bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 2 Evaluation of modulators of cAMP-response in terms of their impact on cell
3 cycle and mitochondrial activity of Leishmania donovani
4 Amrita Saha1, Anindita Bhattacharjee2, Amit Vij1, Pijush K. Das1, Arijit Bhattacharya3*,
5 Arunima Biswas1*
6 1 Infectious Diseases and Immunology, CSIR-Indian Institute of Chemical Biology, Kolkata, INDIA,2
7 Department of Zoology, Cell and Molecular Biology Laboratory, University of Kalyani, Kalyani, INDIA,
8 3 Department of Microbiology, School of Life Sciences and Biotechnology, Adamas University,
9 Kolkata, INDIA
10 *Correspondence
11 Dr.Arunima Biswas
12 Cell and Molecular Biology Laboratory
13 Department of Zoology
14 University of Kalyani
15 Kalyani, Nadia 741235
16 INDIA
17 email: [email protected], [email protected]
18 Phone: 9836793528
19 Dr. Arijit Bhattacharya
20 Department of Microbiology
21 School of Life Sciences and Biotechnology
22 Adamas University, Kolkata-700126, INDIA
23 email: [email protected], [email protected]
24 Phone: 9830248166
25 Running title: Profiling cAMP-modulators against Leishmania
26 Key words: Leishmania; cAMP; Phosphodiesterase; cell cycle; motility; etazolate
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1 Abstract
2 With the identification of novel cAMP binding effecter molecules in Trypanosoma,
3 role of cAMP in kinetopalstida parasites gained an intriguing break through. Despite
4 earlier demonstrations of role of cAMP in survival of Leishmania during macrophage
5 infection, there is essential need to specifically clarify involvement of cAMP in
6 various cellular processes in the parasite. In this context, we sought to gain a
7 comprehensive understanding of the effect of cAMPanalogs and cAMP-
8 phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites.
9 Administration of both hydrolysable (8-pCPT-cAMP) and non-hydrolysable analogs
10 (Sp-8-pCPT-cAMPS) of cAMP resulted in significant decrease of Leishmania
11 proliferation. Amongst the various PDE inhibitors, etazolate was found to be potently
12 anti-proliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study
13 revealed that both cAMP analogues and selective PDE inhibitors resulted in
14 significant cell cycle arrest at G1 phase with reduced S-phase population.
15 Furthermore, careful examination of the flagellar motility patterns revealed
16 significantly reduced coordinated forward flagellar movement of the promastigotes
17 with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and
18 PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial
19 membrane potential. Treatment of etazolate at subcytotoxic concentration to infected
20 macrophages significantly reduced parasite burden and administration of etazolate
21 to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite
22 burden. Collectively, these results imply involvement of cAMP in various crucial
23 processes paving the avenue for developing potent anti-leishmanial agent.
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1 Author Summary
2 Leishmania donovani is the causative agent of fatal Visceral Leishmaniasis. The 3 current available medications are toxic, expensive and require long term daily 4 administrations. With an aim to develop improved therapeutic, components of cAMP 5 homeostasis, particularly cAMP-phosphodiesteares, has been targeted for 6 Leishmania and other kinetoplastid pathogens. cAMP plays diverse roles in 7 functional processes involved in cell division, transition into different stages of the 8 life cycle of Leishmania and motility. In this study, the authors found administration of 9 both hydrolysable and non-hydrolysable analogs of cAMP and certain PDE inhibitors 10 resulted in remarkable decrease proliferation with considerable cytopathic impact on 11 promastigotes. The mammalian phosphodiestearse inhibitor etazolate caused 12 significant reduction in parasite load in L. donovani infected macrophages and 13 demonstrated considerable reduction of liver and spleen parasite burden in in vivo 14 mouse infection model. The study suggested that etazolate, with its slightest impact 15 on mammalian host, can be repurposed for developing effective anti-leishmanials.
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3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Introduction
2 Leishmaniasis defines an array of diseases including self-healing cutaneous
3 leishmaniasis and fatal visceral leishmaniasis caused by various species of
4 protozoan parasite Leishmania. Till date, the disease is considered as a neglected
5 tropical disease and urgently requires new therapeutic interventions. For
6 leishmaniasis, pentavalent antimonials have remained the standard medication till
7 recent decades, but drug resistance is becoming an increasing problem in the
8 disease prone areas [1]. Although a number of new compounds, such as miltefosine
9 or amphothericin B, have been developed over the last few years [2–6], effective,
10 safe and cost-efficient chemotherapy of leishmaniases still remains elusive. It has
11 been currently postulated that cAMP plays an important role in various cellular
12 processes linked with survival and virulence of kinetopalstida parasites [7,8]. In
13 Trypanosoma brucei detailed studies revealed that hydrolysed product of cAMP as
14 well as various isoenzymes of cyclic nucleotide phosphodiesterases (PDE) and
15 adenylatecyclase (AC) have an important role in parasite survival, virulence and cell-
16 cell communications [7,8]. Moreover, the isoform PDEC from T. cruzi was shown to
17 be involved in osmoregulation and the potential of it to be a drug target was validated
18 by high throughput screening of a series of PDE inhibitors [9], which led to
19 identification of tetrahydrophthalazinone compound A (Cpd A) [7]. These
20 observations underpin the possibilities of cAMP pathway as a candidate target for
21 drug development [9].
22 Leishmania differs in terms of its repertoire of cAMP-pathway components from T.
23 brucei as the number of AC genes is considerably less. Additionally it encodes one
24 cytosolic heme-containing AC which is absent in Trypansoma, indicating presence of
25 subcellular microdomain specific cAMP signalling in this parasite [10,11]. Elevation
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1 of cAMP was found to be a prerequisite for differentiation condition induced cell cycle
2 arrest (G1) and adaptation against oxidative stress encountered during early
3 macrophage invasion [12,13]. Moreover, the crucial role played by both exogenous
4 and endogenous cAMP in chemotaxis by sensing chemical cues and inducing
5 flagellar wave reversal [14] suggest the existence of definitive cAMP regulated
6 mechanisms in the parasite. Similar to other kinetoplastida parasites, Leishmania
7 encodes at least 7 different cyclic nucleotide targeted phosphodiesterases (PDE). X-
8 ray crystallography structure of L. major PDEB1 and B2 has been determined and
9 it’s superposition with human PDEs revealed presence of a unique sub pocket
10 nearby inhibitor binding sites [15], widening the scope of selective drug designing.
11 The differentially regulated L. donovani PDEA was characterized to be a high KM
12 cytosolic PDE that regulates cytosolic cAMP pool and modulate expression of anti-
13 oxidant genes [16]. The other cytosolic PDE, PDED was found to be a moderate KM
14 enzyme that interact with PKA-catalytic subunit like proteins to regulate cAMP level
15 [17]. Early studies showed that three human PDE inhibitors (etazolate, dipyridamole
16 and trequinsin) inhibit the proliferation of L. major promastigotes and L. infantum
17 amastigotes with IC50 values in the range of 30–100 µM [18]. Moreover,
18 dipyridamole, etazolate and trequinsin were observed to be potent inhibitors of L.
19 donovani PDEA [16] whereas rolipram was observed to inhibit PDED amongst all the
20 mammalian PDE inhibitors [17]. A robust PDE inhibitor library, has been reported
21 emphasizing PDE as exploitable targets for intervention [19]. However, a
22 comprehensive profile for the impact of PDE inhibitors on various cellular processes
23 in Leishmania is still lacking.
24 Although several cAMP and cAMP pathway associated phenotypes has been
25 identified in Leishmania any functional effector for cAMP is yet to be identified in this
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1 parasite. With an intention to assess the potential of cAMP pathway as target for
2 chemotherapeutic intervention, in the present study, we intended to screen various
3 cAMP analogues and PDE inhibitors. It was observed that hydrolysis-resistant cell-
4 permeable cAMP analogues possess potent anti-proliferative effect and show G0/G1
5 phase arrest indicating that unlike in Trypanosoma, where products of cAMP
6 hydrolysis seemed vital for parasite transformation, in Leishmania cAMP itself could
7 induce these effects. Moreover, mammalian PDE4 inhibitor rolipram and etazolate
8 and PDE5 inhibitor dipyridamole showed more anti-proliferative effect and cell cycle
9 arrest compared to the other well-known mammalian PDE inhibitors. Such effects
10 were also found to be associated with slow and sluggish movement of Leishmania
11 promastigotes, a change in ATP level and a striking change in mitochondrial
12 membrane potential, reinforcing the need for further exploration of cAMP pathway in
13 the parasite for drug development. Efficacy of etazolate in mouse model of VL and
14 apparent unresponsiveness of mammalian cells against this PDE inhibitor validate
15 etazolate as a prospective repurposed drug against Leishmania.
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1 2. Results
2 2.1. Effects of cAMP analogues and PDE inhibitors on L. donovanipromastigotes
3 The effect of various cAMP analogues and PDE inhibitors on the growth of log-phase
4 promastigotes of L. donovani was measured by proliferation assays where parasites
5 were grown in presence of various concentrations of these compounds. Intracellular
6 cAMP concentration was modulated by treatment with one hydrolysable analogue, 8-
7 (4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (8-pCPTcAMP), and one
8 non-hydrolysable analogue, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic
9 monophosphorothioate, Sp-isomer (Sp-8-pCPTcAMPS). Both the analogues
10 significantly affected growth with EC50(growth) of 218.22 µM and 189.78 µM
11 respectively (Table I). On the flip-side, non-hydrolysable cGMP-analogue, 8-(4-
12 chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP) failed to
13 show any anti-proliferative effect on the parasites. Inhibitors previously determined to
14 inhibit Leishmania-PDEs including EHNA, etazolate, dipyridamole, zaprinst, rolipram
15 and trequinsin, were profiled for prospective antiprolifearative activity. Dose-
16 response curves were obtained by calculating the percentage proliferation against
17 untreated population after 4 days of growth to determine EC50(growth). Etazolate was
18 found to be potently anti-proliferative with EC50(growth) of 24.1 µM (Table 1).
19 Dipyridamole and rolipram showed anti-proliferative effects to lesser extents
20 (EC50(growth) of 61.3 µM and 29.04 µM respectively). Trequinsin on the other hand
21 showed comparatively lower anti-proliferative effect with EC50(growth) of 79.6 µM . Rest
22 of the PDE inhibitors tested showed little effect on cell proliferation (Table 1).
23 Since cAMP analogues and PDE inhibitors showed anti-proliferative effects on the
24 parasite, we were interested to introspect whether those impart any effect on
25 parasite viability by determining EC50(viability) by MTT assay after 24 h of exposure. 8-
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1 pCPTcAMP and 8-Sp-pCPTcAMPS significantly decreased the parasite viability with
2 EC50(viability) 200.34 µM and 250.23 µM respectively (Figure S1A and S1B; Table 1).
3 8-pCPTcGMP apparently did not affect parasite viability (data not shown).
4 EC50(viability) values were also determined from the dose responsive curves of various
5 PDE inhibitors (Figure S1). Etazolate, with an EC50(viability) of 89.23 µM, appeared as
6 the most promising anti-proliferative of the inhibitors tested. Dipyridamole, trequinsin
7 and rolipram showed EC50 values of 112.33, 101.5 and 123.12 µM respectively
8 (Table 1, Figure S1C-S1F). All the other PDE inhibitors showed higher EC50(viability)
9 values (Table 1). Collectively, these results suggest that cAMP analogues and PDE
10 inhibitors affects parasite viability at higher doses.
11
12 Table-1. Effect of cAMP analogues and PDE=inhibitors on L. donovanipromastigotes. EC50(growth) was
13 calculated after allowing the promastigotes to grow in presence of analogues/ inhibitors for 96h.
14 EC50(viability) was tested by MTT based vital assay after exposing the promastigotes to the analogues
15 and inhibitors for 24h. Data are representative of at least three independent experiments.
cAMP-analogues/ EC50(growth) (µM) EC50(viability) (µM) PDE-inhibitors 8-pCPTcAMP 218.22±37.24 200.34±47.9
Sp-8-pCPTcAMPS 189.78±29.01 250.23±34.4
Etazolate 24.68±6.31 89.23±12.9
Dipyridamole 61.30±11.71 112.33±8.45
Trequinsin 79.59±6.11 101.5±15.56
Rolipram 29.04±4.22 123.12±20.05
EHNA 100.63±14.63 156.77±14.60
Zaprinast 265.15±15.54 147.80±14.9
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1 2.2. Effect of cAMP analogues and PDE inhibitors on the cell cycle
2 To assess the effect of cAMP analogues and PDE inhibitors on the cell cycle,
3 promastigotes were treated with increasing concentrations (10, 50 and 100 µM) for
4 12 h followed by 2 h incubation with BrdU and the percentage BrdU incorporation
5 was studied by BrdU cell proliferation assay. 8-pCPTcAMP and 8-Sp-pCPTcAMPS
6 treatment at 100 µM concentration significantly decreased the number of BrdU-
7 positive cells (34.2% and 31.64% respectively compared to 82.3% in control) (Figure
8 1A), indicating fewer cells progressing into the S-phase of the cell cycle. Treatment
9 with PDE inhibitors etazolate, dipyridamole, trequinsin and rolipram also decreased
10 the BrdU uptake significantly at 50 µM (24.13%, 28.66% , 31.86% and 26.56%
11 compared to untreated control of 82.38%) whereas, PDE inhibitors EHNA and
12 zaprinast showed only 73.76% and 68.5% positives at 50 µM respectively (Figure
13 1B). To further introspect the impact on cellular proliferation, progression of cell
14 division was determined by scoring of the number, dimension and position of the
15 nucleus (N), kinetoplast (K) and flagellum (F) in cAMP analogues and PDE inhibitor-
16 treated cells after labelling with DAPI. Cells with one of each of these organelles - 1
17 kinetoplast/1 nucleus/1 flagella (1N/1K/1F) representing the G0/G1 population enter
18 S phase and duplicate their DNA content (1N*/1K*/1F cells, where * denotes
19 duplicated DNA). Initiation of the growth of the second flagellum then begins
20 (1N*/1K*/2F cells), where upon segregation of the two daughter kinetoplasts is
21 initiated and 2K1N2F represents S phase followed by nuclear segregation (2N/2K/2F
22 cells) which represents G2/M phase population [20]. Treatment with 8-pCPTcAMP
23 and 8-Sp-pCPTcAMPS resulted in a marked increase in the 1K/1N/1F population,
24 from 54.8 to 75.93% and 78.03% respectively after 12 h, which were associated with
25 concomitant decrease in the 2K/2N/2F and 2K/1N/2F populations (Figures 1C and
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1 1E). Exposure to PDE inhibitors etazolate, rolipram, dipyridamole and trequinsin
2 resulted in a significant increase in the 1N/1K/1F cells (73.43% ± 4.3%, 75.16% ±
3 5.1%, 75% ± 4.9% and 71.5% ± 6.4% compared to 51.53% ± 4.7% for control
4 after 12 h) with equivalent decrease in 2N/2K/1F cells and 2N/2K/2F cells (Figures
5 1D and 1E). Treatment with PDE inhibitors zaprinast and EHNA (Figure 1D) showed
6 K/N/F profiles comparable to that of the untreated cells. Taken together, these
7 results suggest a probable G1 arrest of Leishmania promastigotes when they were
8 treated with cAMP analogues and PDE inhibitors.
9
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1
2 Fig.1. Effect of cAMP analogues and PDE inhibitors on the cell cycle. (A and B) Log phase
3 promastigotes were pretreated with various concentrations of cAMP analogues (A) and PDE inhibitors
4 (B) for 6 h followed by BrdU incorporation assay. (C and D) Log phase promastigotes were treated
5 with cAMP analogues (100 μM) (C) and PDE inhibitors (50 μM) (D) for 12 h followed by DAPI staining
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1 and 1K/1N/1F cells were scored for each of the treatment from at least 50 fields. Representative
2 confocal microscopic images of the above experiment and Phase-contrast images of each treatment
3 are shown separately (E). Data are representative of three independent experiments. ***P<0.001;
4 **P<0.01; ns not significant, two tailed unpaired t-test.
5 To confirm the G1 phase cell cycle arrest, G1-phase-synchronized promastigotes
6 were introduced into fresh medium and the cell cycle profiles of cAMP analogue-
7 treated and PDE inhibitor-treated populations were studied for cellular DNA
8 estimation by propidium iodide based FACS analysis. After exposing the G1
9 synchronized cells to 8-pCPTcAMP and Sp-8-pCPT-cAMPS (10 µM , 50 µM 100
10 µM), parasites were tracked over a period of 8, 16 and 24 h for cell cycle progression
11 in G1, S and G2/M phase (Figure 2A and Table S2). Treatment of the cells with 8-
12 pCPT-cAMP reduced the S phase fraction indicating a potent role of 8-pCPTcAMP
13 induced cell cycle arrest at G1 phase, which also indicated by an increase in G1
14 population (Figure 2B and Table S2). Similarly, Sp-8-pCPT-cAMPS reduced the
15 percentages of S phase cells significantly and increased G1 phase population
16 (Figure 2C and Table S2). For treatment with PDE inhibitors etazolate, dipyridamole
17 and trequinsin, inhibitory toward Leishmania PDEA and PDEB [18], and rolipram,
18 inhibitory toward Leishmania PDED [13], showed significant G1 arrest after 24 h
19 while for inhibitors like EHNA and cGMP-PDE specific inhibitor zaprinst, no apparent
20 G1 arrest was observed. Etazolate, dipyridamole, rolipram and trequinsin resulted
21 significantly reduced S phase cell population at 100 µM respectively compared to
22 untreated population (Table-S2 and Figures 2D to I). These results indicate that
23 chemical modulation of intracellular cAMP can affect cell cycle progression.
24
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1
2 Fig.2. Impact of cAMP analogues and PDE inhibitors on cell cycle progression of L. donovani
3 promastigotes.G1-synchronized log phase promastigote populations, either untreated (A) or treated
4 with 8-pCPTcAMP (100 μM) (B), Sp-8-pCPTcAMPS (100 μM) (C); etazolate (D), dipyridamole (E),
5 trequinsin (F), rolipram (G), EHNA (H) or zaprinast(I) (50 μM each) for 8h, 16h and 24h, were
6 analysed for cell cycle progression by propidium iodide staining based FACS analysis(Table S1).
7 Representative histograms from 24h data are presented.
8 2.3. Modulation of parasite motility and ATP level by dose-dependent treatment of
9 cAMP analogues and PDE inhibitors is linked to mitochondrial activity.
10 Since cAMP has a role in flagellar motility of Leishmania and social motility of
11 Trypanosoma [21], an examination of their flagellar motility patterns revealed that at
12 a concentration of 100 µM both 8-pCPTcAMP and Sp-8-pCPTcAMPS affected the
13 coordinated progressive flagellar movement or forward swimming motility of the
14 parasites significantly (Figure 3A). Parasites were also significantly impaired for
15 progressive flagellar movement in presence of etazolate, dipyridamole and rolipram
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1 (Figure 3A). On the other hand, EHNA and zaprinast, did not affect forward flagellar
2 movement (Figure 3A). Flagellar length and ratio of cell body-flagellar length have
3 been associated with flagellar motility [22]. Average flagellar and cell body length
4 were measured from 50 cells each from three independent populations of cells
5 treated with the inhibitors. Apart from minor increase for dipyridamole treatment,
6 none of the cAMP analogues and PDE inhibitors inducted alteration in flagellar
7 length (Figure 3B). Since, one of the factors effecting flagellar motility is intracellular
8 ATP generation, we determined the cellular ATP levels by ATP bio-luminescent
9 assay after treating the parasites with cAMP analogues and PDE inhibitors.
10 Treatment of parasites with various concentrations of cAMP analogues and PDE
11 inhibitors showed that at higher doses of cell permeable cAMP analogues; and PDE
12 inhibitors etazolate, dipyridamole, trequinsin and rolipram significantly reduced the
13 ATP levels (64.8% ± 5.8%, 61.0% ± 5.5%, 49.7% ± 4.1% and 54.7% ± 5.1%
14 respectively) at a concentration of 50 µM whereas 8-pCPTcAMP and Sp-8-
15 pCPTcAMPS caused lesser effect (31.9% ± 2.8% and 26.0% ± 2.2% of reduction
16 respectively) at 100 µM concentration (Figures 3C and 3D). Taking together these
17 results suggest higher concentrations of 8-pCPTcAMP, Sp-8-pCPTcAMPS, rolipram,
18 dipyridamole, etazolate significantly restricts flagellar movement and parasite motility
19 with a concomitant decrease of cellular ATP levels.
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1
2 Fig.3. Inhibition of parasite motility and depletion of ATP-level upon administration of specific cAMP-
3 analogues and PDE inhibitors. Leishmaniapromastigotes were treated with various cAMP-analogues
4 (100 μM) and PDE inhibitors (50 μM) and the flagellar motility patterns of both the untreated and
5 treated parasites were studied under the bright field of confocal microscope (A). From the same set of
6 experiments, flagellar length and ratio of flagellar length and cell body length was calculated (shown
7 above each bar) (B). Levels of cellular ATP generation were studied after treating the parasites with
8 cAMP-analogues (C) and PDE inhibitors (D) by ATP bio-luminescence assay. Results are
9 representative of three individual experiments, and the error bars represent mean ± SEM. **P<0.01;
10 *P<0.05; two tailed paired t-test.
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1
2 Fig.4. Modulation of mitochondrial membrane potential by cAMP analogues and PDE-inhibitors. Log
3 phase promastigotes were incubated without (A) or with 8-pCPTcAMP (100 μM) (B), Sp-8-
4 pCPTcAMPS (100 μM) (C); etazolate (D), dipyridamole (E), trequinsin (F), rolipram (G), EHNA (H) or
5 zaprinast (I) (50 μM each) for 12h and mitochondrial membrane potential in the parasites were
6 analysed by FACS after treating them with using JC-1, a mitochondrial vital dye. Representative
7 scattered plots for three independent experiments are shown.
8 Since the ATP levels of cAMP analogue-treated and PDE inhibitor-treated parasites
9 were depleted, the impact of these on mitochondrial function was further elucidated
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1 by scoring mitochondrial membrane using JC-1. 8-pCPTcAMP induced maximum
2 reduction in mitochondrial membrane potential compared to control cells reduction
3 (26.1% ± 1.9%) whereas Sp-8-pCPTcAMPS induced moderate effect (10.2% ± 0.9%
4 reduction) at 100 µM (Figures 4A - 4C). Amongst the PDE inhibitors, etazolate and
5 dipyridamole showed maximum reduction (26.4% ± 2.2% and 22.5% ± 1.9%
6 respectively) whereas trequinsin and rolipram showed moderate effect. EHNA and
7 zaprinast did not show significant effect.
8 2.4. Etazolate mitigates intra-macrophage survival of parasites
9 Since etazolate showed maximum effect on cell cycle progression and caused
10 mitochondrial membrane depolarization, the inhibitor was further explored as anti-
11 leishmanial by analysing its impact on parasite multiplication within RAW 264.7
12 macrophages. Etazolate apparently had no effect on viability of macrophage, as
13 determined by MTT assay and gross cell morphology analysis as visualized by
14 microscopy up to 200 µM (data not shown). Etazolate treatment resulted in
15 significant reduction in the number of parasites within macrophages with a maximum
16 effect (75.0 % ± 6.7% reduction) observed at 100 M concentration (Figures 5A and
17 5B).
18 Etazolate was tested in BALB/c mice to test its efficacy to clear parasitic burden from
19 spleen and liver in infected groups. A dose of 5 mg/kg/day and 10 mg/kg/day was
20 administered orally over a period of 10 days following 1 month of Leishmania
21 infection in mice. Spleen and liver were removed from treated and untreated 45 day
22 infected mice, and multiple impression smears were prepared and stained with
23 Giemsa. Spleen or liver parasite burdens, expressed as Leishman-Donovan units
24 (LDU), were calculated as the number of parasites per 1000 nucleated cells/ organ
25 weight (in grams). A dose-related inhibition was noted at a dose of 5 mg/kg/day and
17 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 10 mg/kg/day with great reduction of liver and spleen parasite burden by 70.7 % ±
2 6.9% and 79.5 % ± 7.2% compared to untreated infected mice (Figures 5C and 5D).
3 These results highlight etazolate as candidate anti-leishmanial.
4
5 Fig.5. Effect of etazolate on parasite survival. Macrophages were pre-incubated with either increasing
6 concentrations of rolipram and etazolate (1, 10 and 20 μM) or sodium stibogluconate (100 µg/ ml) or
7 left untreated for 30 min, followed by infection with L. donovani for another 72 h. The number of
8 parasites per macrophage was scored for untreated (U) and treated systems by DAPI staining (A).
9 Representative microscopic image of infected macrophages either untreated or treated with sodium
10 stibogluconate (100 µg/ ml), rolipram (20 µM) or etazolate (20 µM) are shown (B). BALB/c mice were
11 infected with 107 L. donovanipromastigotes and treated etazolate (0-10mg/kg/day). Etazolate was
12 administered i.p. twice a week for 6 weeks starting at 1 week post-infection. PBS was used as vehicle
13 control. The parasite burden in liver and spleen was determined 6 weeks after infection and
14 expressed as Leishman–Donovan Units (LDU). Disease progression was determined in spleen (C)
15 and liver (D) of mice that received etazolate at either 5 or 10 mg/kg/day twice weekly and parasite
16 burden expressed as LDU at 2, 4 and 6 week post-infection (C and D). Results are representative of
18 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 three individual experiments, and the data represent mean ± SD. ***P<0.001, **P<0.01, *P<0.05;
2 unpaired two tailed t test.
3
4 Discussion:
5 Leishmaniasis represents a significant global burden and poses a great challenge to
6 drug discovery and delivery, because of the intracellular nature and disseminated
7 locations of the parasite. Most of the current modes of treatment available till date
8 are not only toxic but also do not completely cure, i.e., eliminate the parasite, from
9 infected individuals [20]. The problem of increased chemoresistance towards the
10 current set of available drugs is further complicating the treatment for this disease
11 [22,23]. Because treatment is a growing problem, the development of new medicines
12 that can replace or complement the current set of available therapeutic compounds
13 is necessary.
14 cAMP has been emerging as one of the major modulator of cytological events in
15 kinetoplastida parasites [8]. Despite the identification of several cyclic nucleotide
16 binding effector molecules there is severe deficit of comprehensive knowledge on
17 cyclic nucleotide signalling from receptor to effector till date. The candidate
18 regulatory subunits and cyclic nucleotide binding domains binds cAMP/ cGMP with
19 extremely high and beyond physiologically permissible Kd indicating almost absence
20 of effective interaction and to perform as a typical effector under physiological
21 condition [15] is not feasible. Bachmaier et al. [24], recently reported binding of
22 nucleotides such as 7-deazapurine derivatives to PKA-regulatory subunit of T. brucei
23 instead of canonical cAMP or cGMP. While studying resistance against PDE inhibitor
24 cpdA and cpdB, a novel class of cAMP effectors, CARP was identified in T. brucei
19 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 [25], indicating existence of non-canonical cAMP effectors in kinetoplastida
2 parasites. CARP and its orthologues [25] are conserved among several
3 kineoplastida parasites. On the receptor/ cAMP generator side of the pathway, an
4 oxygen stimulated soluble adenylate cyclase has been identified, however since this
5 gene is absent in Trypanosoma, this enzyme probably contributes to some cAMP-
6 dependent function restricted to Leishmania. The activation of conserved receptor
7 adenylate cyclases possibly serves as the major trigger for cAMP, though the
8 “ligands” or specific conditions for triggering such “receptors” are not defined yet.
9 The phenotypic attributes like cell death, mitochondrial depolarization and G1 arrest
10 illustrated in this study or social motility [26], flagellar wave reversal [14] are
11 expected to be linked with it or some unidentified upstream effectors. Interestingly 8-
12 pCPTcAMP is less antiproliferative to L. donovani compared to T. brucei [11,25],
13 indicating variation of response against this cAMP-analogue between
14 trypanosomatids. Moreover, the nonhydrolyzable analogue Sp-8-pCPTcAMPS,
15 elicited comparable response in terms of cell cycle analysis and mitochondrial
16 functioning. Such observations hint that possibly the anti-proliferative/ differentiation
17 inducing action of hydrolysis product 8-pCPTcAMP as observed in T. brucei [27], is
18 not conserved in Leishmania. However the EC50 values for the cAMP-analogues,
19 determined in this study, showed subtle deviation across experiments, suggesting
20 responsiveness against the analogues is delicately linked to physiological state of
21 test L. donovani population. Intensive forward genetic approach combined with
22 systemic analysis might shed light on mechanistic details of cAMP mediated
23 responses and variations among trypanosomatids.
24 Though cAMP has cytoprotective effect against oxidative stress in Leishmania, in
25 Trypanosma cAMP-phosphodiesterase inhibitor cpdA displays potent
20 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 antitrypanosomal activity [25], indicating long term potentiation of cAMP triggering
2 probably induce cell death in kinetoplastida parasites. Such observations provide
3 genetic and pharmacological validation of using PDEs as novel drug targets for
4 diseases caused by the kinetoplastid parasites including Leishmania [19]. Sebastián-
5 Pérez et al. already projected PDE-inhibitory imidazole derivatives as candidate
6 antileishmanial, our study corroborates PDEs as promising targets and explores the
7 possibility of repurposing human PDE inhibitors as the starting framework for the
8 design of evaluating in use inhibitors with substantial data for human application.
9 This proposition is supported by the inhibitory effects of some human PDE inhibitors
10 observed on T.cruzi PDEC e.g. etazolate inhibits human PDE4 and TcrPDEC with
11 the IC50 values of 2 and 0.7 μm [9]. Among the PDE inhibitors used in this study,
12 etazolate, a pyrazolopyridine derivative showed maximum anti-proliferative activity
13 against Leishmania parasites with least cytotoxic effect on macrophage cells
14 cultured in vitro. Further it was observed that it significantly affected the cell cycle
15 progression and mitochondrial membrane potential of the parasite, therefore we
16 assessed in vitro for their ability to clear parasite load within the macrophage cells.
17 Though initial reports in T. brucei suggested that etazolate inhibits growth
18 substantially though it does not affect intracellular cAMP pool in the parasite, a
19 number of studies suggested that it can potentially inhibit specific PDEs from T. cruzi
20 and at least two PDE from Leishmania [9,17]. Since etazolate apparently have
21 noimpact of total cellular cAMP pool [5]), it is anticipated that anti-parasitic activity of
22 etazolate is due to microdomain-specific modulation of cAMP response or by binding
23 to some unidentified effector(s). Etazolate, primarily a PDE4 inhibitor [24,28], is a
24 well-established drug of choice with no major side effects reported [28] in preclinical
25 studies as well as pharmacokinetic and safety profiles in Phase I and Phase
21 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 IIaclinical studies. Etazolate produced antidepressant like effects in animal models of
2 depression and at the same time it could be used in the treatment of Alzheimer’s
3 disease. Moreover, chronic etazolate treatment at a dose between 0.5 and 1 mg/kg
4 treatment significantly diminished traumatic brain injury induced anomalies [29].
5 Earlier the drug was demonstrated to be well-tolerated and devoid of side effects by
6 Marcade et al. (2008) based on their study on guanea pigs where they administered
7 the drug at a dose of 10 mg/kg for 15 days [30,31]. In coherence to these reports
8 and based on our in vitro cytotoxicity analysis on RAW264.7 cell line in mice, we
9 tested dose range between 5 to 20 mg/kg which appeared to be well tolerated by the
10 mice. With its potential inhibitory impact on amastigote proliferation in vitro and in
11 experimental animal models for VL, etazolate can be considered a drug for
12 repurposing against VL and can serve as a candidate molecule for synthesizing
13 more selective drugs. Bearing in mind the urgent need for new therapies and the
14 desirability of a relatively short duration of treatment, the requirement for very
15 stringent selectivity might be relaxed somewhat if the drug was efficacious and its
16 side effects were acceptable and reduced compared to current available treatments.
17 Devoid of gross side effects, etazolate, as suggested by the observations can be
18 exploited for developing intervention strategies against leishmaniasis.
19 Materials and methods:
20 1. Ethics Statement
21 The entire study was in strict accordance with the recommendations in the Guide for
22 the Care and Use of Laboratory Animals of the Committee for the Purpose of Control
23 and Supervision of Experiments on Animals (CPCSEA). The protocol was approved
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 by the Institutional Animal Ethics Committee on Animal Experiments of the University
2 of Kalyani (892/GO/Re/S/01/CPCSEA).
3 2. Parasite culture and infection
4 The pathogenic strain of Leishmania donovani AG83 (MHOM/IN/1993/Ag83)
5 (obtained from Dr. Pijush K. Das) was maintained in BALB/c mice and promastigotes
6 were cultured in medium 199 (M199; Invitrogen, Carlsbad, CA, USA) with Hank’s salt
7 which contains Hepes buffer (12mM), L-glutamine (20mM), 10% heat-inactivated
8 fetal calf serum, 50 U/ml penicillin and 50 µg/ml streptomycin. The promastigotes
9 were isolated from infected spleens of BALB/c mice by culturing the respective
10 spleens in M199 at 22oC for 5 days. Simultaneously, RAW 264.7 (obtained from
11 NCCS, Pune, India), adherent murine macrophage cell line was cultured in RPMI
12 1640 (Invitrogen) supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin
o 13 and 100 µg/ml streptomycin at 37 C with 5% CO2. For in vivo infection, BALB/c mice
14 (Saha Enterprise, CPCSEA Regd. No: 1828/PO/Bt/S/15/CPCSEA) of approximately
15 20 g of body weight were injected with 1 x 107 stationary phase promastigotes of L.
16 donovani via the tail vein. Etazolate at a dose of 10 mg/kg/day was administered
17 daily over a period of 30 days following 2 week of post-infection. Parasites were
18 isolated by aseptically removing the spleen and liver of the infected mice and
19 parasite burdens were assessed by Leishman-Donovan units (LDUs) by calculating
20 the number of parasites per 1000 nucleated cells x organ weight (g).
21 3. Parasite viability assay
22 Parasite viability was measured by incubating the treated and control cells in 0.5
23 mg/ml 3-(4,5- dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) for 3
24 hours followed by subsequent addition of 100 µl of 0.04 N HCl in isopropyl alcohol.
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 The principle underlying the MTT assay is that living mitochondria convert MTT to a
2 dark blue compound, formazan, which is soluble in acid isopropyl alcohol. Formazan
3 was detected at 570 nm on the microplate reader. The percentage viability was
4 obtained by calculating the ratio of OD values in wells with treated cells versus the
5 wells with controls multiplied by 100.
6 4. Cell motility
7 Live Leishmania cells were visualized under bright field of confocal microscope on
8 grooved slides which provides the freedom of movement to the parasites. Flagellar
9 movements of both treated and control cells were analysed. Numerous fields were
10 used and the distribution of counting areas was chosen in a non-biased manner
11 along the total surface area of the cover-slip for graphical representation of the
12 motile parasites.
13 5. Flow cytometry and cell cycle analysis
14 5 ml of parasite (0.5-1x107 cells/ml) was centrifuged, washed twice in PBS and re-
15 suspended in 70% ice-cold methanol for the purpose of fixation and the cells were
16 stored at -20oC for future use. The cells were treated with 20 mg/ml of RNase A and
17 incubated at 37oC for 1 hour before proceeding to analysis. Followed by this,
18 propidium iodide was added for staining the DNA and 20,000 cells were analysed for
19 DNA content using BD FACS Aria III and the distribution of G1, S and G2/M phases
20 were then calculated from each histogram in BD FACS Diva Software.
21 6. Macrophage infectivity assay
22 RAW 264.7 cells were cultured in RPMI-1640 and 10% FCS, counted, centrifuged
23 and re-suspended in culture medium and distributed over 18 mm2 coverslips and
24 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 cultured overnight. Cells were treated with PDE inhibitors as mentioned in the result
2 section and infected with L. donovani promastigotes at a ratio of 10 parasites per
3 macrophage and the infection was allowed to proceed for overnight. For the
4 determination of parasite number within the macrophage, cells were fixed in
5 methanol and then stained with DAPI (4’,6- diamidino-2-phenylindole) (1 µg/ml) in
6 PBS containing 10 µg/ml of RNase A (Sigma-Aldrich). Cells were visualized using a
7 Olympus IX 81 microscope with FV1000 confocal system using a 100X oil immersion
8 Plan Apo (N.A. 1.45) objective and analysed by Olympus Fluoview Software (version
9 3.1a, Tokyo, Japan).
10 7. BrdU incorporation assay and 1N/1K/1F enumeration assay
11 Promastigotes were treated with different concentrations of cAMP analogues and
12 PDE inhibitors followed by 2 hours incubation with 100 µM of 5-bromo-2-
13 Deoxyuridine (BrdU) as per standard protocol and the percentage BrdU
14 incorporation was studied by BrdU cell proliferation assay (Millipore). For the
15 determination of number and position of nucleus (N), kinetoplast (K) and flagellum
16 (F) in both treated and non-treated cells, parasites were harvested by centrifugation
17 at 1000 g for 10 minutes at 4oC, washed with 1x PBS and fixed on slide with
18 methanol followed by DAPI (4’,6- diamidino-2-phenylindole) (1 µg/ml) in PBS
19 containing 10 µg/ml of RNase A (Sigma-Aldrich) to stain nuclear and kinetoplast
20 DNA and analysed immediately. Images were acquired using an Olympus BX61
21 microscope, at 1000X magnification, and an Olympus DP71 digital camera and were
22 analysed using Image Pro Plus Software (Media Cybrernetics).
23
24
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 8. ATP bio-luminescent assay and measurement of mitochondrial membrane
2 potential
3 Parasites were centrifuged, resuspended in medium and treated with cAMPanalogs
4 and PDE inhibitors and levels of cellular ATP production was studied by ATP bio-
5 luminescent assay (Cayman Chemicals) as per kit’s protocol.
6 Mitochondrial membrane potential was checked by JC-1 FACS analysis (BD
7 Mitoscreen Mitochondrial Membrane Potential Detection Kit, BD Biosciences).
8 Treated and non-treated cells were incubated with JC-1 for 15-20 minutes, a
9 mitochondrial vital dye, washed with PBS and prepared for flow cytometry. Cells
10 were studied in BD FACS Canto and BD FACS Diva software was used for studying
11 the dot plots.
12 9. Statistical analysis
13 All the results shown are representatives of at least three independent experiments.
14 Cultures were set in triplicate and the results are mean + SD. Statistical differences
15 among the sets of data were determined by Student’s t-test with a P-value <0.05
16 considered to be significant.
26 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.14.906321; this version posted January 14, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Acknowledgment: We would like to acknowledge DST-INSPIRE Faculty Programme,
2 DST-PURSE Programme, University of Kalyani, DST-FIST, Department of Zoology,
3 University of Kalyani, PRG, University of Kalyani, NASI Senior Scientist Fellowship.
4 Funding: AB received grant from DST-INSPIRE Faculty Programme (IFA-12 LSBM-
5 22), Govt. of India.
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1 Supporting Information Legends
2 Figure S1. Dose responsive viability assay for cAMP analogue and PDE inhibitor
3 treatments. Log phase promastigotes were pretreated with various concentrations of 8-
4 pCPTcAMP (A) and Sp-8-pCPTcAMPS (B); etazolate (C), dipyridamole (D), trequinsin (E),
5 rolipram (F), EHNA (G) or zaprinast (H) for 24h followed by MTT assay. Dotted lines
6 represent untreated promastigotes analyzed in parallel to treated population.
7 Table-S1. Cell cycle progression after cAMP-analogue treatment. G1 synchronized L.
8 donovani promastigotes were exposed to various doses of cAMP analogues. Cell cycle
9 stage specific distribution of the populations were analyzed by propidium iodide nuclear
10 staining based FACS analysis after various time periods.
11 Table-S2. Cell cycle progression after PDE inhibitor treatment. G1 synchronized L.
12 donovani promastigotes were exposed to various doses of PDE inhibitors. Cell cycle stage
13 specific distribution of the populations were analyzed by propidium iodide nuclear staining
14 based FACS analysis after various time periods.
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