Leishmanicidal Activity of an In Silico-Screened Novel Inhibitor against Ascorbate Peroxidase of Leishmania donovani

Peroxidases are a heterogeneous family of enzymes that have diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defense system of Leishmania. In view of developing new and novel therapeutics, we performed in silico studies in order to search for a ligand library and identify new drug candidates and their physiological roles against promastigotes and intracellular amastigotes of Leishmania donovani.

ABSTRACT

Peroxidases are a heterogeneous family of enzymes that have diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defense system of Leishmania. In view of developing new and novel therapeutics, we performed in silico studies in order to search for a ligand library and identify new drug candidates and their physiological roles against promastigotes and intracellular amastigotes of Leishmania donovani. Our results demonstrated that the selected inhibitor ZINC96021026 has significant antileishmanial effect and effectively killed both free and intracellular forms of the parasite. ZINC96021026 was found to be identical to ML-240, a selective inhibitor of valosin-containing protein (VCP), or p97, a member of the AAA-ATPase protein family which was derived from the scaffold of N²,N⁴-dibenzylquinazoline-2,4-diamine (DBeQ), targeting the D2-ATPase domain of the enzyme. ZINC96021026 (ML-240) thus has a broad range of cellular functions, thought to be derived from its ability to unfold proteins or disassemble protein complexes, besides inhibiting the ascorbate peroxidase activity. ML-240 may inhibit the parasite’s ascorbate peroxidase, leading to extensive apoptosis and inducing generation of reactive oxygen species. Taken together, our results demonstrated that ML-240 could be an attractive therapeutic option for treatment against leishmaniasis.

INTRODUCTION

Oxidative stress is one crucial mechanism of host defense which macrophages employ to destroy foreign organisms. It protects the cells from pathogens by upregulating the antioxidant moieties. The host macrophage produces H₂O₂ to kill the invading parasites, such as Leishmania donovani, which in turn, detoxifies the effects of H₂O₂ through its unique redox enzyme, ascorbate peroxidase (APX). The parasite lacks catalase and glutathione (GSH) peroxidase, and hence, the removal or detoxification of H₂O₂ relies on the tryparedoxin pathway (1). It has been reported that the overexpression of ascorbate peroxidase in Leishmania major (Lm-APX) enhanced tolerance to mitochondrial dysfunction and apoptosis, mediated by oxidative stress (2). The deleterious effects of H₂O₂ in host macrophages were overcome by ascorbate peroxidase (APX), which suggested that it helps in the differentiation and survival of the parasites inside the host macrophages (3, 4). Hence, the importance of APX to the parasite’s survival as well as the absence of this enzyme in the human host makes it a potential drug target that could be exploited for therapeutic benefits. Inhibition of L. donovani APX (Ld-APX) by occupation of the ligand site (pocket) by a novel inhibitor may increase the chances of altering the parasite’s oxidative stress for escape mechanisms. Since the crystal structure of Ld-APX protein is not available in the Research Collaboratory for Structural Bioinformatics (RCSB), we modeled and screened it with the prepared ligand library for identification of new drug candidates. The selected compound showed better interaction with the protein as suggested by docking and molecular dynamics simulation studies.

The selected inhibitor ZINC96021026 was found to be identical to the commercially available drug ML-240, which inhibits p97 ATPase activity with a 50% inhibitory concentration (IC₅₀) of approximately 100 nM (5). ML-240 also blocks degradation of p97-dependent proteasome substrate with an IC₅₀ of approximately 900 nM (6). The AAA-ATPase p97 is a critical factor in maintaining protein homeostasis in eukaryotic cells, through its roles in promoting degradation of ubiquinated proteins by the proteasome and in maturation of auto-phagosomes (7, 8). ML-240 induced caspases 3 and 7, the executioner caspases in HCT15 and SW403 cells, and potently blocked the proliferation of these cells. The inhibitor function of ML-240 against p97 ATPase activity was made via high-throughput screening (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR). Our in silico study suggested that ML-240 inhibited the ascorbate peroxidase of Leishmania. Molecular dynamics simulation studies indicated that the compound can be a selective inhibitor of APX and inhibits the viability and proliferation of the promastigotes. Like the standard drug miltefosine, ML-240 caused loss of viability in promastigotes in a concentration-dependent manner. ML-240 was also effective in restricting the proliferation of the promastigotes and inhibited the growth of the parasites. ML-240 significantly reduced the intracellular amastigotes in RAW 264.7, human monocyte-derived macrophages, and downregulated the phagocytic index compared to the untreated control. Based on the computational data and experimental evaluation, we concluded that inhibitor ZINC96021026 (ML-240) acts as an antileishmanial agent.

RESULTS

Ligands screening against the generated Ld-APX structure.

The generated Ld-APX homology model is shown in Fig. 1A. The protein is depicted in red in the ribbon diagrams. The pocket-making residues are in blue in the stick model, shown with the help of PyMOL. The target Ld-APX protein also showed a much lower root mean square deviation (RMSD) value of 0.215 Å with its template (Lm-APX), suggesting similarity of the modeled protein (Ld-APX) with the template (Fig. 1B). The quality of the model was also checked through various analyses (Fig. 2). The Ramachandran plot depicts 90.8% amino acid residues in most favored regions, 9.1% residues in additionally allowed regions, and only a few residues in disallowed regions, which suggests that the generated model is of excellent quality (Fig. 2A). The ProSA Web server-based Z-scores of the target Ld-APX model were found to be –8.95, which was close to the experimentally solved template structure (PDB ID 3RIV_A) value of –8.97 (data not shown), suggesting that the obtained Ld-APX model is reliable. The knowledge-based energy profile of the target model was also generated, which showed the overall quality of the model (Fig. 2B and C). The topology of the generated model showed alpha helices, beta sheets, and beta turns which elaborate the detailed structural features. We also performed multiple sequence alignment between the target protein sequence, Ld-APX, and the template Lm-APX sequence (see Fig. S1 in the supplemental material).

FIG 1.

(A) Ribbon diagrams of the Ld-APX enzyme generated via homology-based modeling (red) with the predicted active site residues (in stick form and blue) making the binding pocket using PyMOL. (B) Superimposed structure of the target and its corresponding template enzyme.

FIG 2.

(A and B) Quality of the model as analyzed via Ramachandran plot (A), showing that most of the residues are in the allowed regions (B). (C) ProSA Web server image showing that the generated model is reliable.

The COFACTOR server predicted 3RIW_A (crystal structure of L. major peroxidase mutant C197T) as a related protein with a similar binding site for the target Ld-APX enzyme. It shows a confidence score (C-score) of 0.93, a TN-score of 1.00, RMSD of 0.0, identity of 0.955%, and coverage of 1.00×. The active site residues based on the ligand HEM interaction (as predicted by the COFACTOR server) were S27, L28, I29, R30, W33, P128, D129, G130, F141, L154, I155, A157, H158, C160, G161, E162, C163, H164, F167, S168, Y170, W174, L216, S218, F246, and F250. While visualizing Ligplot, HEM made hydrogen bonds with the E162, H164, and S168 residues and showed hydrophobic contacts with those residues, which were predicted by the COFACTOR server (Fig. S2). Those residues also have higher levels of similarity with the template, as shown in the multiple-sequence alignment (MSA) figure (Fig. S1A). It was reported that Cys197 and Trp208, which were conserved at positions C163 and W174 in the homology model, are important for the stability of the structure and function (9). This valuable information indicates that the ligands interacting with these predicted pocket residues may inhibit the action of the enzyme and possibly stall the proliferation of the parasites. The library of about 5,684 ligands of Sigma-Aldrich compounds was prepared for virtual screening as described previously (10, 11). The ligand 3D-MOL2 files were converted into pdbqt files and then subjected to virtual screening against the Ld-APX protein using AutoDock Vina.

Postscreening analysis.

A total of 21 screened compounds with high binding affinity were extracted for further analysis. These compounds showed binding affinity ranging from –13.1 kcal/mol to –11.0 kcal/mol and several hydrogen and hydrophobic bonds with the target protein (Ld-APX) (Table S1). We selected the top 10 compounds for our study based on high binding affinity, number of hydrogen bonds, and low molecular weight, which were ideal criteria for the selection of drug-like compounds (Table 1). The compounds with the lowest binding energies were extracted and aligned with the protein structure for analysis. The top docked complex between Ld-APX and ZINC96021026 showed a binding energy of –13.1 kcal/mol and hydrogen bond-making residues W33, H159, E162, and D219. It also made several hydrophobic contacts with the pocket, forming the residues mentioned above (residues predicted by the COFACTOR server). The screened compounds ZINC43763954, ZINC43061727, and ZINC14951505 have binding energy of –12.4 kcal/mol, –11.9 kcal/mol, and –11.7 kcal/mol, respectively. The docked complex of the top four screened compounds and their interaction plots generated via the Ligplot tool are shown in Fig. 3.

TABLE 1.

List of top 10 inhibitors and identification of the residues making different interactions^a

Compound ID	IUPAC name	Binding affinity (kcal/mol)	Hydrogen bond (≤3.6 A°)	Hydrophobic interactions
ZINC96021026	2-(2-amino benz imidazol-1-yl)-N-benzyl-8-methoxy quinazolin-4-amine	–13.1	W33, D219, H158, E162	F167, E62, G130, S168, Y170, S218, G63, D129, C163, W174, H164, G161, P26, C160, R30, I155, L216, H34, P128
ZINC43763954	(4S)-4-(2-phenyl ethyl)-2-[6-[(4S)-4-(2-phenylethyl)-4, 5-dihydro-1,3-ox azol -2-yl]pyridin-2-yl]-4,5-dihydro-1,3-oxazole	–12.4	D219, H158	F246, L253, W33, A157, L154, R30, I29, H164, C160, P26, G161, E162, W174, C163, Y170, L216, S168, P128, D129, G130, F141, S218, F250, I155
ZINC43061727	4-hydroxy-3-[(1S,3S)-3-[4-[[4-(trifluoromethyl)phenyl]methoxy]phenyl]-1,2,3,4-tetrahydronaphthalen-1-yl]chromen-2-one	–11.9	G125, L127, W33, H34	I155, L216, S218, F141, D219, V137, Y170, P128, D124, F167, R126, G130, E62, G63, D129, S168, E162, W174, C163, R30, H158
ZINC14951505	4-hydroxy-3-[(1R,3R)-3-(4-phenylphenyl)-1,2,3,4-tetrahydronaphthalen-1-yl]chromen-2-one	–11.7	W33, H34, S218, G130	H164, S27, C163, S168, R30, Y170, P128, G63, D129, I155, H158, F141, D219, L216, G161, W174, C160, E23, E162, P26
ZINC68203217	(3S,6R)-1-[6-(3-amino-1H-indazol-6-yl)-2-(methylamino)pyrimidin-4-yl]-N-cyclohexyl-6-methylpiperidine-3-carboxamide	–11.6	W33, H34, C160, E23	V137, I155, F141, S218, A157, I29, P26, W174, G161, S27, L24, I69, G66, F167, E162, C163, R30, H164, S168, P128, G130, L216
ZINC00601301	6-trifluoromethyl-3-benzyl-7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine 1,1-dioxide	–11.5	R30, H34, E162, C163	I155, L253, W33, F141, C160, S27, P26, H158, W174, S168, H164, L216, I29, A157, F246, L154, F250, W33
ZINC03817793	2-[[7-(3,4-dimethoxyphenyl)imidazo[1,2-c]pyrimidin-5-yl]amino]pyridine-3-carboxamide	–11.4	H34, L154, C160, H158	S168, P128, G161, G63, R30, W174, P26, I29, F250, A157, F246, L253, I155, W33, F141
ZINC02383031	4-hydroxy-3-[(1S,3R)-3-[4-[[4-(trifluoromethyl)phenyl]methoxy]phenyl]-1,2,3,4-tetrahydronaphthalen-1-yl]chromen-2-one	–11.3	W33, H34, S218, G130, S131, L202	F141, C163, P128, R30, W174, S168, G63, D219, F167, E162, V137, H158, L216, D219, I155
ZINC04098975	N-[1,5bis[4-(trifluoromethyl)phenyl]penta-1,4-dien-3-ylideneamino]-5,5-dimethyl-4,6-dihydro-1H-pyrimidin-2-amine	–11.2	P128, E162, R30	F167, H158, S168, L202, G63, E62, W170, G161, P26, R30, C160, W174, C163, H34, I155, W33, V137, S218, F141, D219, L216, G130
ZINC00603064	1-(2-bromophenyl)-3-(7-cyano-2H-benzotriazol-4-yl)urea	–11.0	W33, H158, E162	F250, L253, A157, F246, W174, G161, C160, P26, S27, C163, R30, S168, H164, I155, F141, L154

^aLigplot was used for the analysis.

FIG 3.

Postdocking analysis of docked complexes made between the Ld-APX enzyme and the top four compounds. (A) The Ld-APX enzyme is depicted as a cartoon model with brown color and the top screened inhibitor having ID ZINC96021026 is presented as a stick model in red color. (B) Second ranked inhibitor with ID ZINC43763954 is shown in green color inside the cavity of the modeled protein. (C) Inhibitor ZINC43061727 is depicted in blue color and (D) inhibitor ZINC14951505 is shown in purple color inside the cavity of Ld-APX enzyme. Ligplot was used for the representation of the interactions made by the different ligands with the Ld-APX protein.

Molecular dynamics simulation for system stability.

After the docking study, molecular dynamics simulations of all the systems were carried out for a period of 40 nanoseconds (ns). The stability of the backbone was measured using generated RMSD trajectories. The backbone RMSD of all the systems increased in the initial phase of the simulation (Fig. 4A). There was a deflection of around 0.19 to 0.23 nm in the RMSD value of the Ld-APX-ZINC43763954 complex of between 5,000 and 19,000 picoseconds (ps). Further, the plateau proceeded and increased up to 0.30 nm. The highest fluctuation, of 0.33 nm at 39,800 ps, was observed in this system, after which the fluctuation converged at 0.28 nm at 40 ns (Fig. 4A, green line).

FIG 4.

Molecular dynamics simulation analysis during a time span of 40 ns. (A) RMSD plot as a function of time. (B) RMSF of backbone atoms of the apo Ld-APX enzyme and all the docked complexes. (C) Rg values of all the systems. (D) The average distances between the Ld-APX protein and all four inhibitors are plotted against time. Please refer to the supplemental material for color interpretation.

The Ld-APX-ZINC43061727 complex (shown in blue in Fig. 4A) attained the RMSD value of 0.20 nm at 1,730 ps. It then dropped and continued to show deflection at around 0.1 nm. Finally, it converged to 0.22 nm at 40 ns. Similarly, the Ld-APX-ZINC14951505 complex (shown in purple) attained the RMSD value of 0.24 nm at 3,620 ps. Then it dropped a little and continued to maintain its deflection at around 0.20 nm. Later, its RMSD peak reached 0.28 nm at 13,522 ps. Finally, the plateau proceeded and converged at 0.22 nm (Fig. 4A). The complex made by the ligand with the highest binding affinity (Ld-APX-ZINC96021026 complex) is shown in red. The RMSD value of this complex increased initially to 0.20 nm and dropped thereafter, and later it increased to 0.22 nm at 5,134 ps. After that, it continued to maintain the plateau and finally converged at 0.20 nm at 40 ns. The apo protein (Ld-APX) did not show many changes in backbone RMSD values compared to the top four complexes as described above. It varied between 0.15 nm and 0.19 nm up to 27,000 ps. It attained the highest deflection of 0.26 nm at 34,580 ps, and finally, the apoprotein simulation system had an average RMSD of 0.24 nm (Fig. 4A).

The conformation of the protein structure is determined by its amino acids. Any change in the amino acid sequence by methods such as chemical or mechanical methods, may lead to changes in its structure. The interaction of ligands with the active site residues may bring changes in the protein structure and activity. This inhibitor-induced movement during inhibitor binding leads to its conformational changes, which also alter its biological function (12). It is critical to be acquainted with the changes in protein structure in a structure-based drug design study. The average atomic mobility of protein backbone (N-Cα-C) during the molecular dynamics simulation was measured using RMSF. To understand the structural dynamics of all the systems, RMSF was calculated from 40-ns molecular dynamics (MD) trajectories (Fig. 4B). It can be seen from the RMSF plot that the Ld-APX-ZINC96021026 complex attained an overall lower RMSF than other complexes and apo proteins. This suggested the stable binding of the ZINC96021026 inhibitor. Figure 4B shows that the residues interacting with the ZINC96021026 inhibitor (residues discussed in docking studies) have low flexibility compared with other complexes and apo protein fluctuation data. The amino acids E162 (0.07 nm), H164 (0.07 nm), and S168 (0.10 nm) were conserved and demonstrated low mobility in the top docked complex (Ld-APX-ZINC96021026). The residues which are important for stability and functioning of APX protein showed little fluctuation in the Ld-APX-ZINC96021026 complex (C163 [0.07 nm] and W174 [0.06 nm]) compared with apo protein residues (C163 [0.10 nm] and W174 [0.09 nm]). These residues participated in making hydrophobic interactions during docking (Table 1). Therefore, involvement of these residues in docking as well as during simulation is critical for drug design. It was clear from the docking study that the amino acids W33, G125, G130, H158, and S218 make hydrogen bonds with the inhibitors in almost all docked complexes. The RMSF plot showed that the amino acids W33 (0.05 nm), G125 (0.14 nm), G130 (0.07 nm), H158 (0.05 nm), and S218 (0.05 nm) attained and demonstrated low fluctuations in the Ld-APX-ZINC96021026 complex compared to apo protein values (W33 [0.06 nm], G125 [0.15 nm], G130 [0.16 nm], H158 [0.08 nm], and S218 [0.09 nm]) and the rest of the three complex systems. Another complex, Ld-APX-43763954, showed the RMSF values of these residues as 0.07 nm (W33), 0.25 nm (G125), 0.09 nm (G130), 0.07 nm (H158), and 0.08 nm (S218), which were higher than the ZINC96021026 inhibitor data. Similarly, the rest of the two complex systems acquired higher fluctuations than the Ld-APX-ZINC96021026 complex and apo protein system at several amino acid positions, which suggested that the inhibitor ZINC96021026 formed a stable complex compared with the other compounds.

The stability of the protein ligand complex depends on the compactness of the system. The radius of gyration (Rg) is a well-known measure of the compactness of these generated systems (13). This parameter is associated with the volume of the protein tertiary structure and has been applied to study the molecular insight into the stability of the protein in biological environments. The average Rg values for Ld-APX, Ld-APX-ZINC96021026, Ld-APX-ZINC43763954, Ld-APX-ZINC43061727, and Ld-APX-ZINC14951505 were found to be 1.78 nm, 1.72 nm, 1.76 nm, 1.74 nm, and 1.75 nm, respectively. The apo protein is expected to show a higher Rg value because of loose packing. The results shown in Fig. 4C suggested that the Ld-APX protein attained tight packing and a more stable structure following binding with the ZINC96021026 compound. The inhibitor ZINC43763954 exhibits different gyration from the rest of the compounds and reflects unstable Rg deviations compared with other systems (Fig. 4C).

The average distance between all four inhibitors and the Ld-APX protein was calculated from the trajectories of a 40-ns MD simulation. The Ld-APX-ZINC96021026 complex showed the lowest distance value (0.55 nm) compared to the rest of the three complexes in the distance plot (Fig. 4D). In the Ld-APX-ZINC43763954, Ld-APX-ZINC43061727, and Ld-APX-ZINC14951505 complexes, the distances between ligands and protein were found to be 0.65 nm, 1.14 nm, and 0.71 nm, respectively. Therefore, it was observed that the compound ZINC96021026 was highly selective toward the target protein (Ld-APX). A hydrogen bond provides specificity and directionality of interaction, which is a basic aspect of novel molecule detection (14). Hydrogen bonds between the target protein and docked ligands were computed to access the stability of the complex systems. In the Ld-APX-ZINC96021026 complex system, a maximum of two hydrogen bonds and a minimum three to four hydrogen bonds were formed during the 40-ns time period. In terms of ZINC43763954, the Ld-APX-ZINC43763954 complex formed a maximum of two hydrogen bonds. In the Ld-APX-ZINC43061727 complex, a maximum of two and a minimum of three or four hydrogen bonds were observed. The Ld-APX-ZINC14951505 complex was expected to form four hydrogen bonds, but it showed a minimum of five hydrogen bonds at the end of the simulation. It was observed that all four compounds bonded to protein pockets with two to four hydrogen bonds throughout the simulation (Fig. S3).

Docking and MD simulation of the Ld-APX-miltefosine complex.

We used miltefosine as a control drug in the experimental study, and docking of miltefosine was performed using the same docking protocol as that adopted for the screening study. We observed that the miltefosine has very low binding affinity (–5.9 kcal/mol) compared with the top screened inhibitor, ZINC96021026. The interaction with pocket residues of Ld-APX by miltefosine was also unsatisfactory, which suggests that the selected inhibitor (ZINC96021026) has better binding potential to the target Ld-APX enzyme (Fig. S4A). MD simulation analysis showed that miltefosine was not able to interact strongly with Ld-APX. The average RMSD value of miltefosine was converged at 0.26 nm, while the apo protein trajectory converged at 0.24 nm after 40 ns. The Ld-APX-miltefosine complex system also attained higher fluctuation than apo protein. These results suggested that the Ld-APX-miltefosine complex was unstable (Fig. S4B).

Effect of ZINC96021026 against promastigotes and intracellular amastigotes.

We assessed the effect of the top screened inhibitor, ZINC96021026, on the viability and growth inhibition of promastigotes. Following short-term treatment with ZINC96021026, promastigotes of L. donovani significantly lost cell viability as assessed by the XTT assay. A short-term viability study was undertaken to demonstrate the immediate effect of the compound on the promastigotes following treatment (Fig. 5A). Our data suggested that cell viability of the promastigote was compromised in the presence of increasing concentrations of ML-240 (Fig. 5A). ML-240 was found to be growth inhibitory to promastigotes in long-term culture as assessed by the MTT assay. Following exposure to ML-240 (100 μM), the promastigote growth inhibition was 90%, compared to 100% in miltefosine on day 2 (Fig. S5). The patterns of growth inhibition remained similar for days 4 and 6; however, at day 8, the parasites started to grow again, apparently due to the metabolism of the drug (Fig. S5). Starting with initial inoculums of 2 × 10⁶ million cells, the growth of the promastigotes reached to 9 × 10⁶ million over a period of 8 days, which was significantly higher than the treatment with 100 μM ML-240 (P < 0.001). Upon treatment with ML-240, the number of promastigotes plummeted to less than 100,000/ml after 2 days (P < 0.001). At lower concentrations, ML-240 was significantly leishmanicidal, similar to miltefosine (P < 0.01 and P < 0.001, respectively) (Fig. S5). ML-240 at a 100-μM concentration reduced the parasite growth to 98%, 95%, 88%, and 79% on days 2, 4, 6, and 8, respectively (P < 0.001) (Fig. S5). Miltefosine treatment nearly wiped out the promastigotes in culture on day 2, and they had not recovered on day 6, when the last reading was recorded. ML-240 treatment was significantly effective against the promastigotes; however, the parasite tends to return in culture after day 8, suggesting the metabolic degradation of the compound after long-term exposure in culture medium. Our data suggested that ML-240 is extremely potent in antileishmanial activity, similar to miltefosine (Fig. S6). Besides promastigotes, ML-240 was found to be equally potent in inhibiting the growth of intracellular amastigotes in infected RAW 264.7 macrophages with efficacy similar to miltefosine (Fig. 5B). The antiamastigote effects of ML-240 were assessed and were compared with miltefosine with respect to the percentage of infected macrophages and the number of amastigotes per 100 macrophages (Fig. 5C and D). We also quantitated the 50% and 90% effective concentration (EC₅₀ and EC₉₀) values of ML-240 against the promastigotes at different hours of treatment showing significant leishmanicidal effects (Fig. 5E). The EC₅₀ of ML-240 was also assessed against the amastigotes and RAW 264.7. Compared to miltefosine, ML-240 was somewhat less leishmanicidal with respect to its effect (EC₅₀) against the promastigotes and amastigotes (Fig. 5F).

FIG 5.

(A) Viability of promastigotes in the presence of various concentrations of ML-240 for an 18-h XTT assay. (B) Photomicrographs of leishmanicidal effects of increasing concentrations of miltefosine or ML-240. (C and D) Assessment of the percentage of infected macrophages and amastigotes/100 macrophages in the presence of increasing concentrations of (C) miltefosine or (D) ML-240 following coculture for 2 days. (E) Measurements of EC₅₀ and EC₉₀ of ML-240 against the promastigotes of L. donovani for extended periods of time. (F) Measurements of EC₅₀ for miltefosine or ML-240 against promastigotes and amastigotes and toxicity against RAW 264.7 cells. Data are presented as the mean ± SD; n = 5.

In addition to RAW 264.7, ML-240 significantly reduced the intracellular amastigotes in human monocyte-derived macrophages obtained from the peripheral blood (Fig. S7). Following treatment with ML-240 (50 to 200 μM), the percentage of infected macrophages significantly decreased compared with the untreated control (P < 0.001) (Fig. S7A). Treatment of infected macrophages with 50 and 200 μM ML-240 showed significant differences in the percentage of infected macrophages with enhanced killing in the higher concentration compared to the lower concentration tested (P < 0.001) (Fig. S7A). The percentage of infected macrophages decreased from 53% (control) to 11% in the highest concentration tested (200 μM). The number of amastigotes/100 macrophages decreased from 168.43 ± 23.67 in the control group to 30.42 ± 9.05 at the highest concentration tested (200 μM) (P < 0.001) (Fig. S7A). Significant differences were also observed between the highest (30.42 ± 9.05 in 200 μM) and lowest (72.51 ± 7.81 in 50 μM) concentrations tested (P < 0.001) (Fig. S7A). Significant leishmanicidal activity was also reflected in the phagocytic index between the control group (86.76 ± 18.57) and the treated groups (21.03 ± 3.26, 8.14 ± 3.19, and 3.47 ± 1.83 for 50, 100, and 200 μM ML-240, respectively) (P < 0.001) (Fig. S7B). Miltefosine was used as a positive control for the experiment in order to evaluate the leishmanicidal potential of ML-240. The percentages of infected macrophages were reduced to 11.48 ± 7.06 and 4.61 ± 1.27 in 50 and 100 μM miltefosine, respectively, compared to 51.45 ± 5.21 in the untreated control (P < 0.001) (Fig. S7C). The numbers of amastigotes/100 macrophages were reduced from 168.43 ± 23.57 in the control group to 30.89 ± 19.62 and 8.86 ± 4.73 in groups treated with 50 and 100 μM miltefosine, respectively (P < 0.001) (Fig. S7C). The phagocytic index was reduced from 86.76 ± 18.57 in the control untreated group to 4.45 ± 4.17 and 0.41 ± 0.27 in the groups treated with 50 and 100 μM miltefosine, respectively (P < 0.001) (Fig. S7D). We also studied the cytotoxic potential of ML-240 against various human and murine cell lines and determined the IC₅₀ values for individual treatment. ML-240 demonstrated significant cytotoxicity, similar to that of miltefosine at all the concentrations tested (Fig. S8). The effect of miltefosine or ML-240 against the peripheral blood lymphocytes, monocytes, and red blood cells (RBC) was also studied for the toxicity assessment (Fig. S9). Results suggested that both the drugs have significant levels of cytotoxicity against the mononuclear cells (Fig. S9A and B) but are relatively tolerant to RBC at lower concentrations (Fig. S9C). Higher concentrations of the compounds caused notable lysis of the RBC.

Apoptosis in promastigotes by ML-240.

We assessed the apoptosis of the promastigotes using the TUNEL assay, determining the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick end labeling for breaking of DNA following treatment with miltefosine or ML-240. TUNEL-positive cells increased from 9% to 33% following treatment with ML-240 (50 μM), indicating apoptosis of the parasites (Fig. 6). Similar observation was made with miltefosine (Fig. 6). Quantization of the TUNEL-positive cells was performed, and the effect of ML-240 was compared with the similar treatment with miltefosine (Fig. 6H). The DNA degradation pattern suggested breaking of DNA in the presence of increasing concentrations of miltefosine or ML-240. Amphotericin B (10 μM) was used as a positive control in order to demonstrate the comparable effects (Fig. 6I).

FIG 6.

(A to G) TUNEL staining of promastigotes in the presence of increasing concentrations of miltefosine or ML-240. (H) Quantification of TUNEL-positive cells in the presence of the indicated treatment. (I) Degradation pattern of genomic DNA of promastigotes in the presence of the indicated treatment. Data are presented as the mean ± SD; n = 3.

We also performed annexin V staining in the promastigotes following treatment with either miltefosine or ML-240. Promastigotes of Leishmania lack phosphatidylserine but mimic mammalian apoptotic cells by exposing phosphatidylserine (PS) at the cell surface to trigger the phagocytic uptake into host macrophages (15). Our results suggested that the promastigotes are not annexin V-positive, although the promastigotes demonstrated significant levels of TUNEL positivity as presented above (Fig. S10).

Comparative ROS generation in promastigotes treated with ML-240.

Intracellular localization of reactive oxygen species (ROS) generation was demonstrated following dual staining with CellROX Green and MitoTracker Red CMXRos in promastigotes that were untreated (Fig. 7A) or treated with 10 μM miltefosine (Fig. 7B) or 10 μM ML-240 (Fig. 7C) or 25 μM ML-240 (Fig. 7D). The data suggest the occurrence of long ramified staining in the promastigotes, which indicated mitochondrial involvement. Quantitative estimation indicated a concentration-dependent surge in ROS activity in the presence of miltefosine or ML-240 (Fig. 7E and F). Intracellular generation of ROS in promastigotes was determined following treatment with increasing concentrations of miltefosine or ML-240 (Fig. 7G and H). The data suggest that the ROS generation increases significantly in the presence of 50 μm ML-240 compared to the untreated control. In addition, ATP levels in ML-240-treated promastigotes were drastically reduced compared to those in untreated promastigotes in a concentration-dependent manner (Fig. S11). Promastigotes treated with sodium azide (NaN₃) act as a positive control for the indicated experiment (Fig. S11).

FIG 7.

Assessment of mitochondrial ROS generation in promastigotes following the indicated treatment. (A) Untreated; (B) miltefosine (10 μ); (C) ML-240 (10 μ); (D) ML-240 (25 μM) (magnification ×1,000). ML-240 triggers ROS production and increases mitochondrial oxidative stress in L. donovani promastigotes. Following exposure to ML-240 or miltefosine, the cells were stained with CellROX Green and fixed to measure the oxidative stress level. Green signals indicate the presence of oxidative stress in promastigotes. (iii) The percentage of CellROX-positive cells was quantified on 10 random fields per treatment group. (ii) Cells were stained with MitoTracker Red CMXRos to detect mitochondrial ROS (red). (E and F) Flow cytometric determination of intracellular ROS generation in L. donovani promastigotes following treatment with (E) miltefosine or (F) ML-240 using CellROX Green reagent. (G and H) Time-dependent quantization of ROS generation in the presence of various concentrations of (G) miltefosine or (H) ML-240 using CellROX Green. Data are presented as the mean ± SD; n = 5.

Inhibition and estimation of ascorbate peroxidase activity in promastigotes.

We also assessed the ascorbate peroxidase enzyme activity in promastigotes following treatment with increasing concentration of ML-240. A significant drop in time- and concentration-dependent enzyme activity was observed following treatment with ML-240 (Fig. 8A). APX enzyme activity was measured in promastigotes following treatment with either miltefosine or ML-240. Miltefosine treatment did not have a significant effect on the APX activity, while ML-240 treatment reduced the APX activity significantly (Fig. 8B). To determine the inhibitory effect of ML-240, different concentrations of ML-240 (10, 25, and 50 μM) were incubated with a reaction mixture containing 10 μl enzyme extract. Upon addition of H₂O₂, all three concentrations of the inhibitor showed significant inhibition of ascorbate oxidation at 290 nm. The inhibition kinetics of various concentrations of ML-240 was studied by measuring the initial rate of reaction at a fixed inhibitor concentration against various concentrations of the substrate (0 to 500 μM). The mode of enzyme inhibition was determined by fitting experimental values to a Lineweaver-Burk plot. The V_max and K_m values of Ld-APX in the absence of inhibitors were found to be 1.09 μM · min⁻¹ and 62.19 μM, respectively. ML-240 illustrated the enzyme inhibition in a noncompetitive manner, as evaluated by the K_m and V_max values, as shown in Fig. 8C to E. The K_i values of the ML-240 were determined by fitting the data to equation 1 and are as follows: inhibitor, ML-240; substrate, H₂O₂; type of inhibition, noncompetitive; K_m, 62.19 μM; V_max, 1.099 μM · min⁻¹; K_i, 20.39 μM.

$$V_{\text{max}}^{+I}=\frac{V_{\text{max}}^{-I}}{\text{(1+}\frac{I}{K_i}\text{)}}$$ (1)

FIG 8.

(A) Spectrophotometric determination of ascorbate peroxidase activity in the presence of increasing concentrations of ML-240. (B) Comparative analysis of ascorbate peroxidase activity in promastigotes following treatment with miltefosine or ML-240. (C to E) Inhibition mechanism and binding mode of ML-240. The initial enzyme activity of APX was measured as a function of the concentration of H₂O₂ at a fixed concentration of inhibitors. ML-240 acts in a noncompetitive manner as shown in the Lineweaver-Burk plot. (F) Measurement of intracellular H₂O₂ production in promastigotes by a luminescence-based assay in the presence of increasing concentrations of ML-240. Data are presented as the mean ± SD; n = 5.

Here, V_max^+I and, V_max^–I represent the V_max in the presence and absence of inhibitor, respectively, where I is the concentration of inhibitor used and K_i is the inhibitory concentration. H₂O₂ production surged following treatment with increasing concentrations of ML-240, leading to the apoptosis and death of the promastigotes (Fig. 8F).

In vivo susceptibility of Leishmania donovani to ML-240.

Antileishmanial activity of ML-240 against established L. donovani infection in BALB/c mice was determined and compared with that of miltefosine. Our data indicate that ML-240 has a leishmanicidal effect against intracellular amastigotes in infected cells similar to that of miltefosine. At two different concentrations (5 and 10 mg/kg of body weight), ML-240 reduced the body weight of infected animals to a degree similar to the effect of miltefosine (Fig. 9A and B). Spleen and liver weights in treated animals were reduced significantly compared to those of untreated control animals (infected control) (Fig. 9C to F). Intracellular amastigotes in spleen and liver stamp smear preparation from treated animals showed significant reduction following treatment with ML-240 (Fig. 9G and H). Quantitation of L. donovani parasites (LD bodies; LDU) in spleen showed significant reduction between the infection control and the treated groups (P < 0.001) (Fig. 9I). ML-240 at a higher concentration (10 mg/kg body weight) was significantly more leishmanicidal than miltefosine (5 mg/kg body weight) (P < 0.1) (Fig. 9I). A similar observation was made for liver (Fig. 9J). We also showed the histopathological outcome of the liver biopsy following treatment with the indicated drugs. Our data suggest that ML-240 and miltefosine dramatically reduced the inflammatory infiltrates in the periportal areas of the liver compared with the infection control, suggesting significant therapeutic benefits of ML-240 (Fig. 9K). Altogether, our data suggest that ML-240 has significant potential as a therapeutic choice against L. donovani infection, comparable to that of miltefosine.

FIG 9.

Antileishmanial efficacy of ML-240 in established L. donovani infection. Three-week-postinfected BALB/c mice were left either untreated (infection control) or treated intraperitoneally with ML-240 (5 or 10 mg/kg body weight) or miltefosine (5 mg/kg body weight) on alternate days for 10 days as described in Materials and Methods. (A) Phenotypic illustration of hepatosplenomegaly in L. donovani-infected BALB/c mice with enlarged abdomen. (B) Body weights of L. donovani-infected animals compared to treated groups. (C and D) Enlargement of (C) spleen and (D) liver in L. donovani-infected BALB/c mice compared to the control group 10 days posttreatment. (E and F) Weight of (E) spleen and (F) liver in the infected control compared to treated groups. (G and I) Splenic and (H and J) hepatic parasite burdens (LDU) were determined by counting the parasites in stamp smears (magnification, ×1,000). (K) Histopathological alterations in the paraffin section and hematoxylin and eosin (H&E)-stained section in the liver of L. donovani-infected BALB/c mice after treatment with either miltefosine or ML-240. Microscopic observation of inflammatory infiltrates in the periportal areas of liver sections of (i) noninfected animals, (ii) the untreated infected group, (iii) infected animals treated with 5.0 mg/kg/day of miltefosine, and infected animals treated with (iv) 5 mg/kg/day and (v) 10 mg/kg/day of ML-240. Magnification, ×400. Data are presented as the mean ± SE for five animals per group. Data were tested with ANOVA. Differences between the means were assessed for statistical significance with Tukey’s test (**, P ≤ 0.01; ***, P ≤ 0.001). Results are from one of three representative experiments.

DISCUSSION

Chemotherapy against leishmaniasis is still the main treatment option, although it is plagued by ever-increasing drug resistance. Increased drug resistance of the disease in various countries, including India, specifically against pentavalent antimony (SbV)-based compounds, has caused concern. Several mechanisms of resistance to these drugs have been reported, including reduced drug reduction/activation, decreased uptake, and an increased efflux/sequestration of active molecules. In addition, gene amplification and higher activity of repair mechanisms due to the drug-induced damage also play significant roles. Thus, finding a new drug with unique properties could provide new breakthroughs and free the immune system and body physiology from the shackles of failure which obstruct the therapeutic success. This becomes more urgent since no vaccine or alternative therapy is available. In kinetoplastids such as Trypanosoma, Leishmania, etc. catalase and glutathione (GSH) peroxidase are absent, and removal of the hydroperoxidase in these parasites relies on the tryparedoxin pathway to regulate oxidative stress (16, 17). Ascorbate peroxidase (APX) comprises a key component in the glutathione ascorbate cycle. Glutathione is reported to maintain the reducing environment in the cells and thus maintain the reduced state of the many cellular components (18, 19). A single-copy L. major ascorbate peroxidase gene reported to perform a critical role in the detoxification of H₂O₂ was generated via endogenous processes on account of external interferences such as oxidative burst of the infected macrophages or drug metabolism by the parasite (20). Biosynthesis of ascorbate in kinetoplastids takes place within the glycosomal compartment. Treatment with aminotriazole or sodium azide, an inhibitor of heme-containing enzymes, e.g., catalase and peroxidase, hinders the removal of H₂O₂ from amastigotes (17). It has been reported that overexpressing ascorbate peroxidase in L. major promastigotes enhanced tolerance to apoptosis, mediated by oxidative stress. APX, overexpressed in the mitochondria of L. major (Lm-APX), protects the parasites from the detrimental effects of oxidative stress, which includes mitochondrial dysfunction, cell death, and cellular redox equilibrium (21). Knockdown of the APX gene subjected the parasite to constant exposure to oxidative stress, causing elevated intracellular H₂O₂ content (21). Loss of the APX gene in L. major could also lead to secondary effects on gene expression, including lipophosphoglycan (LPG) and metacyclogenesis, which likely were instigated by changes in the redox equilibrium in the parasites. Our in silico search for a new and novel inhibitor against the APX of L. donovani revealed that valosin-containing protein (VCP), or p97, a member of AAA-ATPase protein inhibitor ML-240, could be a potential choice for leishmanicidal activity. The AAA-ATPase protein family is associated with a variety of cellular functions, including endoplasmic reticulum-associated degradation (ERAD), reassembly of the Golgi membrane, DNA repair, cell division, and autophagy (22–25). Our results demonstrated that ML-240 inhibits the ascorbate peroxidase of promastigotes, resulting in apoptosis and killing of the parasites. ML-240 enhanced the ROS generation in treated promastigotes, involving a major role of mitochondria. Besides promastigotes, the p97 inhibitor also showed leishmanicidal activities against intracellular amastigotes. ML-240 treatment also significantly affected the ultrastructure of the promastigotes and dramatically reduced the ATP levels. Treatment of L. donovani-infected BALB/c mice with ML-240 significantly reduced the parasite burden in vascularized organs such as spleen and liver in a concentration-dependent manner. ML-240 treatment performed at par with miltefosine, which is widely considered the best choice for treatment against kala-azar (i.e., leishmaniasis).

Conclusions.

Treatment of visceral leishmaniasis is solely based on chemotherapy. The pentavalent antimonials amphotericin B and miltefosine are the mainstays in chemotherapeutics targeting leishmaniasis. Most of these synthetic formulations are expensive and have created widespread resistance and toxicity in patients in regions of endemicity, such as India. This poses serious challenges for patient management and elimination of the disease. We undertook a unique approach to identify and test a novel drug using bioinformatics and biological assessment as possible therapeutics against the parasites. We identified that the AAA-ATPase p97 inhibitor ML-240 has significant antileishmanial activity against promastigotes and amastigotes. ML-240 downregulated peroxidases in the promastigotes and induced apoptosis in the parasites. The probe compound (ML-240) demonstrated that it can have effects comparable to those of miltefosine with respect to leishmanicidal activity and can be an alternative choice for therapeutics. ML-240 is effective against ovarian cancers in addition to its role against other cancer cells. Our observation suggested that ML-240 is effective in downregulating the viability of human T cell lymphoma (JE 6.1) and erythromyeloma cells (K-562) in addition to reducing the proliferation significantly in a concentration-dependent manner. The leishmanicidal effects of ML-240 were also explored in the context of apoptosis of the promastigotes in a concentration-dependent manner. Similar to miltefosine, ML-240 induces apoptosis and killing of the promastigotes, likely via inhibiting the ascorbate peroxidase and thus severely jeopardizing survival of the parasites. Therapy with ML-240 against established L. donovani infection in experimental animals showed a dramatic and significant reduction in parasite burden, comparable to that of miltefosine. Taken together, our results demonstrated that the novel inhibitor could be a new chemotherapeutic alternative for the treatment of leishmaniasis.

MATERIALS AND METHODS

We submitted the L. donovani ascorbate peroxidase (Ld-APX) protein sequence (UniProt ID E9BQG1) to the Phyre2 Web server, which generated a homology model using template 3RIV_A (crystal structure of Leishmania major peroxidase; resolution, 1.76 Å) (26). The same template was also predicted by NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), which showed 96% identity, 0.0 E value, and 88% query coverage, suggesting that the template selected by the Phyre2 server holds well for homology modeling. The modeled Ld-APX protein was also validated using a Ramachandran plot for its stereochemical analysis. the ProSA-web tool was used to determine the local model quality (27). The PDBsum program generated the topology of the Ld-APX model (28).

The generated Ld-APX model was submitted to COFACTOR server (29) for ligand binding site prediction. This tool predicted 3RIV as the protein with similar structure, with a template modeling (TM) score of 1.00, RMSD of 0.26, and identity of 0.959. This server predicted the active site residues of the Ld-APX protein, probably making the binding cavity. The coordinates of the cavity were identified using the LIGSITEcsc Web server (30). The AutoDock tool kit was used to prepare the protein pdbqt file for performing docking (31).

Chemical library screening suggests that the bioactive compounds exhibit some binding and inhibition properties (10, 11). We used the ZINC database to download 5,684 Sigma-Aldrich ligands as the library compound for virtual screening, similar to the work we did in a previous study (32). These downloaded molecules were ready to use and were available in 3D-MOL2 file format. Furthermore, these ligands were converted all together into pdbqt files using the AutoDock tool kit (31).

AutoDock Vina was employed for virtual screening (33). AutoDock Tools (ADT) was used for intermediate steps such as protein and ligand pdbqt file preparation and grid box creation. The grid size was set to 50 × 50 × 50 xyz points with grid spacing of 0.375 Å. The grid center was set at dimensions –17.975, 15.113, and –34.738 (x, y, and z, respectively). These coordinates were predicted using the LIGSITEcsc server.

Molecular dynamics (MD) simulations of all four complexes as well as unbound apo-Ld-APX were performed for a period of 40 ns using the GROMOS96 43a1 force field, implemented in the GROMACS 4.5.5 package (34). The PRODRG server was utilized for generating topology files of the ligands (35). The modeled protein (Ld-APX) has significant similarity with the template protein (Lm-APX). Hence, we decided to simulate the protein for an optimal time period. Performing the simulation for a longer time period might have produced similar or identical findings. Furthermore, the computational cost would have been increased. Therefore, we performed MD simulation for all the samples for 40 ns only. The protocol for performing molecular dynamics simulation comprised three steps: (i) the energy minimization, (ii) restraining of the position, and (iii) the production run. The Ld-APX protein was placed in a cubic periodic box, and its dimensions were set in such a manner that the minimum distance between the box walls and the protein was 2.5 nm. For salvation, a simple point charge (SPCE) water molecule was used, and Na⁺Cl⁻ counterions were added to satisfy the electro-neutrality (36). The MD simulation of all the docked complexes and apo protein systems were executed with the same simulating conditions as performed earlier (11). The steepest descent method was used for the energy minimization process for each system. Temperature coupling (using a V-rescale thermostat) and pressure coupling (using a Parrinello-Rahman barostat) were performed to keep the system in a stable environment (300 k, 10⁵ pa), where coupling constants were set to 0.1 and 2.0 ps for temperature and pressure, respectively. The particle mesh Ewald (PME) method for electrostatic and Van der Waals interactions was employed during all simulations. The cutoff distances for the calculation of Van der Waals and Coulomb interactions were set as 1.4 and 0.9 nm, respectively. The linear constraint solver (LINCS) algorithm was employed to constrain all bond lengths. Finally, a 40-ns molecular dynamics simulation was carried out using these well-equilibrated systems. The conformational changes and structural analyses, such as root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg), were analyzed using g_rmsd, g_rmsf, and g_gyrate, respectively. For another application of GROMACS, g_dist was used to calculate the distances between the compounds and the protein. The Graphing Advanced Computation and Exploration (GRACE) program was implemented for plotting the graphs.

Ethics statement.

All animal experiments were conducted in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPSEA), Ministry of Environment and Forests (MoEF), Government of India (GOI). The protocol was approved by the Institutional Ethics Committee of Banaras Hindu University. Mice were euthanized by cervical dislocation to reduce suffering to a minimum and were euthanized as per the American Veterinary Medical Association (AVMA) 2013 guidelines on euthanasia.

Parasites, cell lines, and cell culture details are given in the supplemental materials. Viability and growth kinetics of promastigotes and tumor cells (RAW 264.7, JE6.1, and K-562) were performed in the presence of various concentrations of ZINC96021026 or miltefosine (37). Details are given in the supplemental material.

Human peripheral blood monocyte-derived macrophages and mouse macrophage (RAW 264.7) cells (5 × 10⁴) were cultured on sterile coverslips in culture medium for 3 to 4 days (38). The infection in macrophages and leishmanicidal effects of ZINC96021026 and miltefosine against the infected macrophages are described in the supplemental material (37).

Details of the TUNEL assay, ROS generation, and measurement of intracellular ascorbate peroxidase and H₂O₂ are given in the supplemental material.

Antileishmanial efficacy of ML-240 on established L. donovani infection.

Three-week-infected BALB/c mice were either left untreated (infection control) or treated for 10 days with ML-240 (5 or 10 mg/kg body weight [bw], alternate days, intraperitoneally [i.p.]) or Miltefosine (5 mg/kg bw, alternate days, i.p.) as detailed in the supplemental material.

Data are presented as the mean ± standard deviation (SD) for multiple experiments (n = 3 to 5). Unpaired t test and one- or two-way analysis of variance (ANOVA) and multiple group comparison tests were performed using PRISM software (Graph Pad). Statistical significance was analyzed by log-rank (Mantel Cox) test. A P value of <0.05 was considered statistically significant. *, P < 0.05; **, P < 0.01; ***, P < 0.001.