**4. Sphingosine Kinase**

Sphingosine-1-phosphate (S1P) is a bioactive SL that regulates the growth, survival, and migration of several cell types. S1P is a ligand for five transmembrane G-proteincoupled receptors, S1P1-5, and for several intracellular targets such as histone deacetylases 1 and 2 [48]. Cellular biosynthesis of S1P occurs through phosphorylation of Sphingosine (Sph) catalyzed by SKs. Sphingosine, an effector molecule is biosynthesized by ceramidase activity on the central lipid Cer. SKs exist in two isoforms, SK1 and SK2, encoded by unlinked genes.

The crystal structure of SK1 from the Protein Data Bank is shown in Figure 4, and was generated using ezCADD [13,49]. The active site has the ligand D-erythro-sphingosine. Using ezCADD computer modelling software, we have identified the surrounding amino acids around this ligand. The polar head group is closer to Asp341 and Asp178. The lipophilic tail portion is surrounded by nonpolar amino acids [13]. Pharmacologically, Cer and Sph are associated with growth arrest and apoptosis. On the contrary, S1P is associated with prosurvival roles [50]. SKs and S1P have been implicated in a variety of disease states including cancer [51,52], sickle cell disease [53,54], atherosclerosis [55,56], asthma [57,58], diabetes, fibrosis [59], etc.

Although SK1 and SK2 share a high degree of homology, they differ in size, localization, distribution, and intracellular roles [60,61]. While double-knockout studies in mice suggest that SKs are the sole source of S1P, some functional redundancy exists, as SK1 or SK2 null mice are viable and fertile [62].

The biological significance of SKs has encouraged academia and pharmaceutical companies to target SKs for their therapeutic value. Initial drug discovery efforts resulted in SK1 potent inhibitors, complimented by the availability of the SK1 crystal structure. Based on a Protein Data Bank search, no crystal structure for SK2 has been reported. Potent, selective SK2 inhibitors have been developed through homology modeling of SK1. Competitive inhibition strategies have been reported, and strategies aimed toward competitive inhibition remain a focus. Shown in Figure 5 are the representative SK 2 inhibitors with moderate potency and selectivity. ABC294640 was the first SK2 inhibitor with *K*<sup>i</sup> 10 μM [63] that has been deployed in a variety of disease models, which include ulcerative colitis [64] Crohn's disease [65] ischemia/reperfusion injury [66] osteoarthritis [65] colon cancer [67] and colorectal cancer [68]. However, ABC294640 has recently been reported to have an off-target effect of acting as a tamoxifen-like molecule with the estrogen receptor [69]. A recent study has shown that the sensitivity of BRAFV600E mutant colon cancer cells to Vemurafenib can be increased by reducing the AKT-mediated expression of nucleophosmin and translationally controlled tumor protein [70]. This study highlights the significance of sphingolipid biochemistry and targeting multiple pathways in combination in order to achieve effective cancer therapies. ABC294640 was the first SK2 inhibitor, and when utilizing this as a biological probe, several outcomes were reported in terms of S1P-mediated signaling. For example, mitophagy-mediated apoptosis was unraveled in a multiple myeloma cell line [71]. Other inhibitors, namely SG-12 [72], ®-FTY720-OMe [73], K145 [74], and VT-ME6 [75] exhibited optimal potency and selectivity. K145 has a lipophilic phenoxy ether with a polar head group exhibiting structural similarity to R-FTY720-OMe. K145 is an SK2 inhibitor under investigation for treating leukemia.

**Figure 4.** Crystal structure of Sphingosine Kinase 1. PDB ID: 3VZB. Protein is shown in rainbow color scheme, the ligand D-*erythro* sphingosine is shown in element color scheme with gray, red, and blue for carbon, oxygen, and nitrogen, respectively, and sulfur ion is also shown in crystal structure with element color scheme yellow for sulfur. Active site is located near α8 and 9 helix and β10 sheet.

Based on structure–activity relationship (SAR) studies, isoform-selective SK2 inhibitors with improved potency and half-life in mice were developed. These inhibitors include SLR080811, [76] SLP120701, [77] SLM6031434, [78] SLC5091592 [79], and VT 20dd [80] and are depicted in Figure 6. SLR080811, with a Ki of 13.3 μM and 1.3 μM for SK1 and SK2, respectively, is under study for colorectal cancers with resistance to 5-fluorouracil. [76]. An important finding from these studies was the observation of elevated S1P levels in the mice upon pharmacological inhibition of SK2. Extensive SAR studies of SLR080811 resulted in an azetidine, SLP120701, with an improved half-life of 8 hours in mice [77]. Modifications in the tail region further improved SK2 selectivity as seen with the analog SLM6031434 [78], and a lipophilic-tail-substituted naphthalene-oxy analog, as seen with SLC5091592 [79]. These analogs showed improved SK2 selectivity and potency, comparable to second-generation SK2 inhibitors presented in Figure 6.

**Figure 6.** Other Sphingosine Kinase inhibitors.

As evidenced, sphingosine kinase tail regions can be modified to improve kinase specificity and selectivity. This statement is highlighted by a study that took scaffold of aminothiazole and developed the SK2 inhibitor 20dd (Figure 6). 20dd demonstrated improved potency, selectivity, and in vivo outcomes [80]. A PubMed search for probes of the anticancer potential for these analogs has not been reported so far, but with favorable pharmacological features such as selectivity and specificity towards SKs, further exploration into 20dd and its analogs is warranted.
