Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase

Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani.

ABSTRACT

Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani. Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro. The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

INTRODUCTION

Leishmaniasis manifests itself in several forms, such as cutaneous, mucocutaneous, and visceral leishmaniasis, with the last type being the potentially fatal form caused by Leishmania donovani. The currently available treatment options for leishmaniasis (pentavalent antimonials, miltefosine, amphotericin B) either manifest severe side effects or lead to drug resistance in the parasite (1). Hence, there is an urgent need to identify novel drugs to control this disease.

The aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (aaRSs) occurs in two steps, viz., the formation of an enzyme-bound aminoacyl-adenylate moiety, followed by the transfer of the activated amino acid to either the 2′- or 3′-hydroxyl group on the 3′-terminal adenosine of the tRNA (2). aaRSs constitute a novel class of promising drug targets which, when inhibited, lead to cell death in pathogenic organisms; therefore, inhibitors of aaRSs are being explored as antifungal, antibacterial, and antiparasitic drugs (3, 4).

Leucyl-tRNA synthetase (LRS) belongs to class Ia of the tRNA synthetases (5). The overall architecture of LRS is conserved across different species, as deciphered by the crystal structures of the bacterial and the archaeal LRS (6–8). LRS consists of a specific Rossmann-fold catalytic domain, an appended anticodon-binding domain, a C-terminal extension, and a connective peptide 1 (CP1) editing domain, which is characteristic of class Ia enzymes (9). CP1 consists of about 200 to 300 conserved amino acids and is involved in posttransfer editing (10). CP1 is connected to the bulk of the synthetase by a flexible β-ribbon and undergoes significant rotation during the aminoacylation or editing cycle (6), depending on its specific point of insertion in the primary sequence. Two discrete points of insertion for CP1 within class Ia synthetases have been reported. One of them is observed only in the bacterial LRS, and the other is found in the eukaryotic/archaeal LRS, isoleucyl- and valyl-tRNA synthetases (11). Posttransfer editing by CP1 helps in maintaining the fidelity of an aminoacylation reaction and is dependent on a conserved aspartic acid residue which interacts with the α-amino group of the noncognate amino acid and positions it for hydrolysis by a water molecule (12).

Leucyl-tRNA synthetase (LRS) is a validated antimicrobial target that has been successfully exploited for the development of an antifungal agent, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690) (13, 14). An adduct formation mediated by the boron atom of AN2690 and the 2′- and 3′-oxygen atoms of the tRNA's 3′-terminal adenosine occurs, thus trapping the LRS-bound tRNA^Leu in the editing site, which prevents the catalytic turnover and which stalls protein synthesis (13). Another compound, ZCL039, a benzoxaborole-based derivative of AN2690, was found to be potent against leucyl-tRNA synthetase of Streptococcus pneumoniae (15). A structure-guided drug design approach using Candida albicans LRS was used to design benzoxaborole-derived inhibitors active against the Trypanosoma brucei LRS editing site, and their structure-activity relationship has been established (16). The benzoxaborole scaffold in AN6426 was found to be effective in inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by the formation of a covalent adduct with tRNA^Leu in the LRS editing site (17).

Our earlier comprehensive bioinformatics survey of the Leishmania genome led to the identification of a putative single copy of leucyl-tRNA synthetase (18). The present study for the first time reports the molecular and enzymatic characterization of LRS from Leishmania donovani. Gene replacement studies suggest that this enzyme is indispensable for the survival of this pathogenic organism in vitro. The compound AN2690 showed antileishmanial activity against the promastigote and amastigote stages both in vitro and in vivo in BALB/c mice. AN2690 was also effective in inhibiting the aminoacylation activity of recombinant Leishmania donovani LRS (LdLRS). The data show that LdLRS appears to be essential for the survival of L. donovani and is a suitable target for drug development in the parasite.

RESULTS

The leucyl-tRNA synthetase of Leishmania is phylogenetically closer to plant homologs.

Multiple-sequence alignment of the kinetoplastid leucyl-tRNA synthetase sequences along with representative sequences from eukaryotes and prokaryotes were generated using the T-Coffee multiple-sequence alignment tool (Fig. 1). These data suggest the conservation of the HLGH, KMSKS, and DWLISR motifs characteristic of class Ia tRNA synthetases (11) in the catalytic domain and the GTG and T-rich motifs in the CP1 editing domain. A catalytically essential aspartic acid in the CP1 editing domain is also conserved in all the represented leucyl-tRNA synthetases. While the bacterial enzymes contain zinc-binding motifs and are known to bind to a single zinc ion (11), these motifs are absent in the kinetoplastid and human (cytosolic) leucyl-tRNA synthetases, suggesting the absence of metal ion in these enzymes (Fig. 1). However, the Escherichia coli and human mitochondrial leucyl-tRNA synthetases are known to bind a single zinc ion (11), while the Thermus thermophilus leucyl-tRNA synthetase binds two zinc ions, as shown by the characteristic motifs (Fig. 1). Trypanosoma brucei leucyl-tRNA synthetase (TbLRS) has been explored extensively both in the editing and synthetase catalytic domains for the inhibitor binding studies (9, 16, 19). On the basis of this analysis of TbLRS, the equivalent inhibitor binding residues in LdLRS were analyzed. Although TbLRS and LdLRS share an amino acid sequence identity of only ∼60%, the inhibitor binding residues are completely conserved (Fig. 1).

FIG 1.

Multiple-sequence alignment of kinetoplastid leucyl-tRNA synthetases along with representative enzymes from other eukaryotes and bacteria generated using the T-Coffee tool. The signature motifs of class Ia synthetases are indicated in red bold font. The GTG and T-rich motifs are highlighted in yellow. The catalytic aspartic acid residue is highlighted in blue. The CP1 domain is highlighted in light gray. The zinc-binding cysteine motifs are shaded in cyan. The inhibitor binding residues are highlighted in green. The conserved residues are shaded in black, and the conservative mutations are shaded in dark gray. The GenBank accession numbers of the aligned sequences are as follows: Leishmania donovani, XP_003859311.1; Leishmania major, XP_001681854.1; Trypanosoma brucei, XP_828454.1; Trypanosoma cruzi, XP_805515.1; Candida albicans, EEQ43286.1; Homo sapiens, BAA95667.1; Escherichia coli, EFI86361.1; Thermus thermophilus, WP_014628983.1.

Next, the MEGA (v5) program (20) was utilized for the construction of the neighbor-joining bootstrap tree using the multiple-sequence alignment generated by the CLUSTAL W program (21). The phylogenetic tree (Fig. 2) clearly shows that the kinetoplastid leucyl-tRNA synthetases are closer to the plant homologs, while the mammalian enzymes are closer to the insect enzymes. Plasmodial leucyl-tRNA synthetases are closer to the bacterial homologs (Fig. 2).

FIG 2.

Sequence-based phylogeny of leucyl-tRNA synthetase (LRS) homologs from kinetoplastids, mammals, plants, insects, plasmodia, fungi, bacteria, and archaea. The neighbor-joining bootstrap tree was constructed using MEGA (v5) software. Bootstrap values of >90 are shown in the phylogenetic tree. For analysis, the following sequences were used. The following accession numbers correspond to the species listed from top to bottom, respectively: for Mammalia. BAA95667.1, XP_018883476.1, XP_014996611.1, XP_014710275.1, XP_007080026.1, XP_011962813.1, NP_001095962.1, XP_010848946.1, NP_598898.2, and NP_001009637.1; for Insecta, XP_004930202.1, XP_013140644.1, XP_395743.2, XP_019551695.1, ETN58342.1, XP_011294281.1, JAC42657.1, XP_017855445.1, and AAM50317.1; for Fungi, AAA33599.1, EEQ43286.1, and AAA34805.1; for Plantae, XP_017630544.1, XP_020404072, AAD36946.1, XP_009118303.1, and XP_018483207.1; for Kinetoplastida, XP_009310821.1, XP_805515.1, XP_828454.1, XP_001563232.2, XP_003873311.1, XP_001681854.1, XP_003859311.1, and XP_001464129.1; for Zoomastigophora, EFO65558.1; for Bacteria, NP_213240.1, WP_014628983.1, EFI86361.1, and AMP26935.1; for Plasmodiidae, XP_001349244.1, XP_012762667.2, XP_001614881.1, SCA48531.1, and CDU17148.1; and for Archaea, WP_018153661.1, WP_064496564.1, WP_007043736.1, WP_055430094.1, WP_010885055.1, and WP_010867994.1.

Characterization of LdLRS.

The full-length L. donovani leucyl-tRNA synthetase (LdLRS) gene was cloned and expressed as explained in Materials and Methods. The protein was purified to homogeneity using metal affinity chromatography, and the identity of the protein was confirmed using matrix-assisted laser desorption ionization–time of flight/time of flight (MALDI-TOF/TOF) mass spectroscopy (data not shown) as well as Western blotting (Fig. 3A and B). The Western blot analysis with polyclonal antiserum to recombinant LdLRS recognized a single specific band of ∼122 kDa in the L. donovani whole-cell lysate (Fig. 3B). Detection of the band corresponding to the recombinant LdLRS protein was used as a control (Fig. 3B).

FIG 3.

(A) Purification of the recombinant LdLRS (rLdLRS) protein on Ni²⁺-NTA acid affinity resin. Lane M, molecular weight marker; lane 1, uninduced cell lysate; lane 2, induced cell lysate; lane 3, fraction eluted with 100 mM imidazole showing purified LdLRS. (B) Immunoblotting analysis of the WT promastigote cell lysate (lane 1) using anti-LdLRS antibody. Two different concentrations (0.5 and 1.0 μg) of the recombinant LdLRS protein were used as a control (lanes 2 and 3). Lane M, molecular weight marker. (C to E) Michaelis-Menten plots and Lineweaver-Burk plots (insets) for aminoacylation kinetics catalyzed by the LdLRS enzyme. The enzyme assay was carried out as described in the Materials and Methods section. The kinetic constants for the utilization of tRNA^Leu, l-leucine, and ATP by the LdLRS enzyme were computed using the Michaelis-Menten algorithm within GraphPad Prism (v5.0) software. Results are representative data from three separate experiments and are represented as the mean ± SD. V, velocity.

The purified recombinant enzyme was found to be active and able to catalyze the aminoacylation reaction (Fig. 3C). The kinetic parameters and specificity of LdLRS were determined in vitro using l-leucine, tRNA^Leu, and ATP as the substrates. LdLRS showed a K_m value of 21 ± 6 μM for l-leucine and a V_max value of 42 ± 6 pmol min⁻¹ μg of protein⁻¹ (Fig. 3C). The kinetic parameters obtained for the utilization of ATP were a V_max value of 41.3 ± 2 pmol min⁻¹ μg of protein⁻¹ and a K_m value of 122.2 ± 9 μM (Fig. 3D). Thus, the calculated K_m value is approximately similar to the K_m values for ATP in the case of the human cytoplasmic LRS (112.1 ± 2.1 μM) and human mitochondrial LRS (90 μM) (22). The K_m value deciphered for tRNA^Leu was 2.5 ± 3 μM, and V_max was 39.87 ± 2 pmol min⁻¹ μg of protein⁻¹ (Fig. 3E). This K_m value for tRNA^Leu is closer to that reported for human cytoplasmic LRS (1.02 ± 0.04 μM) (23) and E. coli LRS (1.5 μM) (24).

Isothermal titration calorimetry (ITC) studies were performed to investigate the binding affinities of various substrates with LdLRS. The binding of leucine with LdLRS resulted in positive enthalpy (binding enthalpy change [ΔH] = +4.6 kcal/mol) and positive entropy (entropy change [TΔS] = +10.6 kcal/mol), which suggests that the binding reaction is mainly driven by entropy and opposed by enthalpy (Fig. 4A and Table 1). Weak binding was observed in the case of norvaline and isoleucine (Fig. 4B and C), suggesting the selectivity of LdLRS for leucine over other substrates.

FIG 4.

Binding of LdLRS with leucine (A), norvaline (B), and isoleucine (C). (Top) The raw calorimetric data, denoting the amount of heat (endothermic peaks) produced following each injection of leucine. Upon each titration, as the active site of protein gets saturated with ligand, the area under the peaks gradually becomes smaller. (Bottom) The amount of heat generated per injection as a function of the molar ratio of leucine to enzyme. The experiment was performed in 10 mM phosphate buffer (pH 7.5) at 25°C; the concentration of protein used was 50 μM, and that of the substrate was 400 μM.

TABLE 1.

ITC data showing binding of leucine and AN2690 to LdLRS

Ligand	Kd (μM)	ΔH (kcal/mol)	TΔS (kcal/mol)	ΔG (kcal/mol)	N valuea
Leucine	43 ± 5	+4.6 ± 0.5	+10.6 ± 0.35	−6.0	1.06
AN2690	231 ± 45	+4.3 ± 0.24	+9.2 ± 0.81	−4.9	0.98

^aThe N value from the one-site model.

LdLRS appears to be essential for the viability of the parasite.

In order to determine the dispensability of LdLRS for the parasite, a targeted gene replacement strategy was employed to replace both the wild-type alleles with cassettes harboring drug resistance marker genes, as described in Materials and Methods. The genotype of the LRS/HYG and LRS/NEO heterozygous parasites was confirmed by PCR-based analysis by using primers specific for regions external to the transfected inactivation cassette (Fig. 5A). The 1.7- and 1.4-kb bands in the case of the hygromycin phosphotransferase gene (HYG) cassette and the 1.6- and 1.46-kb bands in the case of the neomycin phosphotransferase gene (NEO) cassette, along with the bands corresponding to the wild-type (WT) allele (1.7 and 1.5 kb), were obtained (Fig. 5B).

FIG 5.

(A) Restriction map of the LdLRS genomic locus and the locations of the primers used for confirmation by PCR-based analysis along with the expected band sizes. Primer 4 was designed as a forward primer to match the upstream region of the LdLRS gene, and primers 8, 3, and 6 were designed to be specific for regions internal to the LRS, HYG, and NEO coding regions, respectively. Primer 2 was designed as a reverse primer to match the downstream region of the LdLRS gene, and primers 7, 1, and 5 were designed as forward primers to be specific for regions internal to the LRS, HYG, and NEO coding regions, respectively. (B, C) Genomic DNA from LRS/HYG or LRS/NEO parasites (B) and ΔLRS(pLRS⁺) parasites (C) was used as a template for PCR analysis. The specific integration of the replacement cassette(s) was checked with HYG, NEO, and LdLRS (WT) gene-specific primers. ZEO, amplification using zeocin resistance cassette-specific primers for detection of the pSP72α-zeo-α-LRS episome. The numbers above the gel refer to the primers. Lane M, molecular size markers. (D and E) Genomic DNA was digested with MluI and separated on a 0.6% agarose gel for Southern blot analysis of wild-type (WT) strain Bob, single transfectant LRS/NEO, and double transfectant LRS/NEO/HYG parasites probed with the 5′ UTR of the LdLRS gene (D) and WT, LRS/NEO(pLRS⁺), and ΔLRS(pLRS⁺) parasites probed with the 370-bp zeocin resistance gene (E). The pSP72α-zeo-α-LRS plasmid digested with MluI was used as a control. Molecular weight markers are indicated to the right of the blots.

LdLRS heterozygotes were subsequently transfected with a second cassette to replace the second copy of the gene. PCR analysis demonstrated that cells selected in the double-antibiotic medium showed HYG and NEO replacement cassettes at the LdLRS gene locus, but these transfectants also showed 1.7- and 1.5-kb bands corresponding to the WT LdLRS gene (data not shown). This result indicates the presence of the wild-type gene in the double transfectants.

The presence of the WT LdLRS gene along with HYG and NEO replacement cassettes in the gene locus was further confirmed by Southern blotting. In the WT cells, digestion of the LdLRS gene locus with MluI was expected to yield a 2.3-kb band after probing with the 5′ untranslated region (UTR) of the LdLRS gene (Fig. 5A). Integration of the HYG or NEO cassette was expected to yield 1.8- and 1.6 kb 5′ UTR-hybridizing bands, respectively, after MluI digestion (Fig. 5A). Bands of the expected sizes after probing with the LdLRS 5′ UTR were obtained in the case of WT and heterozygous parasites (Fig. 5D). In the case of double transfectants (LRS/NEO/HYG parasites), a band corresponding to the WT LdLRS (2.3 kb) was found, in addition to the 1.8- and 1.6 kb bands corresponding to the HYG and NEO cassettes, respectively, indicating the presence of the WT gene in these transfectants (Fig. 5D, lane 3). Even after several attempts, LRS homozygous null mutants could not be obtained, indicating the essentiality of LRS for the Leishmania parasite. However, the overall DNA content of WT, LRS/NEO, and LRS/NEO/HYG parasites, as determined by flow cytometry analysis, indicated that all the selected cell lines had similar and normal ploidy levels (data not shown).

To further test the possibility that LdLRS is an essential gene, chromosomal LRS gene deletion was attempted in the presence of a rescuing episome that contained the LdLRS gene (pSP72α-zeo-α-LRS). LRS/NEO heterozygous parasites containing pSP72α-zeo-α-LRS [LRS/NEO(pLRS⁺) parasites] were transfected with the 5′ UTR-HYG-3′ UTR gene deletion construct. PCR analysis was performed to investigate the genotype of these triple-drug-resistant parasites. PCR analysis revealed the absence of LdLRS from the WT gene locus in the genomes of these parasites, and bands corresponding to the integration of the HYG and NEO cassettes at this locus could be detected, notably, only in the presence of a complementing plasmid (pSP72α-zeo-α-LRS), detected using zeocin-specific primers (Fig. 5C). Further, after MluI digestion a 6.3-kb fragment was detected using the zeocin resistance-coding region as a probe, corresponding to the pSP72α-zeo-α-LRS plasmid in the LRS/NEO(pLRS⁺) and ΔLRS(pLRS⁺) parasites (Fig. 5E). The pSP72α-zeo-α-LRS plasmid digested with MluI was taken as a control (Fig. 5E). Thus, a chromosomal knockout of LRS was achieved only when the gene was provided on a rescuing plasmid. These data provide evidence that LRS appears to be an essential gene in Leishmania.

Genetically manipulated parasites show reduced growth and virulence in vitro.

Leishmania has been reported to possess a single copy of leucyl-tRNA synthetase (18), which is an essential component of the protein translation machinery. Our data suggest that LdLRS targeted for gene disruption encodes a functional enzyme in the parasite. The LRS/NEO heterozygous parasites (doubling time, ∼16.5 h) showed a consistent growth delay compared to their wild-type counterparts (doubling time, ∼12 h), and this delay was found to be rescued in LRS/NEO(pLRS⁺) parasites complemented with an episomal copy of LRS (Fig. 6A). This result may be attributed to a gene dosage effect, leading to the production of the smaller amount of the LRS protein and leading to suboptimal cell proliferation.

FIG 6.

(A) Comparison of the growth curve characteristics of WT, LRS/NEO, and LRS/NEO(pLRS⁺) promastigotes in M199 medium. The experiment was repeated thrice independently in triplicate. Representative data from one experiment are shown. (B) Comparison of the infectivity profile of L. donovani WT, LRS/NEO, and LRS/NEO(pLRS⁺) parasites in the J774A.1 murine macrophage cell line. The murine macrophage cell line J774A.1 was infected with stationary-phase promastigotes at a multiplicity of infection of 20:1. Cells were stained after 12 h and 24 h, and amastigotes were enumerated visually. (C) Aminoacylation activity of LdLRS in the cell lysates of L. donovani WT and heterozygous (LRS/NEO) and LRS/NEO(pLRS⁺) parasites. The results represent the mean ± SD (n = 3). **, a statistically significant difference (P < 0.01) from the wild-type control; ns, nonsignificant.

Virulence studies were carried out in the J774.A1 murine macrophage cell line with the WT and mutant parasites to determine the infectivity of the host cells. The stationary-phase culture of the wild-type and mutant promastigotes was used to infect the murine macrophage cell line, and the parasite load was evaluated at 12 h and 24 h postinfection (p.i.). No significant difference in the parasite load could be detected at 12 h p.i. for the WT and mutant strains. However, the parasite load at 24 h p.i. was significantly greater for the wild-type strain (15/macrophage) than for the LRS/NEO heterozygous parasites (6/macrophage) (Fig. 6B). The parasitemia of the LRS/NEO heterozygous mutants was reduced by ∼40% relative to that of the wild-type parasites at 24 h p.i., and this effect was found to be rescued in LRS/NEO(pLRS⁺) parasites (Fig. 6B). This result suggests that the disruption of a single LRS allele from the parasite has the potency of reducing the ability of amastigotes to multiply in vitro. This may be partially attributed to the slow growth phenotype of the heterozygous mutants compared to their wild-type counterparts, as was observed at the promastigote stage.

In order to evaluate the impact of depletion of LRS in the cell, an in vitro aminoacylation assay was performed with WT, heterozygous (LRS/NEO), and rescued mutant [LRS/NEO(pLRS⁺)] parasites. The aminoacylation activity in the cell lysate of the LRS/NEO heterozygous parasites was found to be ∼2.8-fold lower than that of the WT cells (Fig. 6C). This loss was compensated in the case of the episomally complemented LRS/NEO(pLRS⁺) parasites, which demonstrated aminoacylation efficiency at par with that of the WT cells (Fig. 6C).

The antifungal AN2690 binds to recombinant LdLRS and inhibits its aminoacylation potential.

AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) is a boron-containing small-molecule antifungal agent with broad-spectrum activity against filamentous fungi (Fig. 7A). It is known to act by inhibiting fungal cytoplasmic leucyl-tRNA synthetase by covalently trapping tRNA^Leu in its editing site, thus preventing catalytic turnover and, consequentially, blocking protein synthesis. In order to assess whether AN2690 can act on Leishmania donovani leucyl-tRNA synthetase, an ITC experiment was carried out to study the interaction of AN2690 with LdLRS. Figure 7B (top) shows a steady decrease in the area under the peak as the inhibitor binds and saturates the LdLRS binding site. Analysis of the data indicates the optimal affinity (K_d [dissociation constant] = 231 ± 45 μM) of AN2690 for LdLRS (Fig. 7B, bottom, and Table 1). The binding reaction resulted in endothermic (ΔH = +4.3 ± 0.24 kcal/mol) heat with a favorable entropy change (TΔS = +9.2 ± 0.81 kcal/mol), which denotes that the binding is considerably driven by a hydrophobic interaction (Table 1).

FIG 7.

(A) Structure of AN2690. (B) ITC binding of AN2690 with LRS. The inhibitor (800 μM AN2690) was titrated into 100 μM protein in phosphate buffer (pH 7.5) at 25°C. The data were fitted with a single-site model available in the Origin program. (C) Dose-response inhibition of the aminoacylation activity of LdLRS in the presence of the inhibitor AN2690. Inhibitor concentrations are plotted on the log scale on the x axis. The experiment was performed with 0.1 to 10 μM AN2690.

Further, we wanted to investigate if the observed binding capacity of AN2690 is sufficient to inhibit the aminoacylation potential of the recombinant LdLRS enzyme. Aminoacylation assays were performed in the presence of AN2690 over a concentration range of 0.1 μM to 10 μM, and dose-dependent inhibition was assessed. The 50% inhibitory concentration (IC₅₀) value in the presence of AN2690 was found to be ∼0.83 ± 0.2 μM (Fig. 7C).

AN2690 inhibited L. donovani growth both in vitro and in vivo.

The effect of AN2690 on the growth of WT, LRS/NEO heterozygous, and episomally LRS-complemented heterozygous [LRS/NEO(pLRS⁺)] promastigotes was assessed (Fig. 8A). The effective concentration that inhibited parasite growth by 50% (EC₅₀) of the LRS/NEO heterozygous parasites (∼1.3 μM) was 2-fold less than that of the WT cells (∼2.67 μM) (Fig. 8A). The EC₅₀ of the rescue mutants [LRS/NEO(pLRS⁺)] was found to be similar to that of the WT cells (Fig. 8A). The increased susceptibility of the heterozygous parasites to AN2690 may be attributed to the reduced levels of LdLRS expression in these cells. Further, the potency of AN2690 to act against the amastigote stage was also evaluated in an intracellular amastigote-macrophage model by infecting the J774.A1 murine macrophage cell line with the WT parasites. An EC₅₀ of 2.38 ± 2 μM was obtained for the WT cells after 3 days of treatment with AN2690 (Fig. 8B).

FIG 8.

(A and C) The shift in the growth inhibition profile of AN2690 and miltefosine according to the EC₅₀ values for L. donovani LRS heterozygous mutants compared to those for WT cells. LRS/NEO(pLRS⁺) promastigotes served as a control. L. donovani log-phase (WT) and genetically manipulated promastigotes were seeded in a 96-well flat-bottom plate and incubated with the indicated concentration of AN2690 and miltefosine. The graphs show the cell concentrations relative to those for the untreated controls that were reached after 72 h of incubation at 22°C, as determined by the MTT assay. The experiments were done in triplicate; the graphs depict the mean for the relative cell concentrations and include the standard errors. (B and D) The intracellular parasite load was determined using Giemsa staining of infected J774.A1 murine macrophages at 72 h after treatment with the indicated concentration of AN2690 and miltefosine. The graphs depict the parasite loads relative to those for the untreated controls. The results were obtained in duplicate as representatives of two independent experiments.

As a positive control, the inhibitory effect of miltefosine (hexadecylphosphocholine [HePC]), an alkyl phospholipid compound that was the first oral drug registered for use for the treatment of visceral leishmaniasis, on the growth of WT, LRS/NEO heterozygous, and episomally LRS-complemented heterozygous [LRS/NEO(pLRS⁺)] promastigotes (Fig. 8C) was also studied along with its inhibitory effect against amastigotes (Fig. 8D). No detectable cytotoxicity of AN2690 to the J774.A1 murine macrophage cell line after 48 h of treatment at concentrations up to 200 μM was observed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Next, the efficacy of AN2690 against L. donovani in a murine model was studied. Treatment of infected BALB/c mice began after 7 days of infection by stationary-phase promastigotes injected intravenously into the tail vein. AN2690 was administered orally (11 mg/kg of body weight in 2% [vol/vol] ethanol–5% [wt/vol] dextrose in sterile water) once daily for 5 consecutive days. As a positive control, miltefosine was also administered orally (25 mg/kg in sterile water) to a group of infected mice once daily for 5 consecutive days. Both miltefosine and AN2690 were tolerated very well at the given doses, as evident by the lack of any signs of toxicity.

On the 14th day, i.e., 3 days after the termination of treatment, all the mice were humanely sacrificed, and their spleens were removed aseptically and weighed. Impression smears were prepared, fixed in methanol, and Giemsa stained to determine the parasitic burdens in spleen tissues, and the burdens between the treated and the untreated groups were compared. The parasite load in the case of the control mice was found to be significantly higher than that in the case of the mice treated with miltefosine or AN2690 (Fig. 9A). In the case of miltefosine-treated mice, the splenic amastigote load was suppressed by 75% compared to that in the untreated infected controls. The suppression of the parasitic load in the case of AN2690-treated mouse spleen tissues was about 55% in comparison to that in the untreated infected controls.

FIG 9.

(A) In vivo efficacy of AN2690 against Leishmania donovani in BALB/c mice infected with 1 × 10⁸ stationary-phase L. donovani promastigotes and treated orally after day 7 for 5 days with miltefosine (25 mg/kg) or AN2690 (11 mg/kg) or left untreated. The parasite burden in the spleen, expressed in LDU, was determined 3 days after the completion of the treatment. (B) Real-time PCR of Leishmania donovani in infected mouse spleen tissues showing the fold change in the kDNA level for treated and untreated mice. Ten nanograms of total DNA isolated from the infected and uninfected mouse spleen tissues was used as the template for quantitative PCRs. kDNA levels were quantitated using the 2^-ΔΔCT (comparative C_T) method. The results for all the infected samples (from vehicular control-, miltefosine-, and AN2690-treated mice) were normalized to those for the uninfected samples. The results are the means from two replicate experiments with five mice in each group. Error bars indicate the standard error. The statistical significance was determined using Student's t test (*, P < 0.05).

Also, additional confirmation of the presence of a parasite burden in mouse spleen tissues was done by detection and amplification of a 120-bp fragment of kinetoplast DNA (kDNA) of L. donovani from the total DNA isolated from the spleen tissues. As depicted in Fig. 9B, the parasite burden was reduced 6-fold in the mice treated with miltefosine compared to that in the untreated control mice, while in the case of the AN2690-treated mice, the parasite burden was reduced by 3-fold compared to that in the untreated control mice. Also, the threshold cycle (C_T) values of all the samples were compared (Table 2). The C_T value is inversely proportional to the abundance or relative expression level of the gene of interest. The C_T value was found to be the highest for the uninfected samples, indicating the absence of any parasite DNA in the mouse spleen tissues. On the other hand, the C_T values for the infected samples were less, indicating amplification of parasite-specific kinetoplastid minicircle DNA. A nontemplate control and a positive control with Leishmania donovani genomic DNA as the template were also tested.

TABLE 2.

Real-time PCR confirmation of Leishmania donovani in infected mouse spleen tissues for treated and untreated groups^a

Group and serial no.	Description	CT value
Treated groups
1	Naive mice	31.859 ± 0.24
2	Vehicular control group	14.799 ± 0.02
3	Miltefosine treatment	23.160 ± 0.08
4	AN2690 treatment	19.621 ± 0.88
Controls
1	Nontemplate control	30.953 ± 0.94
2	Positive control (10 ng DNA from L. donovani parasites)	12.492 ± 0.32

^aThe data presented are representative of those from two experiments with similar results and represent the mean ± SEM for 5 mice in each group.

DISCUSSION

The biological pathway of protein synthesis has been thoroughly validated as a target for antimicrobial compounds. Aminoacyl-tRNA synthetases are components of the protein translation machinery which ensure fidelity in the translation of mRNA and may be explored as potent antiparasitic targets (4). The present study reports on the molecular characterization of leucyl-tRNA synthetase from L. donovani. It provides a genetic validation of leucyl-tRNA synthetase as a potential drug target in Leishmania, with the LdLRS gene appearing to be indispensable for parasite survival. The LRS/NEO heterozygous promastigotes showed a growth defect and reduced parasitemia in virulence studies in the J774.A1 murine macrophage cell line compared to their wild-type counterparts.

The open reading frame (ORF) of Leishmania donovani leucyl-tRNA synthetase (LdLRS) encodes a polypeptide of 1,075 amino acids. Kinetic analysis of recombinant LdLRS demonstrated that the enzyme exhibited a catalytic efficiency similar to that reported in the case of other species. The binding data for LdLRS with various substrates obtained in ITC studies suggests a net loss of noncovalent interactions in LdLRS-leucine complex formation. Overall, interactions in LdLRS-leucine complex formation appear to be driven by positive entropy, suggesting an important contribution from hydrophobic interactions. Leucine contains a hydrophobic side chain, which can play an important role in forming hydrophobic interactions upon binding in the enzyme pocket (K_d = 43 μM, change in Gibbs free energy [ΔG] = −6.0 kcal/mol). As a result of these hydrophobic interactions, leucine may displace water molecules in the binding pocket, resulting in favorable entropy. Norvaline contains a small hydrophobic side chain (with one less methyl group than the side chain of leucine), resulting in its weak binding. Isoleucine, which has a similar structure as leucine but a different stereochemical arrangement of its side chain, may not result in effective hydrophobic interactions in the pocket, resulting in poor binding with LdLRS.

The efficacy of benzoxaboroles as potent leucyl-tRNA synthetase inhibitors has been reported in the case of Gram-negative bacteria (25), fungi (14), and apicomplexan parasites, such as Plasmodium falciparum (26), Cryptosporidium, and Toxoplasma (17). A series of benzoxaborole compounds which are orally active in murine models of human African trypanosomiasis against Trypanosoma brucei has also been reported (27). Our results suggest that the benzoxaborole AN2690 efficiently blocks aminoacylation by Leishmania LRS in vitro (IC₅₀, ∼0.83 ± 0.2 μM). The mechanism of action of the benzoxaborole compounds has been demonstrated to involve the formation of a stable covalent adduct with the tRNA^Leu 3′ acceptor end in the LRS editing site that can block aminoacylation (13). AN2690 was found to be effective against the WT promastigote and amastigote stages with an EC₅₀ of ∼2.67 and ∼2.38 μM, respectively. The parasite burden in spleen tissues of AN2690-treated mice was also found to be reduced by 3-fold compared to that in untreated control mice. These findings, coupled with the reported excellent oral bioavailability (25) and low toxicity (28) of benzoxaborole compounds, merit their further exploration as drugs against Leishmania parasites.

The establishment of new drug targets in Leishmania through genetic and chemical validation may help in advancing the development of drugs against the related trypanosomatids T. brucei and Trypanosoma cruzi because of their genomic similarities (29). The Leishmania LRS sequence is highly conserved in T. cruzi and T. brucei, and it may be speculated that this gene is also likely to be indispensable for their survival. Further, this study also provides an insight into the feasibility of targeting single-copy tRNA synthetases in these parasites, wherein the loss of function conferred due to targeting with specific inhibitors will prove detrimental to the parasite's survival.

In this study, although AN2690 was found to be less potent against Leishmania than the drug miltefosine, the reported increases in the numbers of cases of miltefosine resistance in L. donovani warrant the search for new drugs active against this parasite. In conclusion, we have validated the Leishmania LRS enzyme to be a potential target for drug development and also identified an inhibitor which is potent against both the parasite and the recombinant protein. The inhibitor (AN2690) identified here may provide a scaffold for the development of a new class of drugs against Leishmania parasite.

MATERIALS AND METHODS

Cell lines and culture conditions.

L. donovani Bob (LdBob; a derivative of strain MHOM/SD/62/1SCL2D) promastigotes, originally obtained from Stephen Beverley (Washington University, St. Louis, MO), were cultured at 22°C in M199 medium (Sigma) supplemented with 100 units/ml penicillin (Sigma), 100 μg/ml streptomycin (Sigma), and 5% heat-inactivated fetal bovine serum (FBS; Gibco). Wild-type (WT) parasites were routinely cultured in medium with no drug supplementation, whereas the genetically manipulated LRS heterozygotes (LRS/HYG and LRS/NEO parasites) and double transfectants (LRS/NEO/HYG parasites) were maintained in either 200 μg/ml hygromycin (Sigma-Aldrich) or 300 μg/ml paromomycin (Sigma-Aldrich), or both, respectively. The episomally LRS-complemented LRS/HYG(pLRS⁺) and LRS/NEO(pLRS⁺) heterozygous promastigotes were grown in 200 μg/ml hygromycin or 300 μg/ml paromomycin, respectively, and 800 μg/ml zeocin (Sigma-Aldrich). ΔLRS(pLRS⁺) parasites were grown in 800 μg/ml zeocin, 200 μg/ml hygromycin, and 300 μg/ml paromomycin. WT(pLRS⁺) parasites were grown in 800 μg/ml zeocin. For characterization of the mutant parasites phenotypically, cells were subcultured without the selection marker prior to the experiments.

The mouse monocyte-macrophage-like cell line J774.A1, obtained from ATCC, was cultured in RPMI 1640 (Sigma) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO₂.

Sequence and phylogenetic analysis.

For the purpose of multiple-sequence alignment, leucyl-tRNA synthetase (LRS) sequences were retrieved from TritrypDB (30), Swiss-Prot/UniProtKB (31), and PlasmoDB (32). The T-Coffee multiple-sequence alignment tool (33) was used for sequence analysis. A complete multiple-sequence alignment of these sequences was generated using the CLUSTAL W program (21) with default parameters and used as the seed alignment for phylogenetic tree generation using the Jones-Taylor-Thornton (JTT) model (34). The MEGA (v5) program (20) was used for analysis, and the Interactive Tree of Life, a web-based tool at EMBL (itol.embl.de) (35), was used for visualization of the phylogenetic tree.

Construction of expression vector and purification of recombinant protein.

The gene for LdLRS (LdBPK_131000.1) was PCR amplified using a forward primer with a flanking NdeI site (5′-TTTCATATGATGTCCACGGCACGTCGCGATG-3′) and a reverse primer with a flanking BamHI site (5′-TTTGGATCCCTACTCGCGCTTCACTACTGG-3′). The 3,228-bp amplification product encompassing the entire LdLRS open reading frame (ORF) was cloned into the pET28a vector (Novagen). This construct containing an N-terminal His₆ tag was transformed into the E. coli BL21(DE3) strain, and protein expression was induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 16°C for 16 h. The bacteria were then harvested by centrifugation at 5,000 × g for 15 min, and the cell pellet was suspended in lysis buffer (50 mM Tris-Cl, pH 7.4, 10 mM imidazole, 300 mM sodium chloride, 2 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture). Protein was purified using Ni²⁺-nitrilotriacetic acid (NTA)-agarose resin (Qiagen) by eluting with increasing concentrations of imidazole. The purest fractions, as judged by SDS-PAGE, were concentrated by using a 30-kDa-cutoff Centricon centrifugal device (Millipore). The concentration of the purified protein was determined using the Bradford reagent (Sigma). Purified protein was found to be >95% pure, as judged by SDS-PAGE.

Antibody production and Western blot analysis.

Purified recombinant LdLRS protein (50 μg) in Freund's complete adjuvant (Sigma) was subcutaneously injected into mice, followed by three booster doses of the recombinant protein (20 μg) in Freund's incomplete adjuvant (Sigma) at 2-week intervals. The mice were sacrificed after the last booster, and serum was collected for Western blot analysis. Wild-type early-log-phase promastigotes were harvested, and the resultant cell pellets were resuspended in lysis buffer (10 mM Tris-Cl, pH 8.0, 5 mM dithiothreitol [DTT], 10 mM NaCl, 1.5 mM MgCl₂, 0.1 mM EDTA, 0.5% Triton X-100, 0.3 mM phenylmethylsulfonyl fluoride). The cell pellets were lysed by freeze-thaw cycles and sonication on ice, followed by centrifugation at 20,000 × g. Recombinant LdLRS protein and 30 μg of the total soluble promastigote cell extracts were fractionated on a 10% SDS-PAGE gel and blotted onto a nitrocellulose membrane using an electrophoretic transfer cell (Bio-Rad). After blocking with 5% skimmed milk, the membrane was incubated for 2 h at room temperature (RT) with an anti-LdLRS antibody (1:1,000) generated in mice as described above. The membrane was then washed with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS-T) and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin antibody (1:5,000; catalog number 7076S; Cell Signaling Technology). The blot was developed using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences) according to the manufacturer's protocol.

Preparation of tRNA substrate.

L. donovani tRNA^Leu (TritrypDB accession number LdBPK_36tRNA4) was produced as previously described (36). Briefly, the template tRNA^Leu was amplified by PCR from genomic DNA using a T7 promoter sequence (indicated in boldface) at the 5′ end of the forward primer (5′-TAATACGACTCACTATAGGGGTGAGATGGTCGAGTGGTCTA-3′) and a TGG sequence (indicated in boldface) at the 5′ end of the reverse primer (5′-TGGTGATGAGAGTGGGGTTTGAAC-3′). An in vitro transcription reaction using this template was performed using a MEGAscript T7 polymerase kit (Ambion, Life Technologies), and the reaction mixtures were extracted with acid phenol-chloroform (5:1) solution, pH 4.5 (Ambion, Life Technologies). tRNAs were precipitated with isopropanol (Sigma-Aldrich) and resuspended in diethyl pyrocarbonate-treated water. tRNAs were folded prior to the aminoacylation reactions by heating at 70°C for 10 min, followed by addition of 10 mM MgCl₂ and slow cooling at RT.

Aminoacylation assays.

Aminoacylation assays were performed as previously described (36) in aminoacylation buffer (30 mM HEPES buffer, 140 mM NaCl, 30 mM KCl, 40 mM MgCl₂) with 1 mM DTT, 200 μM ATP, 2 U/ml inorganic pyrophosphatase (PPiase; Sigma), 1 mM l-leucine (Sigma), 8 μM tRNA^Leu, and 60 μg/ml recombinant protein or 50 μg of the respective total promastigote cell extracts at 37°C. The reactions (in which each reaction mixture had a 50-μl volume) were performed in clear, flat-bottom 96-well plates (Nunc), and the reaction mixtures were incubated for 30 min at 37°C. In the case of time course aminoacylation reactions, incubation was carried out at 37°C for 5, 10, 15, and 20 min. Liberated inorganic phosphate was detected by addition of 12 μl of the malachite green solution (37) and incubation for 30 min in the dark. The absorbance at 620 nm was measured using a microplate reader (SpectraMax M2; Molecular Devices). Reactions without enzyme or l-leucine were performed as background controls.

In order to determine the kinetic constants, assays were performed and the concentrations of the substrates were varied over the following ranges: 0.1 to 20 mM for l-leucine, 0.5 to 25 μM for tRNA^Leu, and 0.1 to 20 mM for ATP. The remaining substrate concentrations were maintained in excess. A phosphate standard (0 to 5,000 pmol) was used for product quantification. Measurements were performed in triplicate. The reaction velocities were plotted against the substrate concentrations, and the Michaelis-Menten constants were calculated by curve fitting using GraphPad Prism (v5.0) software.

The effect of AN2690 (ApexBio, Houston, TX) on the aminoacylation activity of LdLRS was determined by preincubation of recombinant LdLRS (60 μg/ml) with the inhibitor (0.1 μM to 10 μM) for 20 min at 37°C in the aminoacylation reaction mixture devoid of tRNA, ATP, and PPiase. Subsequently, the reaction mixture (final volume, 50 μl) was supplemented with 8 μM tRNA^Leu, 200 μM ATP, and 2 U/ml PPiase to initiate the process of aminoacylation. After 30 min of further incubation at 37°C, the malachite green reagent was added and the absorbance was recorded as described above. The reactions performed in the absence of inhibitor were taken as a control.

Molecular constructs for the replacement of LRS alleles.

For the inactivation of the LdLRS gene, a targeted gene replacement strategy based on PCR fusion was employed (38). Briefly, LRS gene flanking regions were amplified and fused, using PCR, to the hygromycin phosphotransferase gene (HYG) or neomycin phosphotransferase gene (NEO). The 5′ UTR (1.2 kb) of the LdLRS gene was PCR amplified using primers A and B_HYG or A and B_NEO (Table 3). The NEO gene was amplified from pX63-NEO with primers C_NEO and D_NEO. The HYG gene was amplified from pX63-HYG with primers C_HYG and D_HYG (Table 3). The 3′ UTR (912 bp) of the LdLRS gene was obtained by PCR amplification using the primers E_HYG/E_NEO and reverse primer F (Table 3). The 5′ UTR of the L. donovani LRS gene was then ligated to either of the antibiotic resistance marker genes by PCR using primers A and D_HYG or primers A and D_NEO. This fragment (the 5′ UTR marker gene) was then fused with the 3′ UTR using primers A and F, yielding the fragment 5′ UTR-Hyg-3′ UTR or 5′ UTR-Neo-3′ UTR.

TABLE 3.

Primers used for generation of hygromycin- and neomycin-specific linear replacement cassette fragments

Primera	Sequence
A	5′-TTGAAGGCTCACATGTCGCGTA-3′
BHYG	5′-GGTGAGTTCAGGCTTTTTCATGTTCTTCTCCAGCTGCGCCTTCA-3′
BNEO	5′-CAATCCATCTTGTTCAATCATGTTCTTCTCCAGCTGCGCCTTCA-3′
CHYG	5′-TGAAGGCGCAGCTGGAGAAGAACATGAAAAAGCCTGAACTCACC-3′
CNEO	5′-TGAAGGCGCAGCTGGAGAAGAACATGATTGAACAAGATGGATTG-3′
DHYG	5′-GAAGAGGGTAGACACCCCAACGTCTATTCCTTTGCCCTCGGACGAG-3′
DNEO	5′-GAAGAGGGTAGACACCCCAACGTTCAGAAGAACTCGTCAAGAAG-3′
EHYG	5′-CTCGTCCGAGGGCAAAGGAATAGACGTTGGGGTGTCTACCCTCTTC-3′
ENEO	5′-CTTCTTGACGAGTTCTTCTGAACGTTGGGGTGTCTACCCTCTTC-3′
F	5′-ATGCTTGTCCCGTTCGTGTCGT-3′
G	5′-TTTTTCTAGAATGTCCACGGCACGTCGCGATG-3′
H	5′-TTTTTTCATATGCTACTCGCGCTTCACTACTGG-3′

^aHYG, hygromycin; NEO, neomycin.

In order to generate an episomal complementation construct, the full-length LRS coding sequence was amplified with a forward primer harboring the XbaI site (primer G) and a reverse primer harboring the NdeI site (primer H) (Table 3). This amplified product was then cloned into the pSP72α-zeo-α vector to get the pSP72α-zeo-α-LRS complementation construct. All the fragments and constructs were sequenced for confirmation.

Generation of genetically attenuated parasites.

After PCR amplification and purification, ∼2 μg of linear replacement cassette fragment 5′ UTR-Hyg-3′ UTR or 5′ UTR-Neo-3′ UTR was individually transfected by electroporation into the wild-type L. donovani promastigotes (39). Depending on the presence of the marker gene, transfectants were subjected to antibiotic selection. Cells resistant to antibiotic selection were subjected to PCR-based analysis using the primers shown in Table 4 to check for the correct integration of the replacement cassettes. Thereafter, the second round of transfection was initiated to knock out the other copy of the LRS gene. The genotypes of the mutants were confirmed by Southern analysis using standard protocols (40). Transfection of WT and LRS/HYG or LRS/NEO heterozygous parasites with the pSP72α-zeo-α-LRS episome led to the generation of LRS-overexpressing cell line WT(pLRS⁺) and LRS-complemented LRS/HYG(pLRS⁺) or LRS/NEO(pLRS⁺) heterozygous promastigotes, respectively.

TABLE 4.

Primers used for molecular characterization of the genetically manipulated parasites by PCR-based analysis

Primer	Sequence
1	5′-TGTAGAAGTACTCGCCGATAGTGG-3′
2	5′-GTTTCAGGTGGCACCAGAAGA-3′
3	5′-CGCAGCTATTTACCCGCAGGACAT-3′
4	5′-TGGCCTTTAGCGTCTTCAGCGT-3′
5	5′-ATAGCGTTGGCTACCCGTGATATTGC-3′
6	5′-AACACGGCGGCATCAGAGCAGCCGATTG-3′
7	5′-TCATCGACGATGCCGTGATGTT-3′
8	5′-TACATGAACGGCAAGCTTCACCT-3′

Growth and infectivity assay.

The growth rate experiments were conducted by inoculating stationary-phase parasites at a density of 1 × 10⁶ cells/ml in M199 medium with 5% FBS in 25-cm² flasks without a particular selection drug at 22°C. The growth rate of each culture was determined at 24-h intervals by using a Neubauer hemocytometer. Growth studies with individual cell lines were done at least three times, and similar results were consistently obtained.

For the infectivity assay, cells of the J774.A1 murine macrophage cell line were plated at a density of 5 × 10⁵ cells/well in a 6-well flat-bottom plate. The adherent cells were infected with stationary-phase promastigotes at a ratio of 20:1 for 5 h. Excess nonadherent promastigotes were removed by incubation of the cells for 30 s in phosphate-buffered saline (PBS). These were subsequently maintained in RPMI 1640 containing 10% FBS at 37°C with 5% CO₂. Giemsa staining was performed to visualize the intracellular parasite load.

Isothermal titration calorimetry (ITC).

Experiments were performed using an ITC₂₀₀ microcalorimeter (Malvern, UK) with a cell volume of 200 μl and a syringe volume of 40 μl. All the experiments were carried out in 10 mM phosphate buffer (pH 7.5) at 25°C. The syringe was loaded with the ligand (leucine, isoleucine, norvaline, or AN2690) at a concentration of 400 to 800 μM, and the sample cell was filled with protein at 50 to 100 μM. Out of the total of 20 injections, the initial injection was 0.1 μl, followed by 19 identical injections, each of 2 μl. Each experiment was accompanied by a corresponding control experiment with the ligand in the buffer. The data from which the baseline values were subtracted were finally analyzed using Origin software to obtain binding enthalpies (ΔH) and binding constants (K_a) in a fashion similar to that previously described (41). The solid line represents the best fit of the experimental data to the single-site model yielding the association constant (K_a), the enthalpy change (ΔH°), and the molar binding stoichiometry (N). From this, the equilibrium dissociation constant, K_d (where K_d = 1/K_a), was determined.

Inhibitor assays.

In order to determine the susceptibility profile of the L. donovani wild-type and mutant promastigotes to the inhibitors (AN2690 and miltefosine), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay was performed as described previously (42). Briefly, log-phase promastigotes (5 × 10⁴ cells/well) were seeded in a 96-well flat-bottom plate (Nunc) and incubated with different drug concentrations at 22°C. After 72 h of incubation, 10 μl of MTT (5 mg/ml) was added to each well, and the plates were incubated at 37°C for 3 h. The reaction was stopped by the addition of 50 μl of 50% isopropanol and 20% SDS, followed by gentle shaking at 37°C for 30 min to 1 h. The absorbance at 570 nm was measured in a microplate reader (SpectraMax M2; Molecular Devices).

The susceptibility of the wild-type amastigotes to AN2690 and miltefosine was determined by visualization of the intracellular parasite load using Giemsa staining of infected J774.A1 murine macrophages at 72 h after treatment with different concentrations of the drug. Cells of the J774.A1 murine macrophage cell line were plated at a density of 5 × 10⁵ cells/well in a 6-well flat-bottom plate. The adherent cells were infected with stationary-phase promastigotes at a ratio of 20:1 for 5 h. Excess nonadherent promastigotes were removed by incubation of the cells for 30 s in phosphate-buffered saline (PBS). These were subsequently maintained in RPMI 1640 containing 10% FBS at 37°C with 5% CO₂. Giemsa staining was performed to visualize the intracellular parasite load.

In vivo drug sensitivity assay.

Six- to 8-week-old BALB/c mice were injected intravenously with approximately 1 × 10⁸ stationary-phase L. donovani promastigotes and segregated into groups of 5 mice each. Treatment was initiated at 7 days p.i. Groups of mice were treated with either the drug vehicle (2% [vol/vol] ethanol–5% [wt/vol] dextrose in sterile water), miltefosine (25 mg/kg in sterile water), or AN2690 (11 mg/kg in 2% [vol/vol] ethanol–5% [wt/vol] dextrose in sterile water). Both the drugs and the vehicle were administered orally once daily for 5 consecutive days. On the fourth day after completion of the treatments, the animals were humanely euthanized, the spleens were dissected and weighed, and impression smears were made, followed by fixation with methanol and Giemsa staining. The parasite burden was estimated by counting the number of parasites in the splenic smears prepared after euthanasia. Parasite burdens were determined by the number of amastigotes per 500 spleen cells × organ weight (in milligrams) (Leishman-Donovan units [LDU]) and compared to the results for the treated and untreated groups. Percent efficacy was calculated by the equation [1 − (mean number of amastigotes in treated mice/mean number of amastigotes in untreated mice] × 100.

For additional confirmation of the parasite load in mouse spleen tissues, real-time PCR was performed using total DNA isolated from the spleen tissues of each mouse as a template. DNA isolation from the tissue samples was done using a DNeasy tissue kit (Qiagen) in accordance with the manufacturer's protocol. The target was a 120-bp DNA fragment of the minicircle kDNA of L. donovani. Detection was done by amplification of this region using Leishmania-specific primers (43, 44) (forward primer, 5′-CCTATTTTACACCAACCCCCAGT-3′ [JW11]; reverse primer, 5′-GGGTAGGGGCGTTCTGCGAAA-3′ [JW12]). For the real-time PCR assay, 10 ng of template was used in a final reaction volume of 7 μl with 3.5 μl of SYBR green quantitative PCR master mix (Applied Biosystems, CA, USA) and 1 μM forward and reverse primers in an Applied Biosystems 7500 Fast real-time PCR system in 96-well plates. The reaction conditions were as follows: a first step at 50°C for 2 min and an initial activation step at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. After the 40 cycles, melting curves were generated along with the mean C_T values. Results were expressed using the 2^-ΔΔCT method. The experiment was repeated twice in triplicate.

Ethics statement.

Six-week-old female Swiss Albino mice were used for the generation of the anti-LdLRS antibody. Six- to 8-week-old female BALB/c mice were used for the in vivo drug sensitivity assay. Animal experiments were performed according to the guidelines approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forest, Government of India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Jawaharlal Nehru University (JNU) (IAEC code number 15/2017).

Statistical analysis.

Data are represented as the mean ± standard deviation (SD). A P value of <0.05 was accepted as an indication of statistical significance. Results for the infectivity assay and enzyme activity in the cell lysates were entered as column data in GraphPad Prism (version 5.0) software (GraphPad Software, Inc.) and analyzed by Student's two-tailed t test.