Fig 1.
Inhibition of SphK1 by PF-543 selectively impairs Leishmania donovani growth and reduces intracellular parasite load.
(A) Estimation of LdSphK-1-mediated generation of NBD-SIP levels against SphK inhibitors in L. donovani promastigotes. L. donovani Bob promastigotes were cultured in 25 cm2 flasks followed by treatment with SphK inhibitors; PF-543, DMS and SKI-5C for 48h. Inhibition of SphK1 using these SphK inhibitors cause decreased S1P levels. SphK inhibitor treated promastigotes were resuspended in buffer containing fatty acid-free BSA (0.1% (w/v)) followed by resuspension in buffer containing BSA (1% (w/v)) and incubated with NBD-sphingosine (10μM) for 45 min at 37°C. Promastigotes incorporate NBD-sphingosine (NBD-Sph), which are phosphorylated by Sphingosine Kinase (SphK1) to NBD-S1P. Bar graph depicts ELISA-based S1P quantification in promastigotes treated with inhibitors. (B) Effect of SphK1 inhibitor; PF-543 in Leishmania spp and host macrophages. To evaluate the IC50 for PF543, approximately 6 × 103 THP-1 and 5 × 104 Ld Bob cells were seeded in each well of 96-well flat bottom plates and supplemented with RPMI and M199 media containing varying concentration of PF-543 (200µl/well) in each well. For intracellular amastigotes, 1 × 106 THP-1 cells, treated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) were seeded on glass coverslip in a 6-well plate for 48h. They were infected with late log-phase L. donovani promastigotes at 20:1 MOI and simultaneously treated with PF-543. The cells were further incubated for 2 days at 37°C and 5% CO2. To determine, the intracellular parasite burden (mean number of amastigotes per macrophage) were microscopically assessed using Giemsa staining. For axenic amastigotes, the axenically cultured forms grew optimally at a temperature of 32-33°C in a growth media with pH of 5.4. The IC50 were found to be 450.7nM, 17.54μM, 45μM and 90μM for Leishmania donovani promastigotes, intracellular amastigotes axenic amastigotes and THP-1 macrophages respectively. Each experiment was done in triplicates and repeated thrice. (C) Effect of SphK1 inhibition on parasite infectivity in THP-1 macrophages. THP-1 cells grown in RPMI medium were treated with SphK1 inhibitor; PF-543 for 24h followed by infection with L. donovani promastigotes at an MOI of 20:1 for 48h. (i) After 48h of infection, Giemsa staining was performed to assess the parasite burden. THP-1 cells were fixed, Giemsa stained and amastigotes were counted visually under 100X using light microscope (Scale bar - 20 µm). Virulence capacity was determined by calculating infectivity (upper panel) and parasitemia (lower panel). Untreated infected THP-1 cells were used as control. (ii) Total RNA was enriched using TRIzol. Infectivity was validated using qRT-PCR with primer specific marker JW as a molecular indicator. Expression of JW mRNA in infected cells inferred parasite load and represented as percentage infectivity. Data analysis was performed using the 2-ΔΔCTmethod. The results are representative of three independent experiments. Statistical significance was quantified using the unpaired t-test. The results signify mean ± S.D with n = 3, *P < 0.05.
Fig 2.
Purification, subcellular localization, and functional characterization of recombinant rLdSphK1 in Leishmania donovani in the presence and absence of the SphK1 inhibitor PF-543.
(A) Concentrated rLdSphK1 protein. Gel image showing a purified band of 102 kDa recombinant LdSphK1 (B) The subcellular localization of recombinant LdSphK1 was investigated in promastigotes. (i) DeepTMHMM results predicted globular location with no transmembrane domains with a probability of 1. (ii) Panel A: DIC at 60X. Panel B: DAPI. Panel C: anti-LdSphK1 antibody detected using FITC (green)-conjugated secondary antibody. Panel D: merged micrographs (Scale bar - 20 µm). LdSphK1 expression represented in the green channel. Co-localization shown as the merged image. (C) In silico docking of the rLdSphK1 in presence of PF-543. In-silico ligand-substrate interaction was done using Autodock 1.5.7rc1 and Cygwin terminal. The in silico docking generated the complex of rLdSphK1-PF-543 and represented as surface model highlighted with residues involved in the interaction using Chimera, Ligplus, Discovery Studio v19.1.0, and PyMOL v2.3.2 software. (D) Biophysical interaction and functional characterization of the rLdSphK1 (20 µM) in presence of PF-543. MST analysis confirms the interaction of rLdSphK1 with PF-543 and ABC294640. Dose-response curves of rLdSphK1–PF-543 showed KD of 29.3 µM and no interaction between rLdSphK1 and ABC294640. (E) Analysis of functional characterization of the LdSphK1 recombinant protein. (i) Time kinetics of activity of purified rLdSphK1 and NBD-S1P-based fluorometric assay showed increase in NBD-SIP levels under the influence of varying concentrations (20ng - 500ng) of rLdSphK1 protein. (ii) The inset shows inhibition of NBD-SIP levels in the presence of different concentrations of PF-543 (0.01µM - 5µM) using 200ng of recombinant LdSphK1 in view of sharp increase of NBD-S1P levels at 200ng of rLdSphK1 protein observed in previous experiment.
Fig 3.
Purification, Molecular Docking, and Functional Characterization of Recombinant rhSphK1 in the presence of inhibitors.
(A) Purification of Recombinant rhSphK1 from Supernatant. Gel image showing a concentrated rhSphK1 protein band of 50kDa. (B) In silico docking of the rhumanSphK1 in presence of PF-543. In silico ligand-substrate interaction was done using Autodock 1.5.7rc1 and Cygwin terminal. The in silico bound complex of rhSphK1-PF-543 was represented as surface model highlighted with residues involved in the interaction using Chimera, Ligplus, Discovery Studio v19.1.0, and PyMOL v2.3.2 software. The list of residues involved in hydrophobic interaction and hydrogen bond formation were highlighted in the model. (C) Functional characterization of the rhSphK1 in presence of PF-543. MST analysis confirms the biophysical interaction of rhSphK1 with (i) PF-543 and (ii) ABC294640, which was used as a control. Dose-response curves of PF-543 (20µM) and ABC294640 showed KD of 34.3µM and 70.6µM respectively. (D) Analysis of catalytic activity of the rhSphK1 using NBD-S1P-based fluorometric assay. Time kinetics of activity of purified rhSphK1 demonstrated increase in NBD-SIP levels in presence of 200ng of rhSphK1 protein.
Fig 4.
Effect of PF-543 on cytotoxicity and infectivity of L. donovani SphK1 overexpressor promastigote cell line.
(A) Vector map showing the construct designed for pXG plasmid containing SphKa gene and GFP for the overexpression of the gene through transfection in LdBob cells. (B) Confocal microscopy images confirming successful integration of the plasmid. Integration was verified by the presence of GFP fluorescence signal in the transfected parasites. (C) (i) Estimation of LdSphK-1-mediated generation of NBD-SIP levels against PF-543 in L. donovani SphKa promastigotes. L. donovani bob promastigotes were cultured in 25 cm2 flasks followed by treatment with SphK1 inhibitor; PF-543 for 48h. Inhibition of SphK1 using PF-543 had no significant effect on S1P levels. SphK inhibitor treated SphKa promastigotes were resuspended in buffer containing fatty acid-free BSA (0.1% (w/v)) followed by resuspension in buffer containing BSA (1% (w/v)) and incubated with NBD-sphingosine (10μM) for 45 min at 37°C. Promastigotes incorporate NBD-sphingosine (NBD-Sph), which are phosphorylated by Sphingosine Kinase (SphK1) to NBD-S1P. Bar graph depicts ELISA-based S1P quantification in SphKa promastigotes treated with PF-543 using LdBob as control. (ii) Comparative analysis of the half maximal inhibitory concentration of PF543 against the wild type LdBob and LdSphka OE line. (D) Effect of SphK1 inhibitor; PF-543 in Leishmania overexpressor spp. (i) Effect of L. donovani SphK1 overexpression on intracellular parasite clearance. THP-1 macrophages were cultured in six-well plates in the presence or absence of L. donovani (LdBob) infection (MOI, 20:1) for 6h. Further, THP-1 cells were infected with L. donovani SphK1 overexpressor promastigotes at various MOIs (10:1, 20:1, 30:1, 50:1). Infected THP-1 were washed to remove non-internalized parasites. Total RNA was enriched using TRIzol and the resulting cDNA was subjected to real-time PCR analysis using primers specific for infectivity (JW) genes in infected macrophages. RNU6A was used as a housekeeping gene. The results are expressed as fold-change of uninfected THP-1 cells. Statistical significance was quantified using the unpaired t-test. The data is a representation of mean ± SD from three independent experiments *, p < 0.05; ***, p < 0.001. (ii) GFP-based confirmation of SphK1 overexpression in L. donovani promastigotes. Detection of the presence of GFP encoded SphKa and Bob plasmids in Leishmania donovani and Leishmania overexpressor parasites. L. donovani bob and L. donovani overexpressor promastigotes were cultured in 25 cm2 flasks followed by confocal microscopy.
Fig 5.
Assessment of DL-PPMP cytotoxicity and the effect of PF-543 on Caspase-9 activation in Leishmania donovani-infected macrophages.
(A) Determination of in vitro cytotoxicty of DL PPMP threo and DL PPMP erythro. DLPPMP threo and DLPPMP erythro at varying concentrations (2µM, 5µM, 10µM, 20µM and 50µM) were incubated with THP-1 cells seeded 6 × 103 in a 96-well plate for 48h. The cytotoxicity was analysed using colorimetric estimation of formazan crystals formed by the conversion of MTT by live cells. IC50 determined by plotting the non-regression linear curve was 19.53µM and 116µM for DLPPMP threo and erythro respectively. (B) Estimation of Caspase 9 levels upon PF-543 treatment in Leishmania infected macrophages. (i) THP-1 macrophages were cultured in six-well plates in the presence of L. donovani infection (MOI, 20:1) for 6h. Infected THP-1 were washed to remove non-internalized parasites and treated with PF-543 (20μM) or DL-threo-PPMP; glucosylceramide synthase inhibitor (20μM) or SIP (10μM) for next 48h. Caspase-9 levels were analysed using cell lysates for Western blot. (ii) Band intensities were quantified by ImageJ software and were plotted in GraphPad Prism. Data from one of three experiments is shown.
Fig 6.
(A(i), B) Effect of PF-543 on the expression of inflammatory molecules and clearance of intracellular parasite load.
THP-1 macrophages were cultured in six-well plates in the presence or absence of L. donovani infection (MOI, 20:1) for 6h. Infected THP-1 were washed to remove non-internalized parasites and treated with PF-543 for next 48h. Total RNA was enriched using TRIzol and the resulting cDNA was subjected to real-time PCR analysis using primers specific for inflammatory (IL32γ, IL-12, IL-10 and TNF-α) and infectivity (JW) genes in PF-543 treated and untreated infected macrophages. RNU6A was used as a housekeeping gene. The results are expressed as fold-change of uninfected THP-1 cells. Statistical significance was quantified using the unpaired t-test. The data is a representation of mean ± SD from three independent experiments *, p < 0.05; ***, p < 0.001. (A (ii)) Graph represents cytokine levels measured by sandwich ELISA. Statistical significance was quantified using the unpaired t-test with Welch’s correction, in LPS-stimulated, Ld-infected and PF-543 treated macrophages.
Fig 7.
(A-B). Evaluation of PF-543 and Amphotericin B formulation on cytotoxicity or metabolic viability of Leishmania promastigotes and intracellular amastigotes.
(A) To determine the combinatorial impact of PF-543 and Amphotericin B formulation, promastigotes of L. donovani were seeded in 96-well plates (3 × 106 parasite/mL) in exponential growth phase at increasing concentrations (12.5nM–500nM) of Amphotericin B and (150nM- 2000nM) of PF-543 compounds and maintained at 26°C. After 48h of incubation, the IC50 was determined by MTT assay. The absorbance was measured in a MultiskanEX photometric plate reader for microplates at 540 nm. Data were obtained from three independent experiments performed in triplicate. The fractional inhibitory concentration index (FIC) was evaluated to determine the synergistic activity of lead compounds; PF-543 with Amph-B. Finally, FIC < 0.5 represented strong synergistic activity between two drugs. While FIC > 4 is was considered as the antagonistic activity. FIC values were obtained from three independent experiments. (B) For validating the antileishmanial activity by PF-543 and Amphotericin B formulation on intracellular amastigotes, qPCR-based analysis was performed for all the treatments. For the same, THP-1 cells were seeded at a concentration of 8 × 105 cells/mL in 96-well plates and incubated for 24h with PMA (10ng/mL) supplemented with RPMI 1640. Following 24h priming with PMA, the culture medium was removed, and the cells were infected with L. donovani followed by treatment with the formulations having concentrations kept similar as used previously for promastigotes at 37°C and 5% CO2. Total RNA was extracted, and the resulting cDNA was subjected to real-time PCR analysis using primers specific for kinetoplast minicircle DNA (kDNA)/JW of L. donovani. This primer does not match to human genome, thus directly indicates the intracellular parasite load against the treatments. Data analysis was performed using the 2-ΔΔCTmethod.Values are mean ± S.D (n = 3). The results are representative of three independent experiments performed in triplicates.
Fig 8.
Expression of JW and inflammatory markers upon PF-543 treatment in Leishmania-infected Swiss mice.
Evaluation of the (A) immunomodulatory potential and (B) infectivity of PF-543 and Amph-B in Leishmania-infected Swiss mice was performed. 10mg/Kg of PF-543 and 2mg/Kg of Amphotericin B alone were given for individual treatment in Leishmania infected mice. For constituting the synergistic effect of two drug partners; 2mg/Kg (1mM) of PF-543 and 0.4mg/Kg (0.12µM) of Amphotericin B were prepared in 1XPBS and given together to a mouse weighing 30gm. To evaluate the parasitaemia and expression of inflammatory markers in infected mice upon drug administration, spleen was harvested from each group of mice and qRT-PCR analysis was performed using primers specific for inflammatory markers IL-10 and TNFα and parasite specific kinetoplast minicircle gene, JW in PF-543 and Amph-B treated infected mice in respect to the untreated Swiss mice. RNU6A (RNA, U6 small nuclear 1; THP-1 cells) was used as a housekeeping gene. The results were expressed as normalized fold-change of respective control. Statistical significance was quantified using the unpaired t-test. The data is a representation of mean ± SD from three independent experiments *, p < 0.05; ***, p < 0.001.
Fig 9.
Schematic summary of the anti-leishmanial and immunomodulatory effects of SphK1 inhibition by PF-543.
PF-543 inhibited recombinant L. donovani SphK1 activity, reduced parasite infectivity, and lowered amastigote burden (~40%), while shifting the cytokine profile toward a pro-inflammatory state (↑IL-12, ↑ TNF-α, ↓ IL-10). Inhibition of ceramide synthesis and rescue by S1P confirmed the role of the SphK1/S1P axis in parasite survival. Combination treatment with PF-543 and Amphotericin B showed synergistic efficacy (>90% parasite reduction) and enhanced host-protective immune responses, underscoring SphK1 as a promising therapeutic target in visceral leishmaniasis. Figure created in BioRender. Wali, A. (2025) https://BioRender.com/nit9sx0.