Development of a sphingosine kinase 1 specific small-molecule inhibitor

Article abstract of DOI:10.1016/j.bmcl.2010.10.005

The sphingolipid metabolic pathway represents a potential source of new therapeutic targets for numerous hyperproliferative/inflammatory diseases. Targets such as the sphingosine kinases (SphKs) have been extensively studied and numerous strategies have b

Full text of DOI:10.1016/j.bmcl.2010.10.005

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7498–7502
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a sphingosine kinase 1 specific small-molecule inhibitor
⇑
Jeremy A. Hengst, XuJun Wang, Ugir H. Sk, Arun K. Sharma, Shantu Amin, Jong K. Yun
Department of Pharmacology, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033-0850, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The sphingolipid metabolic pathway represents a potential source of new therapeutic targets for numer-
ous hyperproliferative/inflammatory diseases. Targets such as the sphingosine kinases (SphKs) have been
extensively studied and numerous strategies have been employed to develop inhibitors against these
enzymes. Herein, we report on the optimization of our novel small-molecule inhibitor SKI-I
(N0-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide) and the identi-
fication of a SphK1-specific analog, SKI-178, that is active in vitro and in vivo . This SphK1 specific
small-molecule, non-lipid like, inhibitor will be of use to elucidate the roles of SphK1 and SphK2 in the
development/progression of hyperproliferative and/or inflammatory diseases.
Received 16 August 2010
Revised 29 September 2010
Accepted 1 October 2010
Available online 1 November 2010
Keywords:
Sphingosine kinase
Sphingosine
Sphingosine-1-phosphate
Small-molecule inhibitor
Hyperproliferative disease
Inflammatory disease
Ó 2010 Elsevier Ltd. All rights reserved.
Sphingosine kinase (SphK) is an oncogenic lipid kinase and key
regulator of the sphingolipid metabolic pathway (reviewed in 1–
4). The SphK isoforms (SphK1 and SphK2) catalyze the conversion
in a BALB/c immunocompetent mouse JC mammary adenocarci-
noma model.11,12
Herein, we describe our structure–activity relationship (SAR)
studies of our previously identified SphK inhibitor (SKI) chemotype
of the pro-apoptotic substrate D-erythro-sphingosine (Sph) to the
pro-growth/survival product sphingosine-1-phosphate (S1P). Accu-
mulation of S1P, a potent mitogenic/migratory signaling lipid, has
been linked to the development/progression of numerous hyperpro-
liferative and/or inflammatory diseases including, but not limited to,
cancer, asthma, atherosclerosis, inflammatory bowel disease, sepsis,
rheumatoid arthritis and diabetic nephropathy.5–7 Blocking SphK
activity, therefore, has been postulated to inhibit the uncontrolled
growth associated with hyperproliferative/inflammatory diseases
while simultaneously inducing apoptosis of the targeted cells.
Hence, the SphKs have become burgeoning drug targets for a variety
of hyperproliferative/inflammatory diseases.
I
(SKI-I; N0-[(2-hydroxy-1-naphthyl)methylene]-3-(2-naphthyl)-
1H-pyrazole-5-carbohydrazide) lead compound.11 In this study,
we report the identification of a novel SphK1-specific, non-lipid
substrate, small-molecule inhibitor. We believe, this SphK1 specific
compound will enable further quantitative structure–activity rela-
tionship (QSAR) studies aimed at improvement of efficacy and
facilitate the evaluation of therapeutic strategies specifically
targeting SphK1 for hyperproliferative diseases such as cancer.
While a number of sphingosine kinase inhibitors have been
described (extensively reviewed by Pitman and Pitson 20108),
the roles of the SphKs and S1P in the progression and pathophysi-
ology of hyperproliferative diseases were first elucidated by the
discovery of the sphingosine kinase inhibitors N,N-dimethylsp-
hingosine and
D,L
-threo-sphingosine.9,10 However, their lipid
properties made these lipid-substrate analogs less than ideal
‘drug-like’ molecules and limited their therapeutic potential. To
Our initial chemical library screen for small-molecule inhibitors
of SphK1 yielded four distinct classes of ‘lead’ compounds.11 Of these
four lead compounds, SKI-I (N0-[(2-hydroxy-1-naphthyl)methy-
lene]-3-(2-naphthyl)-1H-pyrazole-5-carbohydrazide: 1) was found
to be the most cytotoxic toward a panel of tumor cell lines
overcome these limitations, we conducted
a small-molecule
library screen, identified four classes of specific small-molecule
inhibitors of SphK and demonstrated their in vivo effectiveness
(IC50 ꢀ 1–3
lM) including multi-drug resistant cell lines and active
in a BALB/c immunocompetent mouse JC mammary adenocarci-
⇑
Corresponding author. Tel.: +1 717 531 1508; fax: +1 717 531 5013.
E-mail address: jky1@psu.edu (J.K. Yun).
noma model.11,12 Our preliminary studies also indicate that SKI-I is
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.005

J. A. Hengst et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7498–7502
7499
Table 2
capable of inhibitingSphK2 albeit at slightly lower levels than SphK1
SphK1 inhibition for various R1 substituted phenyl groups of compound 2
(data not shown). However, poor correlation to Lipinski’s Rule-
of-Five (i.e., c Log P = 6.2; ACS/PhysChem13) and poor oral bioavail-
ability dampened the prospects for use of 1 as an anti-cancer
therapeutic agent.
Currently, the X-ray crystal structures/NMR solution structures
of the SphKs are not known. Therefore, rational drug design and/or
molecular modeling strategies for the optimization of SKIs are not
possible. To gain insight into the structural features of 1 critical for
SphK inhibition, we initiated this study by obtaining a series of
SKI-I analogs.14 We first sought to eliminate the naphthyl rings
of 1 (R1 and R2), due to their ease of metabolism and propensity
of their electrophilic epoxy and quinone metabolites to form
DNA adducts.15–18
Compound
R1
SphK1 % inhiba
7
8
9
4-H
4-Me
4-OMe
11.9
9.8
33.5
a
Values are percent inhibition of SphK1 at 10.7
experiments, standard deviations were 5%.
l
M, averages of two separate
To determine whether these naphthyl rings were necessary for
inhibition of SphK1, we tested a series of analogs in which these
rings were replaced with substituted phenyl rings. SphK1 in vitro
activity assays were performed using affinity purified His6X-tagged
SphK1 protein recombinantly over-expressed in HEK293 cells.19 All
Table 3
SphK1 inhibition for various R2 substituted phenol groups of compound 9
data are reported as percent inhibition of SphK1 at 10.7 lM which
is the calculated IC50 of 1 under these assay conditions. As shown
in Table 1, the R2 2,3-phenyl ring was dispensable for inhibition
of SphK1 (2–5). However, the position of the R2-OH group affects
SphK1 inhibition with compound 2 demonstrating that the ortho
position was preferred over meta and para (2: 59.3% inhibition;
R2 = 2-OH). Similarly, the R1 3,4-phenyl ring was not required for
inhibition of SphK1 as demonstrated by compound 6 (6: 52.5%
inhibition; R1 = 4-OMe).
Compound
R2
SphK1 % inhiba
10
11
12
13
14
15
16
17
3-OH
4-OH
4-F
3,5-Di-OH
4-iso-Propy
2,4-Di-OH
4-N,N-diethyl
3,4-Di-OMe
15.5
14.3
27.4
12.5
15.3
30.8
38
Having determined that the naphthyl rings of compound 1 are
not necessary for SphK1 inhibition, we identified 4-OMe and
2-OH as preferable substitutions in the R1 and R2 positions, respec-
tively. We next sought to determine whether the chain length of
the R1 position affected the inhibitory activity toward SphK1. As
shown in Table 2, we directly compared R1 = 4-H, 4-Me and
4-OMe substitutions (7–9). Interestingly, although R1 = 4-OMe
and R2 = 2-OH were identified above (2 and 6) as substitutions that
retained the inhibitory capacity of compound 1, in combination,
they decreased the SphK1 inhibitory activity of compound 9.
We next decided to further optimize R2 while holding R1
constant. As demonstrated in Table 3 and consistent with the
results obtained above (3 and 4), single substitutions of R2 resulted
in lower percent inhibition than 1 (R2 = 3-OH 10: 15.5% inhibition;
R2 = 4-OH or 4-F 11–12: 14.3% and 27.4% inhibition, respectively).
Larger R2 substitutions were, however, more favorable. In particu-
lar, compound 17 (R2 = 3,4-di-OMe: 44.5% inhibition) was similar
to the inhibition observed for compound 1. Thus, while the naph-
thyl rings of compound 1 were not required for SphK1 inhibition,
it appears that larger groups are preferred at the R2 position.
44.5
a
Values are percent inhibition of SphK1 at 10.7
experiments, standard deviations were 5%.
l
M, averages of two separate
Finally, we examined the interconnecting region between the R1
and R2 substituted phenyl rings. To determine whether substitu-
tions within this region would affect SphK1 inhibition, we tested
a series of compounds with a methyl substitution (Table 4; R3).
Addition of a methyl group at R3 resulted in improved SphK1 inhib-
itory activity (46.8% inhibition, 19; versus 27.4% inhibition, 12; and
59.6% inhibition, 20; vs 44.5% inhibition, 17). Also, consistent with
the results obtained in Table 2, R1 = 4-Me substitutions negatively
impact SphK1 inhibitory activity (21.8% inhibition, 18; versus
59.6% inhibition, 20), suggesting that 4-OMe is the preferred sub-
stitution of the R1 phenyl ring. Thus, through SAR studies, we have
identified a promising new ‘lead’ compound (20) with improved
pharmacological properties. Specifically, we have succeeded in
lowering the calculated Log P (c Log P) value from 6.2 (1) to 4.2
(20) while simultaneously retaining approximately equal % inhibi-
tion of SphK1 activity. Importantly, the c Log D of 20 has also been
lowered to 3.2 (pH 7.4) indicating that 20 has an increased likeli-
hood of oral bioavailability.13
Table 1
SphK1 inhibition for naphthyl ring substitutions of compound 1
Having identified 20 as an effective inhibitor of SphK1 under
in vitro assay conditions at 10.7 lM, we next wanted to determine
the Ki and mechanism of inhibition for this compound. We there-
fore developed a chemical synthesis strategy for the production
of 20. The synthesis of 20 was carried out following our recently
reported method20 as outlined in Scheme 1. The reaction of
4-methoxyacetophenone 21 and dimethyl oxalate in the presence
of sodium methoxide led to the formation of ester 22 which on
treatment with anhydrous hydrazine led to the formation of
pyrazole hydrazide 23. The condensation of 23 with 30,40-dime-
thoxyacetophenone in the presence of catalytic amount of acetic
acid furnished the desired product 20 in good yield. Compound
20 and all other intermediates were characterized on the basis of
NMR and mass spectral analysis.21
Compound
R1
R2
SphK1 % inhiba
2
3
4
5
6
3,4-Phenyl
3,4-Phenyl
3,4-Phenyl
3,4-Phenyl
4-OMe
2-OH
3-OH
4-OH
2,4-Di-OH
2,3-Phenyl
59.3
25.1
28
39.5
52.5
a
Values are percent inhibition of SphK1 at 10.7
experiments, standard deviations were 5%.
l
M, averages of two separate

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J. A. Hengst et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7498–7502
Table 4
SphK1 inhibition for various R3 methyl substituted compounds
Compound
R1
R2
R3
SphK1 % inhiba
18
19
20
4-Me
4-OMe
4-OMe
3,4-Di-OMe
4-Cl
3,4-Di-OMe
Me
Me
Me
21.8
46.8
59.6
a
Values are percent inhibition of SphK1 at 10.7
experiments, standard deviations were 5%.
lM, averages of two separate
We performed in vitro SphK1 activity assays with varying con-
centrations of -erythro-sphingosine (Sph) as substrate in the pres-
ence and absence of several concentrations of 20. The calculated Ki
for 20 ranges from 1.26 to 1.55 M and as shown in Figure 1A, 20
D
l
appears to inhibit SphK1 in a competitive manner with respect to
the Sph binding site. The Ki of 20 as obtained above is in agreement
with the Ki value of 20 (1.33
the Cheng–Prussoff equation and compares favorably to the calcu-
lated Ki value of 1 (1.06 M; Km of Sph = 2.75
M22,23). The close
lM) derived from our IC50 studies by
l
l
agreement between the Ki values of 1 and 20 is consistent with
our % inhibition data indicating that 20 is equally effective to 1
at inhibiting SphK1. Importantly, 20 inhibits SphK1 in a non-com-
petitive manner with respect to ATP binding (Fig. 1B). This is also
consistent with the mechanism of inhibition of 111 indicating that
modification of the SKI-I chemotype has not altered the manner in
which the inhibitor binds to SphK1.
As stated above, 1 inhibits both SphK1 and SphK2. To determine
whether the optimization of 1 affected the inhibitory activity to-
ward SphK2, we next determined the SphK2 inhibitory activity of
20. As shown in Figure 2, 20 does not inhibit SphK2 at concentra-
Figure 1. Kinetic evaluation of compound 20. (A) Sphk1 catalytic activity was
measured in the absence (filled circles) or presence of 20 (10 M filled squares or
17 M filled triangles) a varying concentration of Sph. Lineweaver–Burk represen-
tation indicates the 20 is a competitive inhibitor of Sph binding. (B) Sphk1 catalytic
activity was measured in the absence (filled circles) or presence of 20 (10 M filled
squares or 17 M filled triangles) a varying concentration of ATP. Lineweaver-Burk
l
l
l
l
representation indicates the 20 is a non-competitive inhibitor of ATP binding.
tions up to 25 lM which is near the solubility limit of 20 in the
(5) with reduced SphK1 inhibitory activity. This compound was
less cytotoxic than 1 indicating that the increased cytotoxicity of
20 is due to the inhibition of SphK1 activity and not due to
non-specific cytotoxic effects of the pyrazole-5-carbohydrazide
backbone. To further demonstrate that 20 directly inhibits SphK1
catalytic activity in vivo, we examined the cellular levels of S1P
aqueous SphK assay environment. In contrast, the parent
compound SKI-I (1) significantly inhibits SphK2 activity in a dose
dependent manner. This indicates that 20 is the first small-
molecule non-lipid specific inhibitor of SphK1.
The data above indicate that 20 inhibits SphK1 catalytic activity
in vitro. To demonstrate that 20 is also active in vivo, we next
determined the in vivo cytotoxicity of 20 by performing sulforho-
damine B (SRB) cytotoxicity assays in the lung adenocarcinoma cell
line A549. As shown in Figure 3A, treatment of A549 cells with
compound 20 significantly increases the percent cytotoxicity
(1.37-fold; p <0.02) relative to 1. Importantly, to demonstrate the
specificity of this cytotoxic effect we also examined a compound
production in A549 cells treated with 1 or 20 (1 lM) for 48 h, by
thin layer chromatography. As shown in Figure 3B, 1 and 20 both
reduce cellular S1P levels relative to DMSO controls further indi-
cating that 20 directly inhibits SphK1 in vivo. The lower band ob-
served in the control and compound treated lanes likely
represents a lipid kinase present in the cell lysate as confirmed
Scheme 1. Reagents and conditions: (i) sodium, MeOH, dimethyl oxalate, C6H6, 0 °C, rt, overnight; (ii) anhydrous hydrazine, EtOH, reflux, 6 h; (iii) 30,40-dimethoxyace-
tophenone, cat. AcOH, DMSO.

J. A. Hengst et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7498–7502
7501
Table 5
Cytotoxicity of SKI-178 toward a panel of cancer cell lines
Cancer cell line
20 (IC50
l
M)a
Cancer cell line
20 (IC50 l
M)a
MCF-7
MTR-3
NCI/ADR
A549
H460
H226
1.3 0.4
1.8 0.4
1.5 0.3
0.3 0.2
0.3 0.2
0.3 0.1
0.5 0.2
0.6 0.2
Panc-1
Mia-PaCa2
BxPC3
KG1a
Kusami-3
HL-60
0.1 0.1
0.1 0.1
0.2 0.1
0.5 0.2
0.2 0.1
0.2 0.1
0.2 0.1
0.3 0.2
U251
U87-MG
HL60/VCR
SH-N-SY
a
Values are averages of three experiments.
(4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol, CAS 312636-
16-1), has been used extensively in studies of the in vitro and
in vivo action of SphK.28–30 While SKI-II has been independently
validated as a SphK inhibitor, it was not the most potent of the four
classes of lead compounds identified. SKI-I (1) was more potent
in vitro as well as in vivo.11 SKI-I was also highly cytotoxic toward
several multi-drug resistant cell lines making it an attractive scaf-
fold for further drug development. However, due to its high lipo-
Figure 2. Inhibition of SphK2 by 20. Sphk2 catalytic activity was measured in the
presence of increasing concentrations of 1 (black bars) and 20 (white bars). Lack of
inhibition indicates that 20 does not inhibit SphK2 catalytic activity.
philicity it was unsuitable as
a candidate drug target. We
therefore began a SAR study with the hope of optimizing the phar-
macological properties of SKI-I.
The results of this SAR study yielded an improved compound,
20 (hereafter referred to as SKI-178), with enhanced pharmacolog-
ical properties. While the improvement in efficacy was modest, at
best, this new compound was found to be specific for SphK1. The
importance of this finding offsets the modest increase in efficacy
by providing a new SphK1 specific lead compound platform for
further drug development including QSAR studies, which are
currently being performed in our laboratory. Using this new lead
compound as a model for future studies will allow us to simulta-
neously assess whether a modification of the lead compound
affects SphK1 inhibition efficacy and/or SphK1 specificity. Thus
we will be able to identify determinants of inhibitory activity
and isozyme specificity. Identifying the determinants of SphK1
specificity should enable us to rationally design improved general
SphK inhibitors as well as SphK2 specific inhibitors for future test-
ing in hyperproliferative disease models.
There is conflicting evidence that suggests that either SphK1 or
SphK2 would be the preferred targets for disease intervention. In
this regard, a study of glioblastoma patients reported a significant
correlation between poor patient survival and elevated SphK1
levels, but found no correlation with SphK2 or S1P receptor expres-
sion levels.31 These findings are consistent with a clinical study of
breast tumor samples which demonstrated that SphK1 is signifi-
cantly over-expressed in both hormone-independent (i.e., ERꢁ)
and -dependent tumors, and concluded that high SphK1 expression
is a functional prognostic marker for decreased metastasis-free
survival and the overall poor prognosis of patients.32 Interestingly,
a recent study also reported that high SphK1 expression levels are
a good indicator of daunorubicin resistance of leukemia cells.33
Conversely, a recent study of SphK2 deficient breast cancer cells
indicated that SphK2 catalyzed S1P production was required for
xenograft proliferation and that decreased S1P production attenu-
ated tumor-associated macrophage polarization.34 Separately, Hait
et al.35 identified a role for SphK2 in migration of breast cancer
cells toward EGF. Thus, these conflicting results indicate the need
for specific inhibitors of SphK1 and/or SphK2 to determine whether
strategies targeting SphK1 or SphK2 alone or the SphKs in combi-
nation offer the best therapeutic effect. The development of SphK
isoform-specific inhibitors also offer the ability to dissect the
complex signaling pathways associated with S1P production and
to assign those effects to specific SphK isoforms.
Figure 3. In vivo cytotoxicity and inhibition of S1P formation by 20. (A) In vivo
cytotoxicity of A549 cells was determined by SRB assay24 treated with 1
lM
compounds 1, 20 and the inactive conformer 5. Cytotoxicity is reported as the %
increase or decrease in cell cytotoxicity, after 48 h treatment, relative to 1 as 100%.
(B) SphK1 activity assays19 were performed on whole cell lysates of A549 cells
treated with
1 lM 1 or 20 for 48 h. Lipids were separated by thin layer
chromatography as described previously.36
by its absence from the S1P standard lane which was prepared by
in vitro SphK1 activity assay using recombinant SphK1 and
D
-erythro-sphingosine as a substrate.
Our previous studies have demonstrated that
1 induces
apoptosis in T24, MCF7 and NCI-Adr cancer cell lines.11 We next
confirmed that 20 was cytotoxic toward a panel of cancer cell lines,
including multi-drug resistant cell lines, by employing sulforhoda-
mine B cytotoxicity assays.24 Indeed as shown in Table 5, 20 was
cytotoxic at IC50 concentrations ranging from 1.8 to 0.1 lM in both
drug sensitive and multi-drug resistant cancer cell lines (i.e., MTR-
3, NCI-ADR and HL60/VCR25,26). These data also demonstrate that
specific inhibition of SphK1 is sufficient to induce cell death as
has been demonstrated using siRNA approaches.27
We previously reported the identification of four novel classes
of SphK inhibitor ‘lead’ compounds.11 One of these classes, SKI-II

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J. A. Hengst et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7498–7502
spectrometer. Chemical shifts (d) were reported in parts per million downfield
from the internal standard. The signals are quoted as s (singlet), d (doublet), t
(triplet), (multiplet). High-resolution MS (EI) were determined at the
Chemistry Instrumentation Center, State University of New York at Buffalo, NY.
Thin-layer chromatography (TLC) was developed on aluminum-supported pre-
coated silica gel plates (EM industries, Gibbstown, NJ). Column chromatography
was conducted on silica gel (60–200 mesh).
2-Hydroxy-4-(4-methoxyphenyl)-4-oxomethylbutenoate (22). A solution sodium
(0.92 g, 39.9 mmol) in methanol (10 mL) was added to a mixture containing 4-
methoxyacetophenone (21) (5.0 g, 33.33 mmol) and dimethyl oxalate (4.33 g,
36.66 mmol) in benzene (150 mL) dropwise at 0 °C. After the addition was
complete, the mixture was allowed to warm to room temperature, stirred
overnight, quenched with 1 N HCl solution, and filtered. The residue was
There is ample evidence to suggest that SphK derived S1P
production has a vital role in tumor development, progression of
tumors to highly aggressive and metastatic phenotypes (tumori-
genesis) and the development of multi-drug resistance (MDR).
However, it remains unclear whether strategies that target SphK1
or SphK2 specifically or SphKs in general are more efficacious for
the prevention/treatment of hyperproliferative diseases including
cancer. Thus with the identification of SphK1 specific small-mole-
cule non-lipid like inhibitors as reported herein, we may now be
able to address this central question of SphK pathophysiology.
m
purified through silica gel column chromatography using
a mixture of
methylene chloride/hexanes (7:3) as an eluent to yield 6.0 g (87%) of 22 as a
pale-yellow solid; mp 97–98 °C; 1H NMR (CDCl3): d 8.01 (2H, d, J = 7.0 Hz), 7.06
(1H, s), 7.01 (2H, d, J = 7.0 Hz), 3.96 (3H, s), 3.92 (3H, s).
Acknowledgements
5-(4-Methoxyphenyl)-2H-pyrazole-3-carboxylic acid hydrazide (23). To a solution
of methyl ester 22 (5.0 g, 24.0 mmol) in EtOH (150 mL) was added anhydrous
hydrazine (3.1 g, 96.0 mmol) and the reaction mixture was refluxed for 6 h
under nitrogen. The reaction mixture was cooled to room temperature, the
precipitated white solid was filtered and washed with a mixture of ethanol/
hexanes (1:9) to yield 5.0 g (90%) of 23 as a white solid; mp 231–232 °C; 1H
NMR (DMSO-d6): d 13.5 (1H, s), 7.7 (2H, d, J = 8.5 Hz), 7.02 (3H, m), 4.49–4.45
(2H, m), 3.8 (3H, s); MS (ESI) 233 (M++1).
This research was supported by the Jake Gittlen Cancer Research
Institute and by a grant from Susan G. Komen for the Cure.
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19. In vitro SphK activity assays were performed as detailed previously23 with
minor modifications. D-erythro-Sphingosine (Sph; 25 lM) was employed for all
inhibitor screen assays and was delivered in sphingosine kinase activity assay
buffer (SKAAB) without conjugation to BSA or Triton X-100 micelle formation.
All compounds were employed at 10.7 lM for the initial inhibitor screens.
DMSO was employed in control reactions and did not exceed 5% of the total
reaction volume. For SphK2 specific activity assays, 1 M KCl was included in
the standard SKAAB buffer. Ki and inhibitory mechanism determination
analyses were performed by linear regression analysis using GraphPad Prism 5.
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21. Melting points were recorded on a Fischer–Johns melting point apparatus and
are uncorrected. NMR spectra were recorded using a Bruker Avance 500 MHz