General Synthetic Procedures and Materials
All reagents were purchased from commercial vendors and used without further purification. Dowex 50WX8-200 cation exchange column was converted to its Na+ form by treatment with 1N NaOH, followed by washing with water to bring pH to neutrality. 1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC) was used from a previously unopened bottle. Analytical thin layer chromatorgraphy (TLC) was performed on 0.25 mm precoated Merck silica gel (SiO2
) 60 F254
. Column chromatography was performed on Purasil 60A silica gel, 230– 400 mesh (Whatman). 1
H and 31
P NMR were recorded on a Varian Mercury-300 spectrometer. Chemical shifts are reported in ppm relative to residual deuterium oxide (D2
O) or external indicator 85% H3
peaks for 1
H and 31
P NMR, respectively. High-resolution mass spectrometry (HRMS) data were obtained on a Biotof II (Bruker) ESI-MS spectrometer. 7-Bn-guanosine monophosphate (7-BnGMP, disodium salt) was synthesized according to established methods (40
was synthesized as described earlier (34
), and a slight modification of the same procedure, as outlined below, was used in the preparation of compounds 4Ei-2
7-Bn- guanosine monophosphate disodium salt (0.100 g, 0.200 mmol) and the appropriate amino acid methyl ester hydrochloride salt (D-phenylalanine methyl ester.HCl for 4Ei-2, and D-alanine methyl ester.HCl for 4Ei-3, 1.01 mmol, 5 equiv.) were dissolved in H2O (5 mL) and the pH of the solution adjusted to ~7.20 by dropwise addition of dilute aq. NaOH. To the foregoing was added EDC (0.192 g, 1.01 mmol, 5 equiv.) dissolved in 2 mL of 2mM N-methylmorpholine (pH = 7.0) and the resultant solution allowed to stir at room temperature overnight. Upon complete consumption of the nucleotide starting material (TLC and 31P NMR), the product mixture was concentrated in vacuo and the residue chromatographed on Silica gel, eluting with CHCl3/MeOH/H2O (5: 2: 0.25, containing 0.5% NH4OH). The solid obtained— EDC salt of the phosphoramidate— after evaporation of the solvent was passed through an ion-exchange column (Dowex-50WX8-200, Na+ form) and the relevant fractions pooled and lyophilized to give the desired phosphoramidate sodium salts as white amorphous solids in 35–40% yield.
Compound 4Ei-2: 1H NMR (D2O, 300 MHz) δ 7.22- 7.26 (m, 5H), 7.07- 7.06 (m, 3H), 6.86 (d, 2H), 5.81 (d, 1H), 5.38 (dd, 2H), 4. 54 (m, 1H), 4.23 (t, 1H), 4.12 (m, 1H), 3.81 (dd, 2H), 3.65 (t, 1H), 3.31 (s, 3H), 2.61 (m, 2H) ppm. 31P (D2O, 121 MHz) δ 7.11 ppm. HRMS (ESI): m/z calcd for C27H31N6NaO9P+ (M)+ -- 637.1788, found 637.1785.
Compound 4Ei-3: 1H NMR (D2O, 300 MHz) δ 7.25- 7.26 (m, 5H), 5.90 (t, 1H), 5.52 (dd, 2H), 4. 53 (m, 1H), (4.19- 4.27, 2m, 2H), 3.91 (dd, 2H), 3.52 (t, 1H), 3.38 (s, 3H), 1.07 (d, 3H) ppm. 31P (D2O, 121 MHz) δ 7.26 ppm. HRMS (ESI): m/z calcd for C21H27N6NaO9P+ (M)+ -- 561.1469, found 561.1474.
As is the case with most 7-benzylated guanosines, the signal due to the C-8 proton was not observed for either of the two compounds because of its rapid exchange with protons from the NMR solvent.
Fluorescence Titrations and Determination of Kd
The concentration of eIF4E was optimized to 200 nM and was used for all titration experiments, which was duplicated for each compound. Fluorescence spectra were recorded on a Cary Eclipse fluorescence spectrophotometer. Titration experiments were carried out at 22°C with freshly prepared HEPES buffer (50 mM Hepes, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA) at pH 7.2. Nonlinear fitting was carried out using the statistics software JMP IN 4.0 (SAS Institute) in which the following equation was applied:
- Fe = Fluorescence intensity of protein without ligand binding
- Fel = Fluorescence intensity of protein with ligand binding
- Et = Total concentration of the protein
- Totalligand = Total concentration of the ligand
- Kd = Dissociation constant
Each titration was duplicated and two parallel correction experiments were carried out—corresponding to the increase in fluorescence intensity by the intrinsic fluorescence of the cap analogs and decrease in fluorescence due to eIF4E degradation and dilution effect. A series of ligand stocks 20 µM, 50 µM, 100 µM, 250 µM, 500 µM, 1 mM, 2 mM, and 5 mM were prepared in order to obtain smooth titration curves by minimizing titration errors.
Cell-free Translation Assay
To directly assess the level of translation, we employed the dual-luciferase bicistronic reporter construct pcDNA3-rLuc-POLIRES-fLuc (42
), which is designed so that the translation of Renilla reniformis
luciferase (rLUC) is strictly cap-dependent, whereas the translation of firefly luciferase (fLUC) proceeds via an IRES in a cap-independent manner. The template encodes 2 forms of luciferase each producing a product emitting light at a unique wavelength. The plasmid was linearized with XmaI, purified from an agarose gel, and 5’-capped bicistronic luciferase reporter mRNA was generated by in vitro
transcription (mMESSAGE mMACHINE kit, Ambion) using T7 polymerase, according to the manufacturer’s instructions. Reporter mRNA (0.02 µg) was added to the reaction mixture (17 µL Retic lysate, 1 µL 1.25 mM L-methionine, 1.25 µL translation buffer per reaction), as recommended (Retic Lysate IVT™, Ambion). Renilla
luciferase translation was carried out at high ionic strength conditions (High salt mix: 150 mM potassium chloride); whereas firefly luciferase – at medium ionic strength conditions (Low salt mix: 25 mM potassium chloride). Test compounds or nuclease-free water (control) were introduced into the reaction mixture. In vitro
translation was carried out at 30°C for 1h. The reaction was stopped by chilling (5 min on ice) and samples were diluted with 100 µL of nuclease-free water. Renilla
and firefly luciferase activity/abundance was quantified by luminometry using the Dual-Luciferase Reporter Assay System (Promega) exactly as described in the technical manual, in a Lumat LB 9507 Luminometer (EG&G Berthold). Luminescence was measured in relative light units. Reporter translation in the samples was compared to the control, set at 100. Assays were performed in triplicate; the mean ± SEM values were determined for each compound concentration. To calculate IC50
, the compounds were tested at concentrations ranging from 2 to 2000̣ µM. Data in triplicate corresponding to each concentration were plotted and the IC50
values were determined directly from the plot.
Fish and embryos
Adult Zebrafish (Danio rerio
) maintenance, husbandry and embryo collection were performed at the Zebrafish Research Core Facility (Arnold and Mabel Beckman Center for Transposon Research) using standard procedures (53
) with approval from the Institutional Animal Care and Use Committee at the University of Minnesota. Freshly fertilized eggs were obtained through natural spawning. Eggs were staged for microinjections according to earlier precedents (54
). Embryos were kept at 28,5°C (standard temperature) and staged according to Kimmel et al (55
Microinjections, embryo observation and image acquisition were performed on the stage of a Stemi-2000 stereomicroscope (Carl Zeiss MicroImaging, Inc.) equipped with a PowerShot A630 digital camera (Canon) and PLI 100 Pico-injector (Warner Instruments, LLC) at room temperature. Eggs in chorions were lined up and restrained in the agar grooves (53
) with their animal poles upwards. Reporter mRNA solution (3 nL) was injected into the egg cytoplasm in the geometric center of the yolk depression (15 min post fertilization) through a micropipette with a splinted sharp tip of 2–3 µm in diameter. Test compounds were diluted to the final concentration with HBSS (Gibco) containing 0.03% phenol red (Sigma). Compound-containing medium (5 nL) was microinjected into the cytoplasm during furrow progression at the 2-cell stage in the second round of microinjections, performed through the opening in chorions made by the first injection. Sham-treated eggs (negative control) were injected with 5 nL of the carrier (HBSS). Eggs were kept at 28,5°C.
Zebrafish Translation Assay
Reporter mRNA was diluted with HBSS (Gibco) containing 0.03% phenol red (Sigma) to a concentration of 200 ng/µL. Aliquots were stored at −80°C until needed. Luciferase reporters were expressed ectopically after RLUC-POLIRES-FLUC mRNA injection into the single-cell fertilized eggs. We employed reporter in the form of mRNA, because transcripts are evenly distributed among dividing zebrafish embryonic cells after injection into fertilized eggs, and are translated into protein within the first day of development (56
). Eggs were first injected with the bicistronic luciferase mRNA (0.5 ng) and then with test compound in doses ranging from 5 to 25 pmols, with 7-MeGTP (reference compound), or with the carrier (HBSS). To standardize the level of luciferase expression across the injected eggs, we used a stringent microinjection protocol (one micro-needle per series, with no recalibration or readjustment of injection parameters). To ensure stable results, we normalized luciferase readouts to the number of eggs and utilized greater than 20 embryos (n ≥ 20) per sample. In each series of experiments, eggs were injected continuously (groove after groove), and for drug administration - in groups comprising equal numbers of eggs from all grooves. The second injection was performed through the opening in egg’s chorion made by the first injection. In assay validation using CHD (single dose - 0.1 pmol), the estimated error range for precision and accuracy of the Zebrafish translation assay was determined to be within 15%. Normally developing embryos were harvested at the early gastrula stage (28.5° C, 4.5h after injection). Firefly and Renilla
luciferase activity were analyzed using the Luciferase Reporter Assay system (Promega). Luciferase assays were performed in triplicate according to the manufacturer’s instruction in a Lumat LB 9507 (EG&G Berthold).
Ectoderm explant extraction and cultivation
Ectoderm explants (“animal caps”) were surgically removed from the apical region of late blastulae (stages Sphere-Dome, 4–4.3 hpf) at 28,5°C, in sterile Modified Barth’s saline (MBS) using established procedures (57
) with the following modifications: square blocks of superficial apical blastoderm comprised approximately 70 cells in 3 to 4 tiers with a portion of the outer enveloping layer; and explants were cultivated separately in sterile MBS without antibiotics on agar with a 3% methyl cellulose cushion at 28,5°C for up to 48h. Under these carefully controlled conditions, ectoderm blastula explants retain a spherical shape and contain dividing cells destined only to an ectodermal fate (57
). In unfavorable conditions, explants decompose and largely dissipate within 24h in culture. Explants from injected embryos or intact embryos (as a positive control) were harvested at 2 time points corresponding to 1h and 24h post excision and analyzed by RT-PCR for lineage and stage specific transcripts.
Whole-embryo lysates were prepared from normally developing embryos (20 per sample). Eggs were rinsed with HBSS, dispersed in Lysis Buffer (50mM Tris-HCl (pH 7.5), 250mM NaCl, 50mM NaF, 5mM EDTA, 0.2% NP-40) with protease inhibitor cocktail (Complete MINI, Roche) on ice, kept on a shaker for 20 min at 4°C and centrifuged at 16,000g for 15 min with the supernatant retained. Protein concentration in the supernatant was measured using the BCA™ protein assay kit (Pierce). Twenty micrograms of protein was subjected to 8% SDS-PAGE under reducing conditions and transferred to nitrocellulose. Immuno-detection of proteins was carried out using primary antibodies (Rat anti-HA, 1:2000) followed by horseradish peroxidase-conjugated secondary antibodies (anti-rat IgG, 1:500), and incubation with chemiluminescence substrate (Pierce).
RNA was extracted using TRI-reagent (Sigma). The RNA samples were treated with DNAase using Turbo DNA-free kit (Ambion) according to the manufacturer’s directions and converted to cDNA using the TaqMan reverse transriptase kit (Roche). Real-time PCR was performed using the Roche Light-Cycler with SYBR Green dye according to the manufacturer’s protocol (Roche). Amplified fragments were resolved on 1% agarose gels and sized according to standards. The sequences of the primers and the size of amplificate (bp) are provided below for each gene analyzed.
|Oligonucleotide primers used for RT-PCR|
|Marker||Forward primer (5’-3’)||Reverse primer (3’-5’)||Size|
Amplified fragments were resolved on 1% acrylamide gels and visualized by BrdU staining. Gel images were acquired with a UVP BioDoc-it™ System.
HINT activity in the Rabbit reticulocyte and Zebrafish gastrula lysates
Activity of HINT was quantified by individually titrating 20 µL of Rabbit reticulocyte lysate and 50 µL of zebrafish gastrula lysate (both in 600 µL HEPES) against 30 µL of the fluorogenic substrate, AMP-Tryptamine Phosphoramidate (34
). The observed increase in fluorescence intensity was plotted against time, and the slope of the rectilinear plots substituted in the following equation to obtain the value of the enzyme concentrations:
- v = Slope/(Fpro − Fsub) = kcat·[E]t, where
- v = velocity
- Fpro = Fluorescence due to product (tryptamine)
- Fsub = Fluorescence due to substrate (AMP-Tryptamine phosphoramidate)
- [E]t = Total enzyme concentration
- kcat for AMP-Tryptamine phosphoramidate = 2.1 ± 0.1 s−1
The Rabbit reticulocyte and zebrafish embryo lysates contain 23 and 1.95 ng of the enzyme, respectively.
Liquid chromatography/Mass spectrometry (LC-MS/MS) of 4Ei-1 Metabolism
Rabbit reticulocyte lysate (Ambion) and zebrafish embryo lysates were used in studying 4Ei-1 conversion. In the zebrafish lysate preparation, 300 early pre-gastrula eggs were collected, washed with “embryo medium”, dried and mechanically homogenized. The crude homogenate was kept rotating for 30 min at 4°C, centrifuged (15 min, 12,000 rpm at 4 °C) with supernatant fluid retained and designated embryo lysate. All standard solutions and samples were kept on ice before analysis by LC-MS/MS.
N, N-dimethylhexylamine (DMHA), tetrabutyl ammonium acetate (TBAA), and ammonium acetate were purchased from Sigma. Solvents used for LC analysis were HPLC grade. All solutions for instrument analyses were filtered through a 0.22 um membrane filter, and degassed prior to use. Microcon microcentrifuge filter device YM-10 (molecular weight cutoff - 10,000) was purchased from Millipore. Chromatographic separation was achieved using a narrow bore Eclipse XDB-C18 column (2.1 mm × 50 mm, 1.8 um, Agilent Technologies) eluted at a flow rate of 0.125 mL/min. An injection volume of 10 µL was used for standards and lysates. The mobile phase was composed of 50% Solvent A (10 mM ammonium acetate, pH 6.65) and 50% solvent B (methanol). The running time for each sample was 5 minutes. Sample temperature was maintained at 4 °C with a thermostat-controlled sample compartment. A TSQ quantum classic LC-ESI-MS/MS system (Agilent 1200 LC) was employed for all analyses. The mass spectrometer was operated in negative ion mode with nitrogen as a nebulizing and drying gas. 7-BnGMP and 4Ei-1 were directed to the detector for infusion. ESI source parameters and MS/MS parameters were optimized for maximum sensitivity. Negative ion ESI and selective multiple reaction monitoring (MRM) mode was used in all analyses. The [M-H]− ion of 7-BnGMP (m/z 452.10) was isolated and subjected to collision-induced dissociation (CID) to give the product ion (m/z, 79.19, collision energy 52 V) for quantification. 4Ei-1 was analyzed analogously to give a parent ion (m/z, 594.00) and product ion (m/z, 148.84, collision energy 52 V). The standard curves were obtained in respective matrix with a known concentration of 7-BnGMP and 4Ei-1 ranging from 10 to10,000 ng/mL. Quantification of target compounds was carried out with XCalibur software (Thermo Scientific). Rabbit and zebrafish lysates were diluted 64-fold with 10 mM ammonium acetate (pH 6.65) and methanol in a ratio of 1:1, followed by heating at 50 °C for 30 minutes. Precipitates were filtered using microcon YM-10 (Millipore). The filtrate served as matrix for preparing standard solutions. 10 µL of a known concentration (100, 500, 1000, 5000, 10000, 50000, 100000 ng/mL) of both targets dissolved in methanol was added to a HPLC sample vial. Methanol was removed in a SC210A SpeedVac® concentrator (Thermo Scientific). Lysate matrix (100 µL) was added to each concentrated vial to make the corresponding standard solutions (10, 50, 100, 500, 1000, 5000, 10000 ng/mL). For sample preparation, 4Ei-1 (5 µL) at 150 µM (final concentration) was incubated with lysates (17 µL) at 30 °C for 1 hour, followed by similar treatment (with or without heating at 50 °C for 30 minutes) as the standards. Controls were prepared in the same way except that dH2O (Sigma) substituted for 4Ei-1.