A real-time high-throughput fluorescence assay for sphingosine kinases.

Sphingosine kinases (SphKs), of which there are two isoforms, SphK1 and SphK2, have been implicated in regulation of many important cellular processes. We have developed an assay for monitoring SphK1 and SphK2 activity in real time without the need for organic partitioning of products, radioactive materials, or specialized equipment. The assay conveniently follows SphK-dependent changes in 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled sphingosine (Sph) fluorescence and can be easily performed in 384-well plate format with small reaction volumes. We present data showing dose-proportional responses to enzyme, substrate, and inhibitor concentrations. The SphK1 and SphK2 binding affinities for NBD-Sph and the IC50 values of inhibitors determined were consistent with those reported with other methods. Because of the versatility and simplicity of the assay, it should facilitate the routine characterization of inhibitors and SphK mutants and can be readily used for compound library screening in high-throughput format.

Sphingosine kinases (SphK), consisting of two isoforms, SphK1 and SphK2, catalyze the formation of sphingosine-1-phosphate (S1P) by transferring the ␥ -phosphate from ATP to the primary hydroxyl of sphingosine (Sph). S1P is a potent bioactive lipid that can regulate many complex cellular processes, including growth, survival, migration, angiogenesis, and infl ammation, to name a few (1)(2)(3). Accordingly, abnormal regulation of SphKs that leads to elevated levels of S1P has been implicated in the etiology of cancer, as well as autoimmune and cardiovascular diseases ( 3 ). Hence, pharmacologically modulating SphK activity has important therapeutic potential, and technologies that can assist in the discovery of SphK inhibitors are critical tools for their development.

NBD-Sph substrate preparation
A stock solution of NBD-Sph in chloroform-methanol (8:2) was dried under nitrogen in a glass tube, solubilized in 5% Triton X-100, and sonicated extensively in a water bath with alternating vigorous vortexing. Concentrated stocks of 3.5 mM NBD-Sph in 5% Triton X-100 were stored frozen at Ϫ 20°C until use. However, after thawing and prior to use in assays, NBD-Sph solutions were extensively resonicated with alternating vigorous vortexing.

Fluorescence Sph kinase assay
Fluorescence emission was measured with a TECAN Infinite M1000 fl uorescence plate reader (Männedorf, Switzerland) at 37°C in 384-well polypropylene plates (Greiner Bio-One, Frickenhausen, Germany). Excitation wavelength was 550 nm, and emission wavelength was set at 584 nm with a 5 nm band-pass. Reactions analyzed using absorption were performed in quartz cuvettes with a Cary 50 Bio UV-visible spectroscopy (UV/Vis) Spectrophotometer (Agilent, Santa Clara, CA), and data were collected either in scanning kinetics mode (400-600 nm) or at 550 nm. Prior to the initiation of reactions, solutions were allowed to equilibrate, as indicated, at either 25°C or 37°C for 10 min. In most cases, assays were initiated with 20× ATP-Mg (20 mM ATP, 200 mM MgCl 2 , 900 mM Tris-HCl, pH 7.4), after which 384-well plates were shaken or cuvettes were mixed for 15 s for complete mixing. All assays were prepared as master mixes immediately before use in either SphK1 or SphK2 reaction buffer containing enzyme, NBD-Sph, and inhibitors where indicated. SphK1 reaction buffer contained 30 mM Tris-HCl, pH 7.4, 0.05% Triton X-100, 150 mM NaCl, 10% glycerol, 1 mM Na 3 VO 4 , 10 mM NaF, and 10 mM ␤ -glycero-phosphate. SphK2 reaction buffer contained 30 mM Tris-HCl, pH 7.4, 0.05% Triton X-100, 200 mM KCl, and 10% glycerol. Due to the poor solubility of NBD-Sph, optimal assay conditions contain 0.05-0.1% Triton X-100. It was important to add NBD-Sph to the master mix last and for the solution to be immediately and extensively vortexed just prior to use . Concentrations of DMSO up to 2% had no appreciable effects on catalytic rates and were used where indicated.

SphK-dependent changes in visible spectra of NBD-Sph
The 15-NBD-Sph, with a shorter sphingoid base, has previously been used for measurements of SphK1 activity ( 10 ). During the time course of this assay, samples were collected and sphingolipids extracted with chloroform-methanol (2:1) in alkaline conditions ( 10 ). This effectively separated the phosphorylated and nonphosphorylated NBD-labeled Sphs into aqueous and organic phases, respectively. When carrying out this activity assay with recombinant purifi ed SphK1 ( Fig. 1A ) and 18-NBD-Sph, designated hereafter as NBD-Sph, and prior to organic partitioning, we observed a distinct color change from bright yellow to light red. This led us to examine whether these color changes were quantifi able and SphK dependent. In the presence of SphK1 but ( 13,14 ). For many researchers, this is not a cost-effective alternative for high-throughput screening of small-molecule libraries as it also requires highly specialized expertise and equipment.
In this report, we present a novel NBD-Sph-based SphK assay that can be used to monitor enzymatic activity in real time. This method is highly sensitive when used with a fl uorescence plate reader, can be performed in 384-well format with very small reaction volumes, and can be used to examine the activity of both SphK1 and SphK2. Moreover, assays can also be monitored using absorption at 550 nm in a spectrophotometer or a plate reader, signifi cantly extending and simplifying assay capability by eliminating the need for specialized equipment.

Protein expression and purifi cation
Homo sapiens SphK1 (residues 2-384 with an N-terminal MGH-HHHHHHHHHSSG tag) and H. sapiens SphK2 (residues 2-618 with an N-terminal MGHHHHHHHHHHSSGHIEGRQL tag) were cloned into a modifi ed pET16b bacterial expression vector. For expression, transformed E. coli Rossetta-2 cells were grown in Luria broth with appropriate antibiotics at 37°C with shaking until optical density at 600 nm = 0.4, then the temperature was reduced to 18°C. After 3 h at 18°C, expression was induced overnight with 0.1 mM isopropyl ␤ -D -1-thiogalactopyranoside . Cells were harvested by centrifugation and resuspended at a ratio of 10 ml buffer per liter of initial culture in ice-cold buffer: SphK1 resuspension buffer contained 30 mM Tris-HCl [pH 7.4], 300 mM NaCl, 10% glycerol, 5 mM imidazole, 5 mM MgCl 2 , 5 mM ␤ -mercaptoethanol, and 2.5 mM octyl ␤ -D -glucopyranoside; SphK2 resuspension buffer contained 30 mM Tris-HCl [pH 7.4], 1 M KCl, 10% glycerol, 5 mM imidazole, 5 mM ␤ -mercaptoethanol, and 2.5 mM n -octyl-␤ -D -glucopyranoside. Cells were disrupted by sonication and centrifuged for 45 min at 25,000 g to remove cell debris, and the supernatants incubated at 4°C with 100 µl Ni-NTA Agarose (Qiagen, Valencia, CA) slurry per liter of initial culture volume (preequilibrated with resuspension buffer described previously) for 2 h with gentle shaking. The resin was extensively washed in batch with resuspension buffer, followed by washing with 2 ml per liter of initial culture volume with resuspension buffer containing 0.5 mM ATP and 5 mM MgCl 2 . SphKs were eluted with a step gradient containing 20, 30, 50, 60, 70, and 200 mM imidazole. Fractions containing SphK1 were pooled, concentrated, and loaded onto a 16/60 Superdex S75 (GE Healthcare, Pittsburgh, PA) column (120 ml) preequilibrated with 30 mM Tris-HCl [pH 7.4], 300 mM NaCl, 10% glycerol, and 2.5 mM n -octyl-␤ -D -glucopyranoside, and run at a fl ow rate of 0.1 ml/min in an AKTA fast protein LC system (GE Healthcare). SphK1 eluted as two peaks, one at a position expected for a monomer and another in the void volume of the column. Fractions corresponding to the monomeric volume to a red shift in the excitation wavelength is termed red edge excitation shift (REES). The NBD fl uorophore is highly sensitive to its environment, and the REES of NBDlabeled lipids has been used to monitor a variety of biological and biophysical processes in cellular and/or artifi cial lipid membranes ( 16,17 ). Therefore, we next examined whether the NBD-Sph spectral changes induced by SphK1 or SphK2 activity were accompanied by a corresponding shift in fl uorescence emission. To this end, we collected NBD-Sph fl uorescence emission spectra upon excitation at 474 nm (Ex 474 nm) or 550 nm (Ex 550 nm) in the absence or presence of SphKs and/or ATP. As expected, with Ex 474 nm, in the presence of SphK1 without ATP, NBD-Sph fl uorescence emission spectrum was stable with max at 539 nm ( Fig. 2A ), which rapidly decayed upon addition of ATP ( Fig. 2A , inset). Similarly, with Ex 550 nm in the presence of SphK1 or SphK2 but lacking ATP, NBD-Sph had little to no emission at 584 nm (Em 584 nm) ( Figs. 2B and 3A ). However, when NBD-Sph was incubated with SphK1 or SphK2 and ATP for 5 min at 25°C, there was a concomitant rise in Ex 550 /Em 584 nm emission ( Fig. 2B , inset and Fig. 3A , inset). Importantly, with Ex 550 nm, NBD-S1P in reaction buffer (containing 0.05% Triton X-100) had a fl uorescence emission spectrum ( Fig. 2C ) similar to that shown in Figs. 2B and 3A , with a max at 584 nm, and Ex 550 /Em 584 nm emission was proportional to the NBD-S1P concentration over a broad range ( Fig. 2C , inset). Thus, NBD-S1P and the fl uorogenic species that appears in reactions containing NBD-Sph and SphK have identical spectroscopic properties. Furthermore, TLC analysis ( 4 ) of the reactions clearly demonstrates the formation of a product with the same mobility as the NBD-S1P standard ( Fig. 2D , inset).
lacking ATP ( Fig. 1B ), or in the absence of both SphK1 and ATP ( Fig. 1H ), NBD-Sph (30 µM) in 0.05% Triton X-100 had an absorption spectrum with a max around 474 nm that was stable and consistent with the known properties of the NBD fl uorophore. However, upon addition of SphK1 and ATP, the 474 nm absorbance rapidly decayed and was accompanied by a subsequent rise of a new chromogenic peak around 550 nm ( Fig. 1C ). Decay of the 474 nm peak was also accompanied by a transient max shift of the 474 nm peak to 480 nm ( Fig. 1C ). Importantly, the rate of increase in absorbance at 550 nm was proportional to the concentration of SphK1 ( Fig. 1D ), and in control reactions no changes were observed in the absence of either SphK1 or ATP. Similarly, assays with purifi ed recombinant SphK2 ( Fig. 1A ) showed that NBD-Sph absorbance at 474 nm was stable in SphK2 reaction buffer containing SphK2 but lacking ATP ( Fig. 1E ), but decay at 474 nm was observed with a rise in absorbance at 550 nm when ATP was added ( Fig. 1F ). The rate of increase in absorbance at 550 nm was also proportional to SphK2 concentration ( Fig. 1G ). Importantly, in contrast to NBD-Sph, NBD-S1P in reaction buffer ( Fig. 1H ) had spectroscopic properties similar to the new chromogenic peaks observed in Fig. 1C and F , with a max around 550 nm and a shift of the 474 nm peak to 480 nm. However, when similar SphK assays were carried out with the short chain 14-NBD-Sph ( 15 ), no changes in absorption were observed as those seen with 18-NBD-Sph (data not shown).

SphK-dependent changes in NBD-Sph fl uorescence emission
A shift in the wavelength of maximum fl uorescence emission of a fl uorophore toward higher wavelengths due  ( Fig. 3B ) showed higher rates and maximum fl uorescence emission signal at 584 nm. Initial rates estimated from these experiments showed linearity over a wide range of SphK1 ( Fig. 2E , inset) and SphK2 ( Fig. 3B ,  inset) concentrations. There was a short lag in the appearance of Ex 550 /Em 584 nm emission, and initial rates were fi t to the linear portion of progress curves. Because the lag was inversely proportional to enzyme concentration, it is likely due to the requirement of suffi cient formation of NBD-S1P for the red shift to be detectable. Rates of NBD-S1P formation quantifi ed by Ex 550 /Em 584 nm emission normalized with internal NBD-S1P standards were identical to those determined by TLC ( 4 ) or by organic partitioning of NBD-Sph and NBD-S1P ( 10 ) ( Fig. 2D ). Moreover, k cat calculated from Ex 550 /Em 584 nm emission

Fluorescence SphK assays in 384-well plate format
The ability to perform enzymatic assays quickly, quantifi ably, and in high throughput is critical to accurately screen large inhibitor libraries and hence accelerate drug discovery. Moreover, fl uorescence is much more sensitive than UV/Vis absorption, which allows reaction volumes and reagents to be decreased. Because of the greater signal-to-noise ratio, we conducted experiments to validate SphK activity assays in high-throughput 384-well format with Ex 550 /Em 584 nm fl uorescence emission. Reactions in a total volume of 20 µl were initiated by the addition of 1 µl of 20× ATP-Mg. Ex 550 /Em 584 nm fl uorescence emission progress curves were recorded in the presence of increasing amounts of SphK1 ( Fig. 2E ) and SphK2 ( Fig. 3B ). In control reactions without ATP, no changes in Ex 550 /Em 584 nm fl uorescence emission were observed over the time mammalian SphK1, but as expected, no activity was detected in lysates from cells overexpressing mammalian catalytically inactive SphK1 ( Fig. 2H ). However, the signal is too low to accurately measure basal SphK activity. Importantly, overexpressed SphK1 activity was strongly inhibited by PF-543 ( 19 ), showing specifi city to a SphK1 catalyzed reaction ( Fig. 2H ).

Fluorescence assays in 384-well plate format for SphK inhibitor studies
It was next important to determine whether the REES fl uorescence SphK assay could be used to screen for smallmolecule inhibitors of SphK1 or SphK2. As a fi rst approach, we examined SphK1 activity in the presence of the potent SphK1 inhibitor PF-543 (250 nM) ( 19 ), which almost completely suppressed the ATP/SphK1-dependent appearance of the 550 nm peak ( Fig. 4A ). To further evaluate the dynamic range of this assay, we compared the SphK1 inhibitors PF-543 and N , N -dimethylsphingosine (DMS), which have IC 50 values in the nanomolar and (0.16 s Ϫ 1 ) was in good agreement with the previously reported value for 15-NBD-Sph (0.4 s Ϫ 1 ) ( 10 ).
We next performed experiments that examined fl uorescence emission rates at fi xed enzyme/ATP concentration and increasing amounts of NBD-Sph. Progress curves with SphK1 ( Fig. 2F ) or SphK2 ( Fig. 3C ) showed proportional increases in fl uorescence at concentrations below 50 µM, which plateaued at higher concentrations. Data fi tting analysis of initial rates estimated apparent K m values of 38 µM for SphK1 and 44 µM for SphK2, which are consistent with the reported value for SphK1 with 15-NBD-Sph ( 10 ). Both SphK1 ( Fig. 2G ) and SphK2 (data not shown) also showed dose-dependent rate increases as a function of ATP concentration. The apparent calculated K m for ATP with SphK1 was 70 µM ( Fig. 2G , inset), which is in excellent agreement with previous reports ( 18 ).
It was of interest to examine whether this assay could be used to measure endogenous basal or overexpressed SphK activity in mammalian cells. The assay can readily detect NBD-S1P formation in lysates from cells overexpressing  micromolar ranges, respectively. In assays with SphK1, PF-543 showed strong inhibition ( Fig. 4B ), and data analysis ( Fig. 4B , inset) revealed an IC 50 value of 16 nM. A previous study using 1 µM FITC-Sph as a substrate reported an IC 50 of 3.6 nM ( 19 ). SphK1 inhibition kinetics with DMS revealed an IC 50 of 15.2 µM ( Fig. 4C ), which is in excellent agreement with previous reports ( 20 ). We also examined the effect of the SphK2-specifi c inhibitor K145 ( 21 ). As can be seen in Fig. 4D , K145 suppressed SphK2 activity with an IC 50 of 33.7 µM, which is slightly higher than the K i value determined with the conventional radioactive assay ( 21 ). Taken together, these results suggest that our new fl uorescence method is suitable for rapid screening of SphK1 and SphK2 inhibitors.

CONCLUSIONS
In summary, we have developed a simple, convenient, and accurate real-time assay that can be used to rapidly determine SphK1 and SphK2 activity using NBD-Sph as a substrate. Our assay exploits a spectral REES in the environmentally sensitive properties of the NBD fl uor on NBD-Sph compared with NBD-S1P, which was completely dependent on SphK catalyzed phosphorylation of NBD-Sph to NBD-S1P. Our results suggest that NBD-S1P tends to associate in Triton X-100 micelles and that interactions between NBD groups on NBD-S1P are likely responsible for the red shift, similar to that observed with NBD-cholesterol in artifi cial membranes, where increasing concentrations show REES spectral transitions ( 16 ). Our assay does not require radioactive materials or isolation of products and is suffi ciently sensitive for high-throughput screening of chemical libraries and characterization of lead compounds. In addition, it should be an excellent tool to quickly examine the kinetic properties of SphK mutants with minimal labor and as a potential tool for studying presteady state properties and the mechanism of substrate binding of these enzymes ( 22,23 ), which remain poorly characterized. As SphK activity has been implicated in the etiology of many types of aggressive cancers, as well as autoimmune and cardiovascular diseases, this is an important step forward in the development of pharmacological agents that can target these important lipid kinases.