Figure 1.
Chemical structures of SphK2 inhibitors.
Figure 2.
Chemical structures of dual-pathway inhibitors, sphingosine, and K145.
Figure 3.
Chemical synthesis of K145.
Figure 4.
K145 inhibits SphK2 but not SphK1.
A) SphK1 and SphK2 activities were measured with 5 µM sphingosine in the absence or presence of the indicated concentrations of K145 or 10 µM DMS. Data are expressed as percentage SphK activity in the absence of inhibitor; B) SphK2 activity was measured with increasing concentrations of sphingosine and the indicated concentrations of K145. Lineweaver-Burk analysis revealed a Vmax of 10820±210 pmol/min per mg of protein, and a Ki of 6.4±0.7 µM for SphK2; C) Overlay of K145 with sphingosine; D) CERK activities were measured with 12.5 µM ceramide in the absence or presence of the indicated concentrations of K145 or 100 µM of DMS and sphingosine; E) Effect of K145 (10 µM) on activity of the indicated enzymes was tested by SelectScreen Kinase Profiling from Invitrogen. Data are expressed as percentage of control activity averaged from 2 independent experiments. Data are expressed as mean value ± SEM.
Figure 5.
K145 accumulates and suppresses the S1P level.
A and B) U937 cells were treated with K145 at the indicated concentrations for 3 h and the levels of K145 and S1P were measured by ESI-MS/MS. C) HEK293 cells were treated with K145 (10 µM) for 2 h. Lipids were extracted and different chain length species of ceramide were determined by LC-ESI-MS/MS. Numbers indicate chain length followed by the number of double bonds in the fatty acid. Data are averages of triplicate determinations and are expressed as pmol lipid/106 cells. D) U937 cells were treated with or without K145 (10 µM) for 3 h and levels of C1P species were determined by ESI-MS/MS. E) U937 cells were treated with FTY720 (1 µM) in the absence or presence of indicated K145 for 3 h, then FTY720-P was measured by ESI-MS/MS. *P<0.05 compared to control.
Figure 6.
K145 exhibits antiproliferative and apoptotic effects in U937 cells.
A) U937 cells were treated with K145 at indicated concentrations for indicated intervals, the cell viability was determined by MTT assay; B) U937 cells were treated with K145 (10 µM) for 24 h, then the cells were stained with Annexin V/PI and analyzed by flow cytometry; C) U937 cells were treated with K145 at indicated concentrations for 3 h, after which cell lysates were analyzed by Western blot using corresponding primary antibodies. The image of Western blot represents the results from one of two independent experiments. Data are expressed as mean value ± SEM.
Table 1.
HINT scores of the docked molecules into SphK1 and SphK2.
Figure 7.
Docking results of K145 to SphKs.
Binding mode of K145 in SphK1 (A) and SphK2 (B). K145 is shown as sticks with carbon in green, while the interacting residues of both kinases are shown as sticks with carbons depicted in cyan. For simplicity, hydrogens are only shown on residues forming hydrogen-bonding interactions with K145.
Figure 8.
K145 suppresses the growth of U937 xenograft in nude mice.
BALB/c-nu mice (n = 7) with palpable U937 xenograft were treated daily with vehicle, tamibarotene (15 mg/kg), or K145 (15 mg/kg) for 17 days by i.p. injection. A) After treatment, animals were sacrificed and tumors were removed and weighed and the TGI was calculated; B) Tumor volumes were measured every other day during the treatment course; C) Animal weights were measured every other day during treatment course. Data are expressed as mean value ± SD. *P<0.05 compared to control group.
Figure 9.
K145 suppresses the growth of JC xenograft in BALB/c mice.
BALB/c mice (n = 8) with palpable JC xenograft were treated daily with vehicle or K145 (20 mg/kg and 35 mg/kg) for 15 days by i.p. injection. A) Tumor volumes were measured every other day; B) After treatment, animals were sacrificed and tumors were removed and weighed; C) The S1P and K145 levels in the tumor samples from vehicle and treatment (20 mg/kg) groups (n = 4) were measured by ESI-MS/MS; D) Images of tumor samples from control and treatment groups (n = 7 for each group) after the experiments; E) Tumor samples (20 mg/kg and control groups) were analyzed by Western blot. Data are expressed as mean value ± SEM. *P<0.05 compared to control group.
Figure 10.
K145 suppresses the growth of U937 tumors in nude mice by oral administration.
BALB/c-nu mice (n = 7) with palpable U937 xenograft were treated daily with vehicle, tamibarotene (20 mg/kg), or K145 (50 mg/kg) for 15 days by oral gavage. After treatment, animals were sacrificed and tumors were removed, weighed and images were taken. A) Tumor weight and TGI comparison; B) Images of tumor samples from control and treatment groups (n = 7 for each group) after the experiments; C) Tumor volumes were measured every other day; D) Animal weights were measured every other day. Data are expressed as mean value ± SEM. *P<0.05 compared to control group.