Design and Development of Novel Urea, Sulfonyltriurea, and Sulfonamide Derivatives as Potential Inhibitors of Sphingosine Kinase 1

Abstract

Sphingosine kinase 1 (SphK1) is one of the well-studied drug targets for cancer and inflammatory diseases. Recently discovered small-molecule inhibitors of SphK1 have been recommended in cancer therapeutics; however, selectivity and potency of first-generation inhibitors are great challenge. In search of effective SphK1 inhibitors, a set of small molecules have been designed and synthesized bearing urea, sulfonylurea, sulfonamide, and sulfonyltriurea groups. The binding affinity of these inhibitors was measured by fluorescence-binding assay and isothermal titration calorimetry. Compounds 1, 5, 6, and 7 showed an admirable binding affinity to the SphK1 in the sub-micromolar range and significantly inhibited SphK1 activity with admirable IC50 values. Molecular docking studies revealed that these compounds fit well into the sphingosine binding pocket of SphK1 and formed significant number of hydrogen bonds and van der Waals interactions. These molecules may be exploited as potent and selective inhibitors of SphK1 that could be implicated in cancer therapeutics after the required in vivo validation.

Design, synthesis, and biological evaluation of novel benzimidazole derivatives as sphingosine kinase 1 inhibitor

“…The inclusion bodies were solubilized with the help of N ‐lauroyl sarcosine and the solubilized protein was loaded on the Ni‐NTA column and subsequently purified. [ 64‐67 ] The purified SphK1 was analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), which shows a single band of 45 kDa (Figure 3).…”

“…The secondary cultures of SphK1 were induced by 1 mM IPTG for 4 h, followed by centrifugation at 7000 rpm for 15 min to get the cell pellet, which was later resuspended in the lysis buffer, and inclusion bodies were prepared as described. [ 64 ] Finally, inclusion bodies were solubilized in the solubilization buffer (pH 8.0) comprising 0.5% sarcosine, 50 mM Tris, and 150 mM NaCl. SphK1 was purified using Ni‐NTA affinity chromatography, followed by dialysis for 24 h to get the refolded native protein.…”

Design and synthesis of new pyrazolylbenzimidazoles as sphingosine kinase-1 inhibitors

“…The kinase activity of SphK1 is measured in terms of picomolar concentration of phosphate released in the reaction mixture which is represented in Figure 5 (A and B). The absorbance value of the malachite-inorganic phosphate green complex so formed at 620 nm is converted with the help of phosphate standard curve as described [57][58][59][60][61][62][63][64]. The loss in the SphK1 activity followed an inverse relationship between percentage inhibition and an increasing concentration of selected compounds as shown in Figure 5 (C and D) which was used for the calculation of IC 50 values (Table 3).…”

“…The secondary cultures of SphK1 were induced by 1 mM IPTG for 4 h followed by centrifugation at 7000 rpm for 15 minutes to get the cell pellet, which was later resuspended in the lysis buffer and inclusion bodies were prepared as described [57] . Finally, inclusion bodies were solubilized in the solubilization buffer (pH 8.0) comprising 0.5% sarcosine, 50 mMTris and 150 mMNaCl.…”

“…A standard phosphate curve was used to determine the loss in activity of SphK1 in terms of amount of phosphate released on treatment with increasing concentrations of selected compounds. The inhibition in SphK1 activity was plotted for selected compounds in terms of percentage as described [70] .…”