PDF file.

Acknowledgments

This work was supported by the Federal University of São João del-Rei, the Graduate Program in Health Sciences, the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Funding Code 001, and the Foundation for Research Support of the State of Minas Gerais (FAPEMIG) with financial support for the project (APQ-02358-18).

Data Availability Statement

All data required for the analysis and reproduction of the results presented in this study are fully documented in the text. Supplementary datasets derived from equipment-generated tables, which further support the findings, are available upon reasonable request. Interested researchers may contact the corresponding author, subject to applicable institutional or confidentiality restrictions. Formal requests for access to these additional data should be directed to the corresponding author.

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Edited by

• Editor handled this article:
  Paulo Augusto Netz (Associate)

Publication Dates

• Publication in this collection
  10 Nov 2025
• Date of issue
  2025

History

• Received
  19 Aug 2025
• Published
  15 Oct 2025