Discovery of a Small Side Cavity in Sphingosine Kinase 2 that Enhances Inhibitor Potency and Selectivity

Abstract

The sphingosine-1-phosphate (S1P) signaling pathway is an attractive drug target due to its involvement in immune cell chemotaxis and vascular integrity. The formation of S1P is catalyzed by sphingosine kinase 1 or 2 (SphK1 or SphK2) from sphingosine (Sph) and ATP. Inhibition of SphK1 and SphK2 to attenuate levels of S1P has been reported to be efficacious in animal models of diseases such as cancer, sickle cell disease, and renal fibrosis. While inhibitors of both SphKs have been reported, improvements in potency and selectivity are still needed. Toward that end, we performed structure–activity relationship profiling of 8 (SLM6031434) and discovered a heretofore unrecognized side cavity that increased inhibitor potency toward SphK2. Interrogating this region revealed that relatively small hydrophobic moieties are preferred, with 10 being the most potent SphK2-selective inhibitor (Ki = 89 nM, 73-fold SphK2-selective) with validated in vivo activity.

Sphingosine 1-phosphate signaling in perivascular cells enhances inflammation and fibrosis in the kidney

“…This result suggested that Sphk2 deletion in kidney perivascular cells did not affect inflammatory signaling in adjacent tubular epithelial cells on injury. Furthermore, treatment of wild-type kidney perivascular cells with a selective SphK2 inhibitor [SLM6031434 ( 41 )] before LPS treatment recapitulated the phenotype of Sphk2 -deficient cells (Fig. 3G).…”

A Novel Sphingosine Kinase Inhibitor Suppresses Chikungunya Virus Infection

“…Both SK1 and SK2 have about a 70% sequence similarity, with differences in the length of the N-terminal and the central region being the main differentiator in their sequence [53]. Using the SK1 inhibitor structure, it was shown that the sphingosine binding pocket is deeper in SK2 than in SK1 and that amino acid changes in the pocket (I174 to V304) allow for bulkier side groups to be added to the inhibitor backbone for SK2-selective inhibitors [42]. In our study, however, the compounds that potently inhibited CHIKV without significant cytotoxicity also had activity against SK1.…”

“…As we have previously determined sphingosine kinase importance during CHIKV infection, we investigated the effects of a library of sphingosine kinase inhibitors for their activity during CHIKV infection (Figure 1) [39,40,[42][43][44][45]. To assay for their activity against CHIKV, we pre-treated the HeLa cells with the compounds (20 µM) for one hour prior to infection with CHIKV (MOI = 1) for 24 h. We then used the immunofluorescence assay to detect the viral protein (nsP3) expression, followed by the measurement of nsP3 expression as a read-out of anti-CHIKV activity.…”

“…Although the structure of SK2 has not yet been fully resolved, modification of the current SK1 inhibitors has been used as a strategy, leading to increased understanding of the structure of SK2 and allowing for the synthesis of more selective inhibitors against SK2 [37][38][39][40][41][42]. In this study, we screened inhibitors that were optimized for their pharmacokinetic properties and inhibitory effect against both SK1 and SK2 for their activity against CHIKV.…”

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

“…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]. The utility of SK2 inhibitors is exemplified by the synergy observed between bortezomib and the micromolar potent SK2 inhibitor, K145.…”