Regioisomeric Family of Novel Fluorescent Substrates for SHIP2

SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase, which, through its catalytic action on PtdInsP3, regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target, but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest family of inositide-binding/metabolizing proteins, while maintaining the regiochemical properties of bona fide inositide substrates.


1,3-Bis(benzyloxy)benzene-2,4-diol 21
. mCPBA (1.21 g, 7.0 mmol) was added to a solution of 2,4-Bis(benzyloxy)-3-hydroxybenzaldehyde 19 (10 g, 3.29 mmol) in dry CH2Cl2 (50 mL) and the mixture was stirred for 21 h at room temperature. The yellow solution was washed with an aqueous solution of 10% sodium metabisulphite (2 × 50 mL), a saturated solution of sodium hydrogen carbonate (1 × 50 mL), acidified with 1 M aqueous HCl (150 mL) and finally washed with water (50 mL). The organic layer was dried and the solvent was evaporated to give the crude formate ester (Rf = 0.60, ether-hexane, 2:1) as a yellow solid which was purified by flash chromatography (ether-hexane, 2:1) to give the formate ester 15 as a colorless syrup. The formate ester was dissolved in a mixed solvent (MeOH 50 mL) and stirred for 16 h in the presence of Amberlyst (1.0 g). TLC indicated a new compound with a lower Rf = 0.46 (ether-hexane, 2:1, crude). The Amberlyst was filtered off over a bed of celite and the organic solution was concentrated. The title compound 21 was purified by flash chromatography using CH2Cl2 as eluent Rf = 0.40 (CH2Cl2) as a pale yellow solid (m.p. 61-62 ºC, not recrystallized) 824 mg (78 %). A small portion was recrystallized from diisopropyl ether-hexane to give the title compound as an off white solid. 1   Dibenzylphosphite (1.33 mL, 6.0 mmol) was then added dropwise over 5 min at −10 °C (dry ice alone) and the mixture was stirred for a further 1 h under N2. The solvents were evaporated and the remaining yellow syrup was dissolved in dichloromethane (50 mL), washed with water (50 mL), dried, and the title compound was purified by flash chromatography Rf = 0.24 (EtOAc−hexane, 1:1), to give the product as a colorless syrup (658 mg, 78 %) which was triturated with cold ether and hexane to give a white crystalline solid, m.p. 70-71 ºC. Any impurity below the Rf value can be removed using CH2Cl2−CH3CN (5:1). Yield (658 mg, 78%). 1  1,3-Bis(benzyloxy)benzene-2,4bis(dibenzyloxyphosphoryloxy)benzene 22 (169 mg, 200 μmoles), was dissolved in dry CDCl3 (5 mL), dry 2,4,6-collidine (0.39 mL, 3.0 mmol) was then added and the solution was stirred over an atmosphere of nitrogen. Bromotrimethylsilane (0.4 mL, 3 mmol) was also added and the solution was stirred for 3 h at room temperature to give 23. The solvents were evaporated and the reaction mixture was quenched using a mixed solvent of D2O-2 M TEAB (2:1, 3 mL). The mixture was diluted with water (100 mL) and 1,3-dibenzyloxybenzene 2,4-bisphosphate 24 was purified by ion exchange chromatography using Q-Sepharose Fast Flow and a gradient of triethylammonium bicarbonate (TEAB) 0→2.0 M. The product eluted between 1.1 and 1.4 M TEAB buffer and isolated as the triethylammonium salt which was then used to make the target compound. 1,3-Dibenzyloxybenzene 2,4-bisphosphate 24 was dissolved in water (20 mL) and stirred for 18 h in the presence of palladium on carbon (10%, 200 mg) under an atmosphere of hydrogen. The solution was filtered through a PTFE syringe filter to remove the palladium on carbon and the solvents were evaporated to provide the title compound 10 (151 μmol) in 75.5 % yield as quantified by the Briggs test 3

Preparation of SHIP2:
An expression clone containing the catalytic domain of SHIP2 residues 419-832 (SHIP-cd) with an Nterminal 6xHis-tag followed by a TEV protease cleavage site sub-cloned into the vector pNIC-MBP was purchased via the Structural Genomics Consortium (SGC) from Source BioScience (Clone accession TC124029) and was used to transform E. coli Rosetta2 (DE3) cells (Novagen). Cultures were grown in LB medium supplemented with 25 μg ml -1 kanamycin at 37°C with shaking until OD600 reached 0.8. Target expression was induced by addition of 0.5mM IPTG and incubation at 23°C with shaking overnight. Cells were harvested by centrifugation (5,000 × g, 30 min, 4°C) and the pellet re-suspended in lysis buffer (100 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP, pH 8.0 plus completec EDTA-free protease inhibitor (Roche)). Cells were disrupted by French Press (three passes at 17,000 psi) and cell debris removed by centrifugation (42,000 × g, 60 min, 4°C. The filtered lysate was loaded onto a Ni-charged HiTrap chelating HP column (GE Healthcare). The column was washed with buffer A (20 mM HEPES, 500 mM NaCl, 1% glycerol, 500 mM imidazole, 0.5 mM TCEP, pH 7.5) and eluted with an elution gradient 20 mM to 500 mM imidazole over 50 ml, finishing with buffer B (20 mM HEPES, 500 mM NaCl, 1% glycerol, 20 mM imidazole, 0.5 mM TCEP, pH 7.5). The SHIP2-containing fractions were concentrated to 5 ml and loaded onto a 16/60 Superdex 75 gel filtration column, equilibrated and eluted with gel filtration buffer (20 mM HEPES, 300 mM NaCl, 1% glycerol, 20 mM, 0.5 mM TCEP, pH 7.5). The SHIP2-containing fractions were pooled and the N-terminal histidine tag was proteolytically removed by incubating with His-tagged TEV protease (Invitrogen) in a molar ratio of 30:1 at 4°C overnight. SHIP2 was purified from tag and protease by passing the reaction mixture over a buffer A equilibrated Ni-charged HiTrap chelating column and eluted with buffer A. The SHIP2 with His tag removed was concentrated and exchanged into gel filtration buffer using an Amicon concentrator with a 10 KDa MWCO membrane. For aqueous phase experiments, the protein was diluted into experiment buffer; 20 mM HEPES, 1 mM MgCl2, pH 7.3.

IC50 determination by displacement of 2-FAM InsP5:
Fluorescence polarization was determined as previously described 6 using a BMG ClarioStar plate reader with excitation wavelength 485 nm and emission wavelength 520 nm. 384 well plates were used with a volume of 20 μl in each well. For EC50 determination, varying amounts of SHIP2 (in 20 mM HEPES, 1 mM MgCl2, pH 7.3) were added to 2 nM 2-FAM-IP5. Displacement experiments used 2 nM 2-FAM-InsP5 and 1 μM SHIP2 with additions of displacing ligand/substrate ranging from nM to mM.

Phosphate release assay:
SHIP2 was diluted into 200 mM HEPES, 2 mM MgCl2, pH 7.3 and incubated with the individual compounds for a period of 10 mins at 30°C. The mixtures were cooled on ice. The phosphate detection reagent was freshly prepared; 4 parts 12.14 M ammonium molybdate in 1M sulphuric acid to 1 part 388 mM ferrous sulphate 7 . Using 384 well plates, 10 μl each reaction mixture was aliquoted into a well followed by 10 μl phosphate detection reagent. These mixtures were incubated for 10minutes at room temperature before using a Hidex Sense plate reader to determine the absorbance at 700 nm. A calibration curve was constructed, using KH2PO4 standards ranging from 0 to 100 μM, prepared in the same buffer and treated in the same way as the samples.

HPLC:
Reaction products were analyzed on a 3 mm × 250 mm CarboPac PA200 column (Dionex, UK) fitted with a 3 mm × 50 mm guard column of the same material. Samples (20µl) were injected and compounds were eluted with a gradient derived by mixing water (A) and 0.6 M methanesulfonic acid (B) according to the following schedule: time (min), % B; 0, 0; 25, 100; 38, 100. The eluent flow rate was 0.4 mL min -1 . Compounds were detected by fluorescence, excitation at 280 nm, emission at 330 nm, on a Jasco FP-4250 Fluorescence detector.

Fluorescence Spectroscopy:
Excitation and emission spectra of compounds were recorded using a Jasco FP8500 spectrometer in the fluorescence mode and polarizers were used to reduce spectral background arising from scattering of the excitation wavelength.

TD-DFT calculations:
All calculations were performed using the Gaussian 09 set of programs (Revision C. Gaussian, Inc. Wallingford) using the long-range corrected CAM-B3LYP 8 functional with the 6-31+G** basis set without symmetry constraints. Solvent effects treated using the polarizable continuum (PCM) model 9 with all calculations performed in acetonitrile. The geometries of ground and excited states were confirmed as minima by frequency calculation. Excitation and emission wavelengths and oscillator strengths were calculated using state-specific solvation method 10 .

TD-DFT DISCUSSION OF RESULTS:
TD-DFT analysis of weakly fluorescent compound 2 Bz(1,3,5)P3 and compound 5 Bz(1,2,3,5)P4 reveals that excitation from the ground state to the first excited state exhibits highly mixed character. Significant contributions from HOMO→LUMO, HOMO-1→LUMO+1 transitions were noted, and in the case of compound 2 Bz(1,3,5)P3, additional HOMO-1→LUMO transitions were observed, reflected in the weakest oscillator strength (Tables SA and SC). For compound 3 Bz(1,2,4)P3 and compound 6 Bz(1,2,4,5)P4, the transitions from the ground state to first excited state are predominantly of HOMO→LUMO character, but also with notable contributions from HOMO-1→LUMO+2 (Tables SE  and SG) (Tables SI and SK). Electronic transitions for the emission processes are predominantly LUMO→HOMO for benzene phosphates and tryptophan (Tables SB, SD, SF, SH, SJ, SL) and, for the respective classes of molecule, exhibit the same order of oscillator strengths as for the excitation process (Tables S1 and S2). The measured fluorescence of compound 6 Bz(1,2,4,5)P4 was 30 times more intense than that of the next most fluorescent benzene tetrakisphosphate, compound 5 Bz(1,2,3,5)P4, and approximately 10% of that of tryptophan ( Figure 4). These observations match the trends in oscillator strengths predicted by TD-DFT (Tables S1 and S2).
Our TD-DFT analysis systematically underestimates excitation wavelengths for both benzene phosphates and tryptophan compared with experimental results (Table S1 and S2). A similar observation has been noted for TD-DFT analysis using CAM-B3LYP/6-31+G* level of theory 10 . The predicted emission wavelengths also demonstrate an underestimate of up to 40 nm for the benzene phosphates, possibly as the quality of geometry optimization for molecules in excited states is known to be less accurate than for ground states. As the errors in the TD-DFT predictions are systematic, fluorescent properties of different compounds can reasonably be compared in terms of wavelength shifts.