Chapter2 Development and Evaluation of Human SPHK Inhibitors CHAPTER 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 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 32 Chapter2 Development and Evaluation of Human SPHK Inhibitors not specific to SPHK It was found to inhibit not only two isozymes of human SPHKs (SPHK1 & SPHK2), but other kinases such as PKC (Igarashi and Hakomori 1989; Igarashi et al., 1989) Some attempts to produce inhibitors of SPHK have been carried out yielding promising compounds (French et al., 2003, Kim et al., 2005), but they are lack of proper validation In this study, novel inhibitors were designed and synthesized based on the natural substrate of SPHK (D-erythro-Sphingosine) These compounds are aimed to be more specific inhibitors for SPHK, and even isotype-specific (SPHK1 vs SPHK2) With the newly developed inhibitors, a better understanding of the role of SPHK in the process of stem cells differentiation will be studied as well In this chapter, only the compounds synthesis and evaluation are discussed Compounds function on stem cells differentiation will be addressed in Chapter The inhibitors of human SPHK used in this study were designed as the analogues of the natural substrate, D-erythro-sphingosine, by modifying several functional chemical groups of it The synthetic compounds were then evaluated for their efficiency and specificity to inhibit human exogenous and endogenous SPHK; the compounds were also tested for their potential cytotoxicity effects, and counter screened against other kinases including DAGK and PKCα The materials and methods are addressed below, followed by the results and discussion 2.1 MATERIALS AND METHODS Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich, Singapore 2.1.1 Synthesis of Human SPHK Inhibitors 33 Chapter2 Development and Evaluation of Human SPHK Inhibitors 2.1.1.1 Compounds Designed As Analogues of Sphingosine Earlier studies have shown that D-erythro-sphingosine (Figure 2.1a) is a natural substrate for SPHKs The design of the compounds was based on investigations of: 1) changing the fatty acid carbon chain length; 2) converting the existing double bond to either a single or triple bond, or 3) changing the hydroxyl group to other functionalities (Figure 2.1b) Scheme 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 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 is still used here to describe the flow (3) OH OH HO C13H27 D-erythro-sphingosine Figure 2.1 sphingosine C13H27 (1) NH2 NH2 (a) HO (2) The functional groups which would be modified (b) (a) D-erythro-sphingosine (b) Positions of modifications on D-erythro- 34 Chapter2 Development and Evaluation of Human SPHK Inhibitors (a) O H HO C H C R O OH H C O NBoc C R R MnO2 R O BuLi, THF NBoc O + NBoc O CH2Cl2 NBoc o -40/-15 C 2b 2b1: R = C4H9 2a 2a1: R = C4H9 2a2: R = C8H17 2a3: R = C13H27 5a: R = C4H9 5b: R = C8H17 5c: R = C13H27 10% Pd/C, H2, Amberlyst 15, MeOH Ph2S, MeOH OH OH HO O HO NHBoc C R NHBoc 3b 3a R R O Phenol, TMS-Cl, CH2Cl2 OH OH HO O HO NH2 R NH2 4a R HO 4b HO NHBoc 3a or 3b O O OH R CH2Cl2 R NHBoc (b) MnO2 NBoc HO HO NHBoc R 8a: R = C4H9 8b: R = C8H17 8c: R = C13H27 NH2 R Scheme 1: Synthesis of Analogues of Sphingosine 2.1.1.2 Chemical Experimental Procedures for the Synthesis of Compounds 2.1.1.2.1 Starting Materials and Apparatus All chemical reagents and solvents were obtained from Sigma Aldrich, Merck, Lancaster, 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 35 Chapter2 Development and Evaluation of Human SPHK Inhibitors Flash column chromatography was performed with silica (Merck, 70-230 mesh) 1H NMR and 13C 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 1H NMR analysis As described in the Scheme above, starting from S-(-)-1,1-dimethylethyl-4-formyl-2,2dimethyloxazolidine-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 (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 Synthesis of compounds 2a ((S)-tert-Butyl-4-((R)-1-hydroxy-3-alkyl-2-ynyl)-2,2dimethyloxazolidine- 3-carboxylate), and 2b ((S)-tert-Butyl-4-((S)-1-hydroxy-3-alkyl-2ynyl)-2,2-dimethyloxazolidine-3-carboxylate) Garner’s aldehyde (0.432 ml, mmol) in tetrahydrofuran (12 ml) was added, via a cannula, to a solution of the respective alkyne (2.72 mmol) and BuLi (1.45 ml, 2.32 36 Chapter2 Development and Evaluation of Human SPHK Inhibitors mmol) in tetrahydrofuran (25 ml), at -23 ºC The reaction mixture was stirred under nitrogen for hours at -23 ºC When TLC indicated the complete consumption of the starting materials, the reaction mixture was quenched with distilled water (10ml) and extracted with ethyl acetate The combined organic layer was washed with ammonium chloride, dried over magnesium sulfate, filtered, concentrated and purified by column chromatography (ethyl acetate: hexane = 1:8) to yield 2a and 2b Synthesis of Compound 3a (tert-Butyl-(2S,3R)-1,3-dihydroxyalk-4-yn-2-ylcarbamate) Compound 2a (0.2534 mmol) was dissolved in anhydrous methanol (15 ml) Amberlyst 15 resin (1.2 wt eq) was added and the reaction mixture was allowed to shake for 48 hours After which, the reaction mixture was filtered through Celite and evaporated in vacuo, and the residue was purified by filtration through silica gel with Hexane/ethyl acetate 1:1 to give 3a 2.1.2 Evaluation of the Synthetic Human SPHK Inhibitors 2.1.2.1 Compound Function on Exogenous SPHK Activity After the six analogues of sphingosine were synthesized, the inhibitory function of them on SPHK activity was first investigated by an in vitro SPHK assay (addressed in Section 2.1.2.1.5 SPHK Assay) using cell lysate which predominantly contained SPHK1 or SPHK2 protein CHO cells were transfected to over-express SPHK1 or SPHK2, to study the function of the synthetic compounds on the specific inhibitory potential against SPHK1 or SPHK2 activity 37 Chapter2 Development and Evaluation of Human SPHK Inhibitors 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% CO2 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) 38 Chapter2 Development and Evaluation of Human SPHK Inhibitors Over-expression of SPHK1 and SPHK2 was achieved by transfecting the SPHK1 or SPHK2 plasmids into CHO cells using Lipofectamine TM 2000 (Invitrogen), following the instructions provided by the manufacturer Briefly, CHO cells were transfected with 24μg plasmid/DNA using LipofectamineTM 2000 (Invitrogen) as a carrier, following the instructions provided The cells had been plated in 100mm tissue culture dishes Transfection efficiency for SPHK1 was detected by fluorescence microscope (by detecting green fluorescence) and SPHK activity assay For SPHK2, since there is no reporter system (like GFP) in the plasmid, the transfection efficiency was measured by SPHK activity assay comparing transfected to un-transfected cell lysates The SPHK activity assay utilizes P32-γ-ATP to generate a radio-labeled product (S1P), the goldstandard method to measure and quantify SPHK activity in vitro More details about this assay will be addressed below 2.1.2.1.3 Cell lysis CHO cells were collected and re-suspended in suitable amount of SPHK buffer (described below) Cells were lysed by several cycles of freeze-thawing and total cell lysates were obtained from the supernatant after centrifugation Protein concentration in the cell lysates was measured using the Bradford assay 2.1.2.1.4 Protein Quantification by Bradford Assay Bradford assay was used to quantify proteins in cell lysates It is a rapid and accurate method commonly used to determine the total protein concentration of a sample The assay is based on the notion that the absorbance maximum for an acidic solution, of Coomassie Brilliant Blue G-250, shifts from 465 nm to 595 nm when it binds to proteins Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a 39 Chapter2 Development and Evaluation of Human SPHK Inhibitors 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 MgCl2 solution γ[32P]ATP (10mCi/ml) RedivueTM 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 40 Chapter2 Development and Evaluation of Human SPHK Inhibitors enough contact area for sphingosine to react with SPHK as sphingosine alone is not readily phosphorylated by SPHKs Radio-labeled ATP/Mg2+ mixture was fleshly prepared, before each experiment, by mixing 10mCi/ml [32P]-γ-ATP with unlabeled ATP-MgCl2 (5mM ATP “cold”) followed by vortex, to generate a mixture of “hot” and “cold” ATP of 2μCi/sample 2.1.2.1.5.2 Reaction Procedures 80μg of cell lysate, over-expressing SPHK1 or SPHK2, was placed into conical glass tubes and the tubes were placed on ice, 10μl of sphingosine-BSA complex was added, with or without the synthesized compounds Compounds, as well as DMS (used as a control), were tested at five different concentrations: 10μM, 25μM, 50μM, 75μM, and 100μM The reaction mixture was supplemented with SPHK buffer to a final volume of 190μl, finally 10μl of ATP mixture was added and the tubes were then vortexed Reactions were started by placing the samples at 37°C, and reactions were terminated after 30 minutes To terminate the reactions, 20μl of HCl (1N) was added To extract the lipids, 0.8ml of chloroform: methanol: HCl (100:200:1, v/v) mixture was added to the samples, mixed and left to stand at room temperature for 5-10 minutes The organic and aqueous phases were separated by adding 240μl of chloroform and 240 μl of 2N KCl The mixture was then vortexed, and rested at room temperature for 5-10 min, followed by a 5-10 centrifugation at 400g 50μl of samples from the organic phase, which contain radio-labeled S1P, were spotted onto a TLC plate (silica G50, Watman), using a positive displacement pipette After the sample spots were completely dry, the TLC plate was placed into a TLC chamber containing the resolution solvent: 1-butanol:methanol:acetic acid:water (80:20:10:20, v/v) The organic solvent mixture helps to run the lipid 41 Chapter2 Development and Evaluation of Human SPHK Inhibitors the PCR reaction were designed to amplify a 200bp fragment of the SPHK1 cDNA Lane demonstrated the correct size of PCR products using the plasmid as the template Figure 2.3 Amplified EGFP-SPHK1 verification (From left to right) Lane1: 100bp DNA ladder; Lane2: control (without DNA template); Lane3: plasmid amplified; Lane4: stock plasmid 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 49 Chapter2 Development and Evaluation of Human SPHK Inhibitors A B SPHK activity by SPHK assay Transfection efficiency C 2100% 1800% 1500% 1200% 900% 600% 300% 0% untransF EGFP-SPHK1transF SPHK2-transF 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 + the SD of triplicates samples from three independent experiments U U 2.2.2.2 Compounds Functions on SPHK1 and SPHK2 Activity in vitro Transfected CHO cell lysates were used in the validation The most widely used SPHK inhibitor – DMS – was used as a control when compounds were tested in both SPHK1 and SPHK2 assays Figure 2.5A and Figure 2.5B show the six compounds and DMS functions on cell lysates that over-express SPHK1 or SPHK2, respectively DMS showed a clear dose-dependent character in inhibiting both exogenous SPHK1 and SPHK2 activities (Figure 2.5A and 2.5B) In both tests, 100μM DMS was shown to 50 Chapter2 Development and Evaluation of Human SPHK Inhibitors inhibit half amount of SPHK1 or SPHK2 activity, indicating that DMS is not a specific inhibitor for either SPHK1 or SPHK2 A # * * * * * * B ** * 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 + the SD of triplicates samples from three independent experiments (n=3±SD, *PCP6>CP1>CP2 (Figure 2.5A) However, in cells endogenous SPHK1 inhibition test, compounds inhibition efficiency is: CP3 & CP6>CP4>CP1>CP5 (Figure 2.6) One of the possible explanations is that, different inhibitory efficiencies are determined by different compounds capabilities to penetrate into the cells Compounds that showed better inhibitory efficiency in inhibiting cell-endogenous SPHK1 than the in vitro overexpressed SPHK1 may possess better capability to penetrate into the cells It can be deduced from Figure 2.6, that CP3 and CP6 possess better penetration capabilities than the other compounds, even though the other compounds (CP1, CP2, CP4 and CP5) showed good inhibition to endogenous SPHK1 as well Based on the results we obtained, it is obvious that all the six compounds are more specific than DMS to SPHK1, and not inhibit SPHK2, DAGK, or PKC alpha Among all of the compounds, CP3 showed the best inhibitory effect in all tests, which is equally potent as DMS, but it is specific for SPHK1 However, the chemical synthesis procedure gave really low yield of CP3 (12.46%, Table 2.1), which limited further investigations using this compound In contract, CP6, which showed as good as CP3 in inhibiting cell 62 Chapter2 Development and Evaluation of Human SPHK Inhibitors endogenous SPHK1, had a relatively high yield (63.88% Table 2.1) For obvious reasons, for further experimentation, high yield compound is more desirable Therefore, CP6 was chosen as the candidate compound for further experimentation to study the role of SPHK in mediating stem cell differentiation The project for developing new inhibitors of human SPHK is still ongoing For future work, more compounds should be synthesized and evaluated Besides the evaluation assays mentioned in this chapter, compounds could be also screened using perhaps other enzymes, for example other PKC-isoforms and/or Phosphoinositide 3kinase Also, compounds could be tested and validated in animal models of diseases However, due to the limited duration of PhD study, and the requirement of Graduate Program in BioEngineering, investigations of the synthetic compounds were more on their applications in tissue engineering, which is in stem cells differentiation, in this study More studies of compounds function on stem cell differentiation are addressed in the next chapter 63 ... (m, 22 H), 0. 92 (t, 3H); 13C NMR (300 MHz, C6D6) δ 154.8, 94.6, 85.6, 80 .2, 79.7, 79 .2, 65.0, 64 .2, 63.8, 63 .2, 62. 8, 61.9, 32. 0, 29 .8, 29 .6, 29 .4, 29 .2, 28 .9, 28 .7, 28 .0, 25 .8, 25 .4, 23 .2, 22 .7,... 64.44% 72. 98% 63.88% 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... specific inhibitors for SPHK, and even isotype-specific (SPHK1 vs SPHK2) With the newly developed inhibitors, a better understanding of the role of SPHK in the process of stem cells differentiation