Int. J. Mol. Sci.

2017, 18, 2332 6 of 18

Int. J. Mol. Sci. 2017, 18, 2332 6 of 18

A549 HCC827

100 100

80 80

60 60

40 40
8f 8f
5c 5c
20 20
5f 5f
5h 5h
0 0
0 0,5 0,75 1 2,5 5 0 0,5 0,75 1 2,5 5

(A) (B)
H1975

100

80

60

40
8f
5c
20
5f
5h
0
0 0,5 0,75 1 2,5 5

(C)
Figure
Figure 1. The
1. Thecompounds
compounds 5c, 5f,
5c,5h,
5f, and
5h, 8f
and are8fable
are to reduce
able cell density
to reduce of cultured
cell density NSCLCNSCLC
of cultured cell
lines. Percent of cell density of A549 (A), HCC827 (B), and H1975 (C) NSCLC
cell lines. Percent of cell density of A549 (A), HCC827 (B), and H1975 (C) NSCLC cells treated cells treated with
different doses (0.5–5
with different doses µM) of µM)
(0.5–5 the compounds
of the compounds 5c, 5f, 5c,
5h, 5f,
and5h,8fand
for 8f
3 days, as measured
for 3 days, by
as measured
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
bromide (MTT)Data assay.represent the meanthe
Data represent
(±SD) of three independent experiments, each performed in triplicate. Bars show
mean (±SD) of three independent experiments, each performed in triplicate. Bars show SDs. Statistical SDs. Statistical
significance
significance waswasdetermined
determined bybythethe
Student
Studentt-test: p <p0.1
t-test: for for
< 0.1 compounds
compounds 5c (5
5cµM, A549),
(5 µM, 5h 5h
A549), (2.5(2.5
µM, µM,
all all
cellcell
lines),
lines), and 8f (2.5 µM, all cell lines); p < 0.01 for compounds 5h (5 µM, all cell lines) and 8f (5(5
and 8f (2.5 µM, all cell lines); p < 0.01 for compounds 5h (5 µM, all cell lines) and 8f µM,
µM, allall cell
cell lines).
lines).

To verify the selectivity of 5h and 8f towards the two enzymatic isoforms, we performed a
To verify the selectivity of 5h and 8f towards the two enzymatic isoforms, we performed a SphK
SphK activity assay. The principle of the method is to quantify, by luminescence, the remaining
activity assay. The principle of the method is to quantify, by luminescence, the remaining amount
amount of adenosine triphosphate (ATP) in solution following the kinase reaction. The luminescent
of adenosine triphosphate (ATP) in solution following the kinase reaction. The luminescent signal
signal is inversely correlated with the kinase activity. The SphK1 competitive inhibitor PF-543 was
is inversely correlated with the kinase activity. The SphK1 competitive inhibitor PF-543 was used
used as a positive control for SphK1 and as a negative control for SphK2. As shown in Figure 2A,
as a positive control for SphK1 and as a negative control for SphK2. As shown in Figure 2A, PF-543, 5h,
PF-543, 5h, and 8f tested at 10 µM, were able to inhibit SphK1 activity as demonstrated by the higher
and 8f tested at 10 µM, were able to inhibit SphK1 activity as demonstrated by the higher ATP levels
ATP levels in solution when compared to that of the control. When tested on SphK2, only compound
in solution when compared to that of the control. When tested on SphK2, only compound 5h caused
5h caused an increment of ATP levels, with respect to the control, indicating that it is able to inhibit
an increment of ATP levels, with respect to the control, indicating that it is able to inhibit both the
both the enzymes (Figure 2B).
enzymes (Figure 2B).
In order to better display these results, we converted the data to percent of inhibition, obtaining
the graphs reported in Figure 2C,D. As displayed, PF-543 was completely ineffective on SphK2
(Figure 2B) and selectively inhibited SphK1 (Figure 2A), even if it was less effective than expected.
Moreover, when it was tested on A549 cells, it reduced cell density by less than 50% at 5 µM (Figure
S1) showing an antiproliferative effect lower than that of the here reported compounds. Finally,
compound 8f was able to selectively inhibit SphK1, while compound 5h showed a comparable
inhibition of both the enzyme isoforms SphK1 and SphK2. This data proposes 5h as a dual inhibitor
and might explain the higher efficacy of 5h as an antiproliferative agent. Several groups have