Targeting Leishmania donovani Sphingosine Kinase 1 using PF-543 enhances immune response and limits parasite load

Sphingosine-1-phosphate (S1P) is a bioactive lipid mediator regulating apoptosis, proliferation, and immune responses. While S1Ps presence in Leishmania donovani phagolysosomes has been reported, the role of sphingosine kinases, especially SphK1, in parasite survival and host immune modulation remains underexplored. This study investigates the molecular and functional role of L. donovani SphK1 (LdSphK1) and evaluates the antileishmanial potential of PF-543, a specific SphK1 inhibitor.

LdSphK1 and human SphK1 (rhSphK1) were cloned, expressed in E. coli, purified, and analyzed by SDS-PAGE. Enzymatic activity and inhibition by PF-543 were assessed using NBD-S1P-based fluorometric assays. Protein-ligand interactions were analyzed using Microscale Thermophoresis (MST) and validated in silico docking studies, which identified key species-specific differences in the inhibitor's active site. Leishmania promastigotes overexpressing LdSphK1 were studied via confocal microscopy, and their viability and infectivity were assessed in vitro. THP-1 macrophages infected with L. donovani were treated with PF-543 alone or with Amphotericin B and analyzed by MTT assay, RT-PCR, Giemsa staining, ELISA and immunoblotting. In vivo efficacy was tested in L. donovani-infected Swiss mice.

rLdSphK1 (~102 kDa) and rhSphK1 (~50 kDa) were enzymatically active and significantly inhibited by PF-543. MST demonstrated specific, measurable binding of PF-543 to both orthologues (KD ~ 29μM under identical experimental conditions). In L. donovani SphK1 overexpressor (LdSphKa) promastigotes, PF-543 inhibited SphK1 activity and reduced parasite infectivity, more than in wildtype L. donovani promastigotes. Notably, PF-543 treatment reduced parasite infectivity in vitro, lowered amastigote load by ~40%, and promoted a pro-inflammatory cytokine shift (↑IL-12, ↑ TNF-α, ↓ IL-10). Inhibition of ceramide synthesis and S1P supplementation revealed that S1P rescues ceramide-induced parasite death, implicating SphK1 in parasite survival. PF-543 and Amphotericin B demonstrated synergistic anti-parasitic effects both in vitro and in vivo, with >90% reduction in parasite burden in mice.

PF-543 exerts moderate direct inhibition of parasite SphK1 while prominently modulating host SphK1-dependent immune and apoptotic pathways, collectively restricting Leishmania survival. Rather than functioning as a parasite-selective inhibitor, PF-543 acts as a dual host-parasite modulator. These findings provide proof-of-concept evidence that simultaneous targeting of sphingolipid signalling in both host and parasite can enhance anti-leishmanial efficacy and support further exploration of SphK-based combination therapeutic strategies.