Topographical Mapping of Isoform-Selectivity Determinants for J-Channel-Binding Inhibitors of Sphingosine Kinases 1 and 2

Abstract

Sphingosine kinase enzymes (SK1 and SK2) catalyse the conversion of sphingosine into sphingosine 1-phosphate and play a key role in lipid signaling and cellular responses. Mapping of isoform amino acid sequence differences for SK2 onto the recently available crystal structures of SK1 suggests that subtle structural differences exist in the foot of the lipid-binding 'J-channel' in SK2, the structure of which has yet to be defined by structural biology techniques. We have probed these isoform differences with a ligand series derived from the potent SK1-selective inhibitor, PF-543. Here we show how it is possible, even with relatively conservative changes in compound structure, to systematically tune the activity profile of a ligand from ca. 100-fold SK1-selective inhibition, through equipotent SK1/SK2 inhibition, to reversed 100-fold SK2 selectivity, with retention of nanomolar potency. -phosphate (S1P) is a key signalling lipid derived from sphingosine (Sph) by the action of sphingosine kinases (SK1 and SK2). S1P is transported to the extracellular environment, where it functions as a ligand for a family of five S1P-specific G protein coupled receptors (S1P 1-5 ), but it also acts on specific intracellular target proteins, such as histone deacetylases 1/2 (HDAC1/2). 1, 2 Irreversible cleavage of S1P is catalysed by S1P lyase, producing (E)-2hexadecenal and phosphoethanolamine, 3 but S1P is also reversibly dephosphorylated by S1P phosphatase to regenerate Sph, 4-6 levels of which are additionally influenced by flux through the ceramide (Cer) synthesis pathway. 7 The effects of S1P on cell function favour proliferation, migration, differentiation and survival and are generally opposed by Cer, which induces apoptosis, senescence and growth arrest. 8,9 As a consequence the concept of the 'sphingolipid rheostat' was proposed, 10,11 in which the inter-conversion of Cer via Sph to S1P controls cellular fate (recently reviewed 12,13 ). Altered S1P signalling has been associated with numerous pathophysiologies, including cancer, cardiovascular disease, neurodegenerative conditions, diabetes and inflammatory disease. 14 The possibility of manipulating the sphingolipid rheostat for therapeutic purposes has therefore provided a rationale for exploring the development of SK inhibitors and their potential to reduce S1P formation and signalling and to raise pro-apoptotic Cer levels.The SK1 and SK2 enzymes are encoded by distinct genes and differ in their subcellular localisation, biochemical properties and functions; there are three N-terminal variants of SK1 and two N-terminal variants of SK2. 15,16 A number of classes of SK inhibitors have emerged in the last two decades, including the recent discovery of an ATP-competitive SK inhibitor chemotype that decreases cellular S1P levels, elevates Sph/Cer, induces apoptosis, and that inhibits cell proliferation and colony formation. 17 Most work to date, however, has focused on the development Sph-competitive inhibitors. This includes the development of inhibitors selective for SK1 t...

Recent advances in the role of sphingosine 1‐phosphate in cancer

“…In this regard, mapping SK2 amino acid differences onto the SK1 crystal structure indicates subtle differences in the ‘foot’ of ‘J‐channel’ (which accommodates sphingosine) of the two isoforms. Probing these isoform‐specific differences with a chemical series (derived from the potent SK1‐selective inhibitor, PF‐543) demonstrated that it was possible to systematically turn a 100‐fold SK1‐selective inhibitor, through chemical modification, to an equipotent SK1/SK2 inhibitor (Compound 49, pIC 50 7.8) and, with further modification, to a 100‐fold SK2 selective inhibitor, with nanomolar potency (HWG‐35D (Compound 55), pIC 50 7.4) [132]. In addition, structure–activity relationship profiling has identified a side cavity in SK2 that can be exploited to increase inhibitor potency, with relatively small hydrophobic moieties preferred (e.g., SLM6071469, K i = 89 n m , 73‐fold SK2 selective) [133].…”

A Novel Selective Sphingosine Kinase 2 Inhibitor, HWG-35D, Ameliorates the Severity of Imiquimod-Induced Psoriasis Model by Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes

“…The SK2 inhibitor, ABC294640 has been reported to induce the ubiquitin-proteasomal degradation of SK1 and DES1 [16,17]. In contrast, another SK2 inhibitor, (R)-FTY720 methyl ether (ROMe), does not induce the proteasomal degradation of SK1 [25].…”

“…Sci. 2020, 21, x FOR PEER REVIEW 3 of 16 the efficacy of SK2 inhibition in IMQ-induced psoriasis mouse models using a novel specific SK2 inhibitor, HWG-35D [17].…”

“…In addition, ABC294640 induces the ubiquitin-proteasomal degradation of SK1 and dihydroceramide desaturase 1 (DES1) [16]. Some of us recently performed a ligand-based structure activity relationship study to highlight the key determinants for switching between SK1-and SK2-selective inhibition, leading to the design of the novel SK2 inhibitor, HWG-35D (compound 55 in Adams et al [17]), which exhibits an IC 50 of 41 nM and 100-fold selectivity for SK2 over SK1 [17].…”

“…Recently, our group showed the effect of SK2 inhibition in a psoriasis mouse model using the modestly potent SK2 inhibitor, ABC294640, which diminished Th17 differentiation of naïve CD4 + T cells with concomitant reduction in SOCS1 mRNA levels [21]. Due to the potential complicating effect of ABC294640 on SK1 and DES1 [15,24], we have now re-examined the efficacy of SK2 inhibition in IMQ-induced psoriasis mouse models using a novel specific SK2 inhibitor, HWG-35D [17].…”