Chapter2 Development and Evaluation of Human SPHK Inhibitors

32
CHAPTER 2 DEVELOPMENT AND EVALUATION OF HUMAN
SPHK INHIBITORS

As discussed in Chapter 1, SPHK and its product S1P, play important role in many
cellular processes, such as in the regulation of intracellular calcium signals, in
angiogenesis and control of cell adhesion molecule expression, and chemotaxis. More
particularly, in immune cells, it was shown that the SPHK/S1P pathway may promote
inflammation by triggering the release of proinflammatory mediators (Taha et al., 2006,
Melendez, 2008). SPHK and S1P are very tightly involved in several pathological
processes, indicating that the SPHK/S1P pathway represents an interesting target for the
development of novel therapeutics. In particular, compounds having the ability to
modulate the levels of S1P would have a high potential for the treatment of diseases
wherein S1P is believed to be involved, such as cardiovascular diseases-including
atherosclerosis, thrombosis and dyslipidemia, diabetes including type I and type II, stroke,
autoimmune and inflammatory diseases such as multiple sclerosis, psoriasis and
inflammatory arthritis, allergic diseases such as asthma and dermatitis, T helper-1 related
diseases, chronic obstructive pulmonary disease, cancer and neurodegenerative disorders
(Kuokkanen et al., 1997; Nair et al., 1977; Enlund et al., 1999; Hong et al., 1999; Xia et
al. 2000).
Another important potential of SPHK inhibitors is that, they may be capable to promote
the stem cells differentiation, therefore, shortening the incubation duration for stem cells
differentiating into pure subpopulation(s), as discussed in Chapter 1.
Unfortunately, there are no specific inhibitors for SPHK commercially available yet.
DMS is currently the most widely used inhibitor of SPHK, but it has been shown to be
Chapter2 Development and Evaluation of Human SPHK Inhibitors

33
not specific to SPHK. It was found to inhibit not only two isozymes of human SPHKs

Scheme 1 summarises the experimental design, including compounds structures and key
procedures to synthesize them. During the first round testing, only six compounds were
finally synthesized and evaluated.
It should be noticed that Scheme 1 is the synthesis flow for the whole project of
developing SPHK inhibitors, which has been filed as a patent, while this thesis only
covered six analogues described here. However, in order to keep the story as a whole one,
Scheme 1 is still used here to describe the flow.
HO C
13
H
27
OH
NH
2
HO C
13
H
27
OH
NH
2
D-erythro-sphingosine
(a)
The functional groups which would be modified
(b)
(1)
(2)
(3)

Figure 2.1 (a) D-erythro-sphingosine. (b) Positions of modifications on D-erythro-

HO
OH
NH
2
R
HO
NH
2
R
OH
4a 4b
ONBoc
CR
O
5
ONBoc
C
O
6
R
HO
R
O
NHBoc
7
HO
NHBoc
R
8
O

HO
OH
NHBoc
R
3a or 3b
MnO
2
CH
2
Cl
2
(a)
(b)
2a1: R = C
4
H
9
2a2: R = C
8
H
17
2a3: R = C
13
H
27
2b1: R = C
4
H
9
5a: R = C

or Fluka, and were used without further purification. Analytical thin layer
chromatography (TLC) was carried out on pre-coated silica plates (Merck silica gel 60,
F254) and visualized with UV light or stained with phosphomolybdic acid (PMA) stain.
Chapter2 Development and Evaluation of Human SPHK Inhibitors

36
Flash column chromatography was performed with silica (Merck, 70-230 mesh).
1
H
NMR and
13
C NMR spectra were measured on a Bruker ACF 300 or AMX 500 Fourier
Transform spectrometer. Chemical shifts were reported in parts per million (δ), relative to
the internal standard of tetramethylsilane (TMS). The signals observed were described as
follows: s (singlet), d (doublet), t (triplet), m (multiplet). The number of protons (n) for a
given resonance was indicated as nH. Mass spectra were performed on a Finnigan/MAT
LCQ mass spectrometer under electron spray ionization (ESI). Optical rotations were
determined with a JASCO DCP-1000 digital polarimeter and were the average of at least
10 measurements. The purity of all synthesized compounds was >95% as estimated by
1
H
NMR analysis.
As described in the Scheme 1 above, starting from S-(-)-1,1-dimethylethyl-4-formyl-2,2-
dimethyloxazolidine-3-carboxylate, also known as Garner’s aldehyde 1, and using the
synthetic route shown in Scheme 1, six analogues of sphingosine were synthesized.
In the first step, acetylides of various chain lengths, obtained from the treatment of
alkynes with BuLi, were coupled diastereoselectively with 1 (Scheme 1) giving both the
erythro-isomer 2a and the threo-isomer 2b (Garner et al., 1996). Subsequent ring opening
of 2a using Amberlyst 15 yielded 3a, in high yields (Herold et al., 1988).
2.1.1.2.2 Synthesis Procedure for the Six Compounds as described in the Scheme 1
Chapter2 Development and Evaluation of Human SPHK Inhibitors

38
2.1.2.1.1 CHO cells culture
CHO cells were cultured in Dulbeco’s modified Eagle’s medium (DMEM),
supplemented with 10% heat-inactivated FBS (GIBCO), 1% 2mM L-glutamine,
10mg/mL streptomycin and 10U/mL penicillin. The cells were cultured in an incubator at
37°C, 5% CO
2
in a humidified environment.
2.1.2.1.2 Over-expression of SPHK1 and SPHK2 in CHO Cells
A plasmid containing the SPHK1-cDNA fused to an enhanced green fluorescence protein
cDNA (EGFP-SPHK1) has previously been developed in the laboratory (Melendez et al.,
2000). The plasmid details are shown in Figure 2.2. The construct was made using the
restrictive sites for the enzymes NheI and EcoRI, and inserting the SPHK1 cDNA.

Figure 2.2 Map of EGFP-SPHK1 plasmid. The SPHK2 clone was purchased from iDNA Technology (Open Biosystems), with
SPHK2 cDNA was inserted into a pCMV-SPORT6 vector.
Both plasmids were amplified in DH5α (E.coli) system and purified using QIAfilter Midi
Cartridges (QIAGEN).
Chapter2 Development and Evaluation of Human SPHK Inhibitors

39
Over-expression of SPHK1 and SPHK2 was achieved by transfecting the SPHK1 or
SPHK2 plasmids into CHO cells using Lipofectamine

visible color change. Within the linear range of the assay (~5-25μg/mL), the more protein
present, the more the Coomassie Blue binds changing the absorbance of the sample.
After getting cell lysates from the CHO cells, over-expressing SPHK1 or SPHK,
compounds were tested for their inhibitory function on these two proteins using the
SPHK assay (described below).
2.1.2.1.5 SPHK Assay
2.1.2.1.5.1 Buffers, Solutions and Substrate Preparation
D-erythro-sphingosine was prepared in ethanol at 50mM in a screw-capped glass tube
and store at -70°C.
Bovine serum albumin (BSA) (tissue culture grade) was prepared in PBS to generate a
final concentration of 4mg/ml.
20mM ATP was freshly prepared in 200mM MgCl
2
solution.
γ[
32
P]ATP (10mCi/ml) Redivue
TM
was purchased from GE Healthcare Bio-Sciences and
the final working concentration was 2μCi/sample.
SPHK buffer: 20mM Tris-HCl (pH7.4), containing 20% glycerol, 1mM mercaptoethanol,
1mM EDTA, 1mM sodium orthovanadate (SOV), 1mM PMSF, 1mM Aprotinin, 1mM
Leupeptin and 1mM Pepstatin A. The buffer was stored at 4℃ and the protease inhibitors
(Aprotinin, Leupeptin, Pepstatin A, PMSF and SOV) were freshly added each time
before using.
Substrate preparation: 1mM D-erythro-sphingosine was prepared by mixing 20μl
sphingosine (50mM) with 1ml BSA (4mg/ml). This solution was routinely vortexed (1-2
min) before using it, to generate sphingosine-BSA complexes. These complexes ensure
Chapter2 Development and Evaluation of Human SPHK Inhibitors


Chapter2 Development and Evaluation of Human SPHK Inhibitors

42
components in the samples, which are then separated in the TLC plate. The plate was run
in the chamber until the solvent front reached 2cm from the top of the plate, then
removed from the chamber and air dried in a fume hood. Radioactive signal on the TLC
plate was captured by exposing the plate to a Typhoon
TM
scanner and the intensity of the
radioactive signals were visualized and quantified using a Typhoon
TM
phosphor-imager.
2.1.2.2 Compounds Function on Endogenous Human SPHK1 Activity
After detecting compound function on over-expressed SPHK1 and SPHK2 containing
lysates, their inhibitory function on endogenous SPHK activity was investigated, this
assay also helps to evaluate the membrane penetration ability of the compounds.
In this study, U937 cells, differentiated into macrophages, were used, as the lab has
previously shown that the anaphylatoxin C5a stimulates SPHK1 in these cells, without
stimulating SPHK2.
2.1.2.2.1 Human Histiocytic Lymphoma U-937 Cells culture
The U-937 cell line was purchased from ATCC (Rockville, MD). It is a human leukemic
pro-monocyte lymphoma cell line. The cells were cultured in RPMI 1640 supplemented
with 10% FBS (GIBCO, Invitrogen Singapore), 2mM glutamine, 10U/ml penicillin and
10mg/ml streptomycin at 37C, 5% carbon dioxide in a humidified atmosphere.
The cells were differentiated into a more macrophages adding 1mM of dbcAMP to the
culture media and continuing the culture for further 48 hours.
2.1.2.2.2 Compounds Function on endogenous SPHK1 activity
Differentiated U937 cells were pretreated with 10μM of each of the synthesized
compounds, or with 10μM of DMS, for 30 minutes prior to stimulation. Cells were then
stimulated with 5nM of C5a and warmed to 37°C for different the times indicated in the

were tested at five concentrations: 10μM, 25μM, 50μM, 75μM, and 100μM. The lipids
were separated using the TLC method as for the SPHK assay, and the results were
visualized and quantified using Typhoon
TM
phosphor-imager.
2.1.2.3.2 PKC Assay
Another counter screening in this study was to evaluate the compounds on PKC activity.
PepTag
®
assay for non-radioactive detection of PKC (Promega) was used. This PKC
assay is a non-radioactive assay, which utilizes fluorescent peptide substrates that are
highly specific for PKCs. The method is very simple: the active PKC phosphorylates its
specific substrate, and thus the peptide net charge was altered from +1 to -1.
Electrophoresis is then used to separate the phosphorylated and non-phosphorylated
peptides, as the change in the net charge will change the migratory properties of the
peptide during electrophoresis.
In this study, recombinant human PKC alpha was used. Our compounds, as well as DMS
as a control, were tested at five different concentrations: 10μM, 25μM, 50μM, 75μM,
and 100μM in this assay. The assay was carried out following the protocol manufacturer
provided. Briefly, 5μl of PKC reaction 5xBuffer, 5μl of PepTag C1 peptide (0.4μg/μl),
and sonicated PKC activator 5X Solution were mixed with deionized water to make up a
final volume of 25μl, to prepare a reaction solution mixture. Initially, the reaction
mixture was incubated for 2 minutes at 30°C. 5ng human PKC alpha was immediately
added into the pre-warmed reaction mixture and incubated at 30°C for 30 minutes. The
incubation was terminated by placing the reaction tubes in a 95°C heat block for 10
Chapter2 Development and Evaluation of Human SPHK Inhibitors

45
minutes. 1μl of 80% glycerol was then added and mixed to ensure the samples remaining
in the wells when running electrophoresis, and then the samples were loaded on a 0.8%

H NMR (500 MHz, C
6
D
6
) δ4.71
Chapter2 Development and Evaluation of Human SPHK Inhibitors

46
(m, 1H), 4.18 (m, 1H), 3.84 (m, 2H), 2.10 (t, 2H), 1.75 (s, 3H), 1.53 (s, 3H), 1.46 (s, 9H),
1.41 (m, 4H), 0.87 (t, 3H);
13
C NMR (500 MHz, C
6
D
6
) δ 154.8, 95.6, 86.6, 81.3, 80.1,
64.8, 63.8, 63.1, 30.4, 28.9, 27.8, 27.7, 25.6, 25.3, 21.6, 18.2, 13.2; HRMS calculated for
C
17
H
29
O
4
N

+ Na: 334.1994, found 334.1989. Yield: 76.4%.
(S)-tert-Butyl-4-((R)-1-hydroxyundec-2-ynyl)-2,2-dimethyloxazolidine-3-carboxylate
(2a2). [α]
25
= -44.62° (c = 43.5 x 10

25
= -49.16° (c = 84.0 x 10
-3
g/ml, CH
2
Cl
2
);
1
H NMR (300 MHz, C
6
D
6
) δ4.85
(m, 1H), 4.08 (m, 1H), 3.79 (m, 2H), 2.01 (t, 2H), 1.56 (s, 3H), 1.40 (s, 3H), 1.36 (s, 9H),
1.32 (m, 4H), 0.76 (t, 3H);
13
C NMR (300 MHz, C
6
D
6
) δ 154.8, 95.6, 86.6, 81.3, 80.1,
65.9, 64.7, 64.2, 31.6, 29.1, 28.9, 28.8, 26.7, 26.4, 22.8, 19.3, 14.3; HRMS calculated for
C
17
H
29
O
4
N + Na: 334.1994, found 334.1987. Yield: 15%.

H NMR (300 MHz, CDCl
3
) δ4.57 (m, 1H), 4.08 (m, 1H),
Chapter2 Development and Evaluation of Human SPHK Inhibitors

47
3.72 (m, 2H), 2.18 (t, 2H), 1.43 (s, 9H), 1.24 (m, 12H), 0.85 (t, 3H);
13
C NMR (300 MHz,
CDCl
3
) δ 156.2, 87.9, 80.0, 64.3, 62.6, 55.8, 31.7, 29.1, 29.0, 28.8, 28.4, 28.2, 22.5, 18.6,
14.0; HRMS calculated for C
18
H
33
O
4
N + Na: 350.2302, found 350.2314. Yield: 72.98%.
tert-Butyl-(2S,3R)-1,3-dihydroxyoctadec-4-yn-2-ylcarbamate (3a3). [α]
25
= -63.31° (c
= 0.420 x 10
-3
g/ml, DMSO);
1
H NMR (300 MHz, CDCl
3
) δ4.58 (m, 1H), 4.05 (m, 1H),
3.70 (m, 2H), 2.19 (t, 2H), 1.44 (s, 9H), 1.24 (m, 22H), 0.86 (t, 3H);

H
HO

Chapter2 Development and Evaluation of Human SPHK Inhibitors

48
(S)-tert-Butyl-4-((R)-1-hydroxyhexadec-2-ynyl)-2,2-dimethyloxazolidine-3-
carboxylate (2a3): [α]
25
= -40.09° (c = 26.0 x 10
-3
g/ml, CH
2
Cl
2
);
1
H NMR (500 MHz,
C
6
D
6
) δ4.60 (m, 1H), 4.09 (m, 1H), 3.79 (m, 2H), 2.06 (t, 2H), 1.68 (s, 3H), 1.44 (s, 3H),
1.37 (s, 9H), 1.33 (m, 22H), 0.92 (t, 3H);
13
C NMR (300 MHz, C
6
D
6
) δ 154.8, 94.6, 85.6,

Structures of six obtained compounds (analogues of sphingosine)

2.2.2 Compounds Inhibitory Function on SPHK Activity in vitro
2.2.2.1 Over-expression of SPHK1 and SPHK2
2.2.2.1.1 EGFP-SPHK1 Plasmid Verification
The EGFP-SPHK1 was constructed in the laboratory, and was amplified and purified
followed by 1% of agarose-gel electrophoresis verification (Figure 2.3). The primers for
ONBoc
CC
4
H
9
H
HO
ONBoc
CC
4
H
9
OH
H
ONBoc
CC
8
H
17
H
HO
HO
OH

2.2.2.1.2 Over-expression of SPHK1 and SPHK2
As it was described in Section 2.1.2.1 Compound Function on Exogenous SPHK Activity,
EGFP-SPHK1 transfection efficiency was detected by fluorescence microscopy and
SPHK assay.
Figure 2.4 shows the same visual field of the EGFP-SPHK1 transfected CHO cells under
normal and fluorescence microscope. It can be clearly seen that the transfection was very
successful and the over-expression was shown to yield a substantial increase in SPHK
activity, 18-fold for SPHK1 and 5-fold for SPHK2, in transfected cell lysates. The results
are shown in Figure 2.4C. These transfections allow us to differentiate between SPHK1
or SPHK2 activities when testing for compound selectivity. Chapter2 Development and Evaluation of Human SPHK Inhibitors

50

Figure 2.4 Transfection efficiency of SPHK1 and SPHK2. CHO cells transfected
with the EGFP-SPHK1 shown under normal microscope (A) or fluorescence microscope
(B). Results shown are representative of three independent experiments. Figure C, SPHK
activity from CHO cell-lysates that had been transfected with EGFP-SPHK1 and SPHK2,
and compared to un-transfected-control cell lysate. Results are the average U+U the SD of

51
inhibit half amount of SPHK1 or SPHK2 activity, indicating that DMS is not a specific
inhibitor for either SPHK1 or SPHK2. Figure 2.5 Inhibitory functions of DMS and compounds on SPHK1 and SPHK2
activity. A, Inhibition of SPHK1 activity by various concentrations of compounds and
DMS is shown. B, Inhibition of SPHK2 activity by various concentrations of compounds
and DMS is shown. Results are the average U+U the SD of triplicates samples from three
independent experiments. (n=3±SD, *P<0.05, vs. EGFP-SPHK1 transF in A, or vs.
SPHK2-transF in B;
#P<0.05. Student’s t-test).

It is very clear that none of the six compounds showed to inhibit SPHK2 activity (Figure
2.5B). However, all of them showed to inhibit SPHK1 to different degrees (Figure 2.5A).
A
B
*
*
#
*
*
*
*
*
*
*
Chapter2 Development and Evaluation of Human SPHK Inhibitors

52

three independent experiments.

2.2.4 Counter Screening Assays for Compound Specificity
2.2.4.1 Role of the Compounds on DAGK Activity
2.2.4.1.1 Optimization of the amount of DAG used in DAGK Assay
The amount of DAG used in the assay was optimized first.
Five different concentrations of DAG (16nmol, 32nmol, 160nmol, 800nmol, and
4000nmol) were tested and the radio-labeled product signals are shown in Figure 2.7.
Results suggest that under all five concentrations tested, radio-labeled product signals
were detectable and the signals increased when the amount of DAG increased.
70
90
110
130
150
0 5 10 15 20 25 30
C5a
C5a +DMS
C5a + CP1
C5a + CP2
C5a + CP3
C5a + CP4
C5a + CP5
C5a + CP6
Time
SPHK1 activity % over basal
Chapter2 Development and Evaluation of Human SPHK Inhibitors

54
In order to have the best sensitive control for inhibition, the smallest amount of DAG

75μM, and 100μM) of DMS were tested. The PKC inhibitors Bisindolilamide (Bis) and
RO-31-8220 were tested at 100nM as positive controls. Results from DMS, Bis, and RO-
31-8220 are shown in Figure 2.9.
*
*
Chapter2 Development and Evaluation of Human SPHK Inhibitors

56
DMS, Bis and RO-31-8220 function on PKC activity
0.00%
20.00%
40.00%
60.00%
80.00%
100.00%
120.00%
ctrl 10uM
DMS
25uM
DMS
50uM
DMS
75uM
DMS
100uM
DMS
100nM
Bis
100nM
RO-31-