Leishmania donovani Dipeptidylcarboxypeptidase Inhibitor as a Potential Oral Treatment for Visceral Leishmaniasis

ABSTRACT

Chemotherapy is the key intervention to control visceral leishmaniasis (VL), a neglected tropical disease. Current regimens include not only a few drugs but also present several drawbacks, including moderate to severe toxicity, cost, long-term administration, patient compliance, and growing drug resistance. Thus, the need for better treatment options against VL is a priority. In an endeavor to find an orally active and affordable antileishmanial agent, we evaluated the therapeutic potential of compounds belonging to the (2Z,2′Z)-3,3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-ones) series, identified as inhibitor(s) of Leishmania donovani dipeptidylcarboxypeptidase, a novel drug target. Among them, compound 3c exhibited best in vivo antileishmanial efficacy via both intraperitoneal and oral routes. Therefore, the present study led to the identification of compound 3c as the lead candidate for treating VL.

INTRODUCTION

Leishmaniasis, a neglected disease caused by protozoan parasites of the genus Leishmania, is endemic in 98 countries, with a global incidence estimated at approximately 0.9 to 1.6 million cases occurring each year (1). Human visceral leishmaniasis (VL) results from infection with Leishmania donovani and Leishmania infantum and is usually fatal if left untreated, as evident from more than 50,000 deaths per year (2, 3). The control of VL relies mainly on chemotherapy with pentavalent antimonials, pentamidine, paromomycin, amphotericin B (or its lipid formulations), and miltefosine (4). However, the management of the disease control is hindered by high costs, toxicity, long-term administration, and parasite resistance (5, 6). Over the past few years, the lack of new medicines has become a global concern (7). Therefore, there is an urgent need for developing safe, effective, and low-cost oral drugs for the cure of VL.

A rational approach to develop new chemotherapeutic agents is based on the identification of drug targets and their specific inhibitor that can affect parasite survival. Aiming at the identification of compounds as new leads against VL, we focused on the identification of inhibitors of L. donovani dipeptidylcarboxypeptidase (LdDCP) enzyme, a novel drug target. Dipeptidylcarboxypeptidase is an angiotensin-converting enzyme (ACE)-related metallopeptidase (8) that belongs to the M3 family of mono-zinc peptidases and is attributed to cleaving N-benzoyl-l-glycyl-l-histidyl-l-leucine (Hip-His-Leu [HHL]), a substrate required by ACE to release hippuric acid. Interestingly, captopril, a mammalian ACE inhibitor, inhibited not only LdDCP enzyme activity but also in vitro parasite growth. These observations suggested that LdDCP may have a role in parasite growth. A three-dimensional model of LdDCP was generated based on the crystal structure of Escherichia coli DCP by means of comparative modeling (9), and a virtual screening approach was applied to identify potential inhibitors for LdDCP using our chemical library (i.e., the CSIR-Central Drug Research Institute) of 15,452 compounds. Four compounds belonging to two chemical classes were identified as potential LdDCP inhibitors (10). Out of these four compounds, three compounds 3a-3c belonged to the series I (2Z,2′Z)-3,3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-one), whereas compound 4 belonged to series II (3,5-disubstituted isoxazole). Notably, we have earlier reported the antioxidant and hypolipidemic activity of compounds belonging to series I, whereas compounds belonging to the isoxazole series have displayed a antithrombotic effect (11, 12). These chemically diverse compounds not only inhibit parasite enzyme LdDCP but also elicit in vitro antileishmanial activity (10). Series I was further explored to identify a clinical candidate for treating VL.

In the present study, a series of nine compounds belonging to (2Z,2′Z)-3,3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-one) (1–12) were synthesized and evaluated for their antileishmanial activity against L. donovani, an etiologic agent of VL in India (Fig. 1). The in vitro toxicity of these compounds against mammalian macrophage cells was also studied to evaluate the selectivity index (SI). Further, in vivo efficacy of active compounds was determined in the L. donovani/golden hamster chronic disease model. Among them, compound 3c was found to exhibit the best in vivo efficacy when administered by both intraperitoneal and oral routes.

FIG 1.

Synthesis of N,N′-bis(1-aryl-6-hydroxy-hex-2-ene-1-one-3-yl)1,n-alkanediamines.

RESULTS

Earlier reports from our laboratory established that four compounds belonging to two chemical classes exhibited inhibition of both parasite DCP enzyme activity as well as growth (10). Table 1 depicts the in vitro antileishmanial activities of nine newly synthesized compounds belonging to chemical series I and compound 3c (10). Two compounds, 3k and 3l, did not show any in vitro antileishmanial activity against both stages of the parasite. Interestingly, four of five (3c, 3e, 3h, 3i, and 3j) active compounds inhibited the parasite DCP enzyme with K_i values in the range of 22 to 28 nM. However, none of them exhibited any effect on mammalian ACE at the same concentration. The study hence provided the proof of concept that compounds belonging to series (2Z,2′Z)-3,3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-one) are the inhibitors of LdDCP enzyme and have potential for treating visceral leishmaniasis.

TABLE 1.

In vitro evaluation of compound 3c and its analogues^a

CDRI compound	IC50 (μg/mL)		CC50 (μg/mL)	SI	Ki
	Promastigote	Amastigote			DCP (nM)	ACE (μM)
3c	21.77 ± 2.23	14.29 ± 0.83	58.35 ± 2.29	4.08	25 ± 5.00	147.6 ± 6.20
3d	17.61 ± 0.18	24.59 ± 0.11	34.10 ± 4.24	1.38	480 ± 0.05	43 ± 0.28
3e	13.84 ± 1.74	11.86 ± 0.58	46.30 ± 0.51	3.90	270 ± 0.02	12.9 ± 0.28
3f	27.18 ± 1.91	6.69 ± 0.27	40.40 ± 0.75	6.00	ND	ND
3g	6.80 ± 0.04	3.43 ± 0.06	15 ± 0.81	4.37	260 ± 0.01	34.25 ± 2.30
3h	1.57 ± 0.05	8.84 ± 0.51	64.20 ± 3.89	7.26	25 ± 7.07	7 ± 1.40
3i	1.43 ± 0.13	5.16 ± 0.07	62.50 ± 2.64	12.10	23 ± 2.80	15.1 ± 1.40
3j	1.03 ± 0.08	6.36 ± 0.11	77.10 ± 3.20	12.12	28 ± 7.00	39 ± 6.30
3k	>100	>50	ND	ND	ND	ND
3l	>100	>50	ND	ND	ND	ND
Sodium stibogluconate	915.33 ± 3.05	172 ± 2.64	6,020 ± 0.03	35	ND	ND
Miltefosine	1.02 ± 0.14	3.77 ± 0.17	21.73 ± 1.10	5.76	ND	ND

^aIC₅₀, CC₅₀, and K_i values are presented as the means ± the SD of at least three independent experiments. The selectivity index (SI) for each compound was calculated as the ratio between the cytotoxicity (CC₅₀) and activity (IC₅₀) against L. donovani amastigotes. Sodium stibogluconate and Miltefosine were used as standard control drugs. ND, not done.

Except for compound 3d, all the five identified compounds (3e to 3j), along with previous active compounds (3a to 3c) (10), were tested for their in vivo efficacy at 50 mg/kg/day for 5 days via intraperitoneal (i.p.) route (Table 2). Compounds 3a to 3c and compounds 3g to 3j exhibited antileishmanial efficacy in the range of 58 to 91%, whereas compounds 3e and 3f were highly toxic, since the animals did not survive after the last dose administered. Interestingly, compound 3c emerged as the most effective molecule since it reduced the parasitic load in the spleen on day 28 of last administration by 86%, followed by compounds 3j and 3i, which also exhibited comparable efficacies. On the other hand, the standard drug, sodium stibogluconate at 20 mg/kg (i.p.), resulted in an 85% inhibition of the parasite load.

TABLE 2.

In vivo efficacy of compounds in L. donovani/golden hamster model via intraperitoneal route

Compound	Mean % inhibition (50 mg/kg i.p.) ± the SDa
	Day 7	Day 28
3a	63.70 ± 13.60	61.50 ± 26.00
3b	66.50 ± 4.90	39 ± 8.40
3c	91.57 ± 2.61	86.71 ± 7.14
3d	ND	ND
3e	NA	NA
3f	NA	NA
3g	58.50 ± 8.66	69 ± 10.39
3h	73.93 ± 8.87	58.30 ± 4.28
3i	87.63 ± 3.91	77.93 ± 7.66
3j	89.97 ± 3.77	83.20 ± 3.42
Sodium stibogluconateb	81.37 ± 5.49	85.30 ± 9.37

^aND, not done; NA, not available (since the animals did not survive after the last administration).

^bSodium stibogluconate at 20 mg/kg (i.p.) was used as the standard control drug.

Three most active compounds—3c, 3i, and 3j—were tested for their in vivo efficacy via the oral route at a 100-mg/kg dose for five consecutive days (Table 3). Interestingly, compound 3c exhibited a very high efficacy compared to compounds 3i and 3j. Maximum inhibition (91 to 93%) was obtained on day 7 after last administration; this was maintained up to day 28, whereas compounds 3i and 3j exhibited 85 to 90% inhibition of the parasite load on day 7 of the last administration, but inhibition was reduced to 70 and 54%, respectively, on day 28, suggesting recrudescence. The data clearly indicate that compounds 3i and 3j were not able to cure animals. Table 4 depicts the dose-dependent in vivo antileishmanial efficacy of compound 3c. Three dose regimens were tested for both i.p. (50, 25, and 10 mg/kg for 5 days) and oral (100, 50, and 25 mg/kg) routes of administration. The compound exhibited dose-dependent inhibition of the parasite burden after both i.p. or oral administration on day 7 that was maintained up to day 28 of the last administration (Table 4). Therefore, compound 3c was identified as the most active compound in the series that can be taken up for further development as a potent antileishmanial agent. Interestingly, compound 3c antileishmanial efficacy was also comparable to miltefosine, the only available oral antileishmanial drug.

TABLE 3.

In vivo efficacy of compound 3c and analogues in L. donovani/golden hamster model administered via oral route^a

Compound	Dose regimen (mg/kg)	Mean % inhibition ± the SD
		Day 7	Day 28
3c	100	91.22 ± 3.69	93.35 ± 2.32
3i	100	85.54 ± 12.06	69.36 ± 11.86
3j	100	90.22 ± 5.18	54.06 ± 12.81
Miltefosineb	25	91.57 ± 3.20	96.56 ± 1.50

^aSix animals were used in each group.

^bMiltefosine was used as an oral control drug.

TABLE 4.

Dose-dependent in vivo efficacy of compound 3c via intraperitoneal and oral routes^a

Route of administration	Compoundb	Dose (mg/kg)	Mean % inhibition of parasite load ± the SD
			Day 7	Day 28
Intraperitoneal	Compound 3c	50	91.57 ± 2.61	86.71 ± 7.14
		25	78.57 ± 6.65	73.00 ± 5.81
		10	72.23 ± 6.22	70.16 ± 13.64
	Sodium stibogluconate	20	81.37 ± 5.49	85.37 ± 9.37
Oral	Compound 3c	100	92.43 ± 2.41	90.23 ± 4.70
		50	76.16 ± 6.79	72.24 ± 7.60
		25	45.66 ± 3.59	54.59 ± 18.15
	Miltefosine	25	91.57 ± 3.20	96.50 ± 0.55

^aSix animals were used in each group.

^bSodium stibogluconate and miltefosine were used as standard control drugs.

In the survival study, all animals in the infected control group died within 74 days. On the other hand, 100% of infected animals treated with compound 3c and miltefosine remained alive until the termination of the experiment, i.e., 150 days (Fig. 2). No death was also observed in normal healthy controls.

FIG 2.

Survival of animals in groups, uninfected, infected, and treated with 3c and miltefosine. Survival was observed up to 150 days. Six animals in each experimental group were used for the investigation.

DISCUSSION

Leishmaniasis is one of the most neglected tropical diseases in terms of drug discovery and development. Most antileishmanial drugs are either highly toxic or have issues pertaining to resistance and also require hospitalization. Therefore, they are inadequate for use in the field. Recent improvements have been achieved by combination therapy, reducing the time and cost of the treatment. Nonetheless, new orally active drugs are still urgently needed (6, 13). An ACE-related dipeptidylcarboxypeptidase (LdDCP) was identified in L. donovani (8) and was established as a novel drug target for the identification of new chemical entities (9). Using virtual screening approach, three compounds (3a to 3c) belonging to class (2Z,2′Z)-3,3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-one) were identified as potential inhibitors of LdDCP with no effect on mammalian ACE. These compounds appeared to be promising hits against L. donovani with IC₅₀ values that measured in the range of 3.97 to 16.29 μg/mL (10). To demonstrate the proof of concept, nine compounds were synthesized and evaluated in vitro against promastigotes and intracellular amastigotes. Except for two compounds, seven showed potential antileishmanial activity with the IC₅₀ measured in the range of 3.43 to 24.59 μg/mL (Table 1). Three of these compounds also inhibited recombinant LdDCP at nM concentrations (K_i in the range of 25 to 28 nM) comparable to that for compound 3c (10). Inhibition of mammalian ACE by these compounds was achieved at 1,000-fold higher concentrations, i.e., in the μM range (Table 1). These results demonstrate that the identified compounds are selective inhibitors of parasite enzyme.

To establish that the identified compounds indeed have the potential to be developed as antileishmanial agents, these promising compounds were evaluated for their in vivo antileishmanial efficacy in L. donovani/hamster model treated via the i.p. route. The hamster model has been shown to exhibit remarkable resemblance to active human VL (14) in terms of hepatosplenomegaly, a relentless increase in the visceral parasite burden, progressive cachexia, bone marrow dysfunction, immune-depression, and eventually death (15). Since intravenous administration route is not possible in the hamsters because of small tail, i.p. route was followed to evaluate in vivo efficacy.

The main checkpoint safety criterion for a compound is the SI, which should be ≥5.0 (selected on the basis of miltefosine, the only oral drug). The results of the present study demonstrated an interesting deviation from the accepted normal criteria. Compounds 3e with an SI of 3.90 and compound 3f with an SI of 6.0 were found to be very toxic as all animals died before day 7 of the last administration. On the other hand, the most active lead compound 3c with a low SI (~ 4) not only exhibited a significant inhibition in parasite burden in spleen on day 28 of the last treatment but also exhibited an increased life span. No death was observed until the termination of experiment, i.e., at 150 days (Fig. 2). Similarly, no casualty was observed in healthy uninfected controls and miltefosine (SI = 5.0)-treated animals. In accordance with this, animals treated with either compound 3c or miltefosine exhibited weight gain comparable to that of noninfected healthy controls (data not shown). Another two compounds (3i and 3j), like compound 3c, exhibited good efficacy on day 7 of the last administration (85 to 90%) but failed to maintain their antileishmanial activity up to day 28 of the last administration. The observation clearly indicated that animals treated with compounds 3i and 3j not being cured may be due to recrudescence and thus were not suitable for further study. The rank of potency was as follows: 3c > 3j > 3i > 3h. Further, compound 3c also showed a very good dose-dependent antileishmanial efficacy when administered both i.p. and orally on day 7 of the last administration (Table 4), which was maintained on day 28 posttreatment even at lower doses. Therefore, compound 3c was the most potent antileishmanial lead compound. The data clearly suggested that the compounds belonging to (2Z,2′Z)-3,3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-one) could be developed as novel antileishmanial agents.

In conclusion, the (2Z,2′Z)-3, 3′-(ethane-1,2-diylbis(azanediyl))bis(1-(4-halophenyl)-6-hydroxyhex-2-en-1-one) series provided potent antileishmanial agents, and compound 3c showed exciting potential as a clinical candidate to be developed for the treatment of visceral leishmaniasis.

MATERIALS AND METHODS

Parasite, cell lines, and animals.

The L. donovani promastigote Dd8 strain (MHOM/IN/80/Dd8) originally obtained in the form of promastigotes from the late P. C. C. Granham, London, United Kingdom, were used as infective agent. The strain is maintained at the CSIR-Central Drug Research Institute in golden hamsters by regular subpassages. Promastigotes tagged with luciferase gene (16), and maintained at 25 ± 1°C in medium 199 (Sigma Chemical) supplemented with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel) and G418 (100 μg/mL) were used for antipromastigote activity.

Mouse macrophage cell line J774A.1 obtained from National Centre for Cell Sciences (NCCS; Pune, India) were grown and maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic and antimycotic solution (penicillin [10,000 U/mL], streptomycin [10 mg/mL], and amphotericin B [25 μg/mL]; Sigma A5955) at 37°C with 5% CO₂.

Male hamsters (Mesocricetus auratus), bred in-house and weighing 40 to 45 g, were used. All in vivo experiments were carried out under a license from the CSIR-CDRI’s institutional animal ethics committee (approval IAEC/2009/124). The test animals were housed in animal quarters under controlled conditions. They were fed standard rodent pellets and had free access to drinking water. Animals were sacrificed using deep ether anesthesia during or after the studies.

Chemical synthesis of compounds.

The synthesis of compounds belonging to series I was performed according to a previously reported procedure (11). Treating acetophenone with γ-butyrolactone in the presence of sodium methoxide resulted in intermediate 2. The reaction of the intermediate 2 with several diaminoalkanes in the presence of boron trifluoride etherate resulted in the formation of compound 3 (Fig. 1). The physical and spectroscopic details for the synthesized compounds can be found in the supplemental material.

In vitro and in vivo evaluation of the antileishmanial activity.

(i) Antipromastigote activity. The in vitro effects of compounds and the nanoformulation on the growth of promastigotes were assessed by monitoring the luciferase activity of viable cells after treatment (16). Briefly, the transgenic promastigotes of the late log phase were seeded at 5 × 10⁵ cells/well in 96-well plates and incubated for 72 h in medium alone or in the presence of serially diluted compounds (1 to 100 μg) or nanoformulation. After incubation, an aliquot (50 μL) of promastigote suspension was aspirated from each well and mixed with an equal volume of Steady-Glo reagent (Promega), and luminescence was measured. The values are expressed as relative luminescence units (RLU).

(ii) Antiamastigote activity. Macrophage cells (J-774A.1) were seeded in a 96-well plate (5 × 10⁴ cells/200 μL/well) in RPMI 1640 containing 10% fetal calf serum and incubated at 37°C in a CO₂ incubator. After 24 h, the medium was replaced with fresh medium containing stationary-phase promastigotes expressing firefly luciferase (2.5 × 10⁵/200 μL/well) and allowed to infect macrophages for 24 h. The test compound(s) was added at appropriate concentrations (500 ng to 100 μg/mL) in complete medium and then incubated at 37°C in a CO₂ incubator for 48 h. After incubation, the medium was replaced with 50 μL of phosphate-buffered saline and mixed with an equal volume of the Steady-Glo reagent. After gentle shaking for 1 to 2 min, the reading was taken using a luminometer (16).

Cytotoxicity assay.

The macrophage cell viability was determined using the MTT assay (17). Exponentially growing cells (J774A.1; 1 × 10⁵ to 2 × 10⁵ cells/100 μL/well) were incubated with test compounds (at 3-fold dilutions up to 7 points) in complete medium at 37°C with supply of 5% CO_2. Control wells received only vehicle (dimethyl sulfoxide [DMSO]). After 72 h of incubation, 25 μL of MTT reagent (5 mg/mL) was added to each well, followed by mixing and incubation at 37°C for 2 h. The medium was then removed without disturbing the cell layer, and 150 μL of pure DMSO was added. The sample was then mixed for 15 min, and the absorbance was recorded at 544 nm on a microplate reader. The CC₅₀ values were estimated as described by Huber et al. (18). The SI for each compound was calculated as the ratio between the cytotoxicity (CC₅₀) and the activity (IC₅₀) against leishmania amastigotes.

In vitro effect on enzyme.

Recombinant leishmanial DCP was expressed in E. coli BL21(DE3)/pLys and purified to homogeneity as described previously (8, 19). The enzyme activity was measured according to the method of Cushman and Cheung (20) using N-benzoyl-l-glycyl-l-histidyl-l-leucine (HHL) as the substrate. To determine the K_i values of the selected compounds, different concentrations of compounds were in vitro incubated with the enzymes LdDCP and ACE for 5 min prior to the addition of the substrate. Rat serum was taken as the source of mammalian ACE. The protein concentration was determined by the Bradford method using bovine serum albumin as the standard (21).

In vivo antileishmanial efficacy evaluation.

For in vivo evaluation of chemotherapeutic agents, we used a modified method (22), where the delayed action of the drug can also be assessed by conducting repeated spleen biopsies on the same animal at different intervals (days 7 and 28) without sacrifice. This is a more rational approach since it gives comprehensive information regarding cure, toxicity, and survival of the treated animals and explains the sequential effects of the drug in the model (23). Male golden hamsters (inbred strain), weighing 40 to 45 g, were intracardially infected with 1 × 10⁷ amastigotes isolated from the infected spleen (24). Infection (amastigotes/500 cell nuclei) was first monitored in spleen dab smears on day 20 postinfection after Giemsa staining, and then animals with a +1 infection (5 to 15 amastigotes/100 spleen cell nuclei) were randomized into several groups of six animals each for chemotherapeutic trials. Drug treatment, i.p. or orally, was initiated after 2 days after the first biopsy and continued for 5 consecutive days. Posttreatment splenic biopsies were performed on the days 7 and 28 of the last drug administration, and amastigote counts/500 cell nuclei were assessed using spleen dab smears after Giemsa staining. The efficacy was expressed in terms of the percent inhibition as follows: PI = 100 – [ANAT × 100/(INAT × TIUC)], where PI is the percent inhibition of amastigote multiplication, ANAT is an actual number of amastigotes in treated animals, INAT is the initial number of amastigotes in treated animals, and TIUC is the fold increase of parasites in untreated control animals.

To evaluate the effect of the compound on animal survival, four groups (six hamsters per group)—uninfected healthy controls, infected controls, infected animals treated for 5 days with 3c (100 mg/kg), and infected animals treated for 5 days with miltefosine (30 mg/kg)—were used. The physical conditions and survival of individual hamster belonging to all the groups were recorded up to 150 days, and the mean survival period was also calculated.