Fluorogenic metabolic probes for direct activity readout of redox enzymes: Selective measurement of human AKR1C2 in living cells

Yee et al. 10.1073/pnas.0604672103.

Supporting Information

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Fig. 5. Correlation of the metabolic rate of probe 1 (initial 30% of metabolic reduction) to the relative abundance of AKR1C2 RNA transcript. [Probe 1] = 5 mM, rate of probe metabolism quantified relative to the fully reduced product; the relative abundance of RNA was determined by real-time RT-PCR as described in Supporting Text. Data shown are the average ±SD of three independent experiments as described in Supporting Text.

Scheme 1. Synthesis of fluorogenic substrate 1 and product 2.

Supporting Text

Synthesis of Fluorogenic AKR1C Substrate 1 and 3 and Fluorescent Product 2 and 4. The syntheses of 3 and 4 have been described (1). For the synthesis of 1, a solution of 5 (206 mg, 1.09 mmol) in CH₃CN (5 ml) was added dropwise to a solution of 8-hydroxyjulolidine (203 mg, 1.04 mmol) and PPh₃ (273 mg, 1.04 mmol) in CH₃CN (15 ml) at -5°C. After 10 min at -5°C, the resulting mixture was heated in a sealed tube to 120°C and maintained at this temperature for 24 h. The reaction mixture was cooled down, and solvent was removed in vacuo. The residue was subjected to multiple rounds of column chromatography (eluent gradient: CH₂Cl₂-EtOAc 100:0 to 95:5 and hexanes-EtOAc 9:1) and recrystallized from CHCl₃-hexanes to afford 1 (212 mg, 59%).

The synthesis of 2 proceeded with the addition of CeCl₃•7H₂O (48 mg, 0.13 mmol) to a solution of 1 (0.10 mmol) in MeOH-CH₂Cl₂ 2:1 (6 ml) at 0°C, followed by addition of NaBH₄ (20 mg, 0.52 mmol). After 20 min, the reaction was quenched with a saturated aqueous solution of NH₄Cl and extracted with CHCl₃. The organic layer was dried over MgSO₄ and evaporated, and the crude product was purified by column chromatography on silica gel (eluent gradient: CH₂Cl₂-EtOAc 98:2 to 8:2). Recrystallization from CHCl₃-hexanes provided pure alcohol 2 (87%).

Molecular Characterization, Substrate 1. ¹H NMR: (300 MHz, CDCl₃) d ppm: 7.95 (m, 2H); 7.63 (m, 1H); 7.48 (m, 2H); 6.73 (s, 1H); 5.93 (s, 1H); 3.28 (m, 4H); 2.92 (t, 2H, J = 6.5 Hz); 2.63 (t, 2H, J = 6.2 Hz); 1.95 (m, 4H). ¹³C NMR: (75 MHz, CDCl₃) d ppm: 194.3; 161.6; 152.2; 151.9; 146.5; 135.3; 134.6; 130.1; 128.9; 123.1; 118.7; 106.9; 106.1; 105.4; 49.9; 49.5; 27.5; 21.2; 20.4; 20.3. IR: (NaCl, cm^-1) 2936; 2844; 1716; 1670; 1614; 1586; 1547; 1522; 1433; 1371; 1311; 1260; 1166; 728. LRMS (FAB): Expected: 345.14, Found: 346 (C₂₂H₂₀O₃N, M+H).

Molecular Characterization, Product 1. ¹H NMR (300 MHz, CDCl₃) d ppm: 7.35 (m, 5H); 6.86 (s, 1H); 6.37 (s, 1H); 5.96 (d, 1H, J = 3.6 Hz); 3.21 (m, 4H); 2.85 (t, 2H, J = 6.5 Hz); 2.63 (m, 2H); 2.30 (d, 1H, J = 3.6 Hz); 1.91 (m, 4H). ¹³C NMR (75 MHz, CDCl₃) d ppm: 163.1; 156.6; 151.3; 145.5; 140.7; 128.9; 128.4; 127.1; 121.9; 117.9; 106.7; 106.1; 105.7; 72.3; 49.8; 49.4; 27.6; 21.4; 20.5; 20.4. IR: (NaCl, cm^-1) 3385; 2938; 2843; 1685; 1611; 1554; 1521; 1437; 1374; 1311; 1205; 1175; 1119; 732; 700. LRMS (FAB): 348 (C₂₂H₂₂O₃N, M+H).

Kinetics of Metabolism of Radiolabeled Physiological Substrate. To determine the kinetics of DHT metabolism in living cells, a mixture of radioactive and nonradioactive steroid containing 40,000 cpm of [¹⁴C]DHT was dried overnight and redissolved in DMSO to give final concentrations of 0.2-5K_M DHT. Five microliters of the radiolabeled DHT mixture in DMSO was then added to living cells. The final 0.25% (vol/vol) DMSO had no effect on cell viability. Then, 500-ml aliquots of the cell medium were taken from 0 to 3 h after addition of the DHT substrate mixture, the time points varying to allow for a plot of only the initial reaction rate (<30% substrate reduction). The aliquots of medium were then twice extracted by adding 500 ml of water-saturated ethyl acetate followed by 30 s of vortexing (>96% recovery). The ethyl acetate was evaporated to complete dryness using a Sorvall Speed Vacuum and redissolved in methanol containing reference steroids 3a-diol, 3b-diol, androsterone, androstanedione, and DHT. The dissolved steroids were plated on Whatman LK6D Silica TLC plates (Fisher), prerun twice, and developed three times using methylene chloride/diethyl ether (11:1 vol/vol). The TLC plates were analyzed with an automatic TLC-linear analyzer (Bioscan ImagingScanner System 200-IBM with an AutoChanger 3000; Bioscan). The computer-aided software quantitatively determined the radio signals emitted from the TLC plates and calculated the percentages of each metabolic product versus the total radioactivity. The positions of radioactive steroid signals on the TLC plates were verified by staining reference standards and quantified by scintillation counting. The percentages of substrate consumed were corrected for endogenous DHT metabolism and multiplied by the concentration of DHT used to approximate the metabolic rate (pmol/h). Kinetic parameters were subsequently estimated using GraFit (Erithacus Software; www.erithacus.com/grafit). All reported enzymatic kinetic parameters are the average of three independent determinations (performed in duplicate) from three different preparations of substrate and cellular transfections.

Determining the Kinetics of Reversible Inhibition: Fluorimetric Assay. IC₅₀ values for AKR1C2 in vitro and in living cells were measured by varying the inhibitor concentrations 0.125-3´ IC₅₀ while holding fluorogenic substrate 1 concentration equal to its K_M or K_M,app for AKR1C2. Inhibitor-substrate cocktails were dissolved in acetonitrile (in vitro assays) or DMSO (assays in living cells). The final concentration of acetonitrile in the in vitro assays did not exceed 4% (vol/vol), and the final concentration of DMSO in the cell medium did not exceed 0.5% (vol/vol). The presence of the acetonitrile and DMSO had no effect on initial reaction velocities. The reaction was followed by fluorimetric analysis as described above for fluorogenic substrate metabolism experiments. The corresponding inhibition data, which described the fluorimetric rate in the presence and absence of varying inhibitor concentrations, were fit using KaleidaGraph [y = (range)/[1 + (I/IC₅₀)^S] + background] to yield the IC₅₀ value. Then, using the association that exists between the IC₅₀ value, K_M, and the substrate concentration, the K_I or K_I,app values were then estimated for the inhibitors using the Cheng-Prusoff (2) relationship [K_I = (IC₅₀)/(1 + S/K_M)], where the compounds were known to display competitive inhibition kinetics.

Determining the Kinetics of Reversible Inhibition: Conventional Radiochemical Assay. Experiments to determine cellular kinetics of inhibition for AKR1C2 enzyme through a conventional radiochemical assay were performed by varying the inhibitor concentrations 0.125-3 ´ IC₅₀ while holding the DHT concentration equal to the K_M,app for AKR1C2. Inhibitor-substrate cocktails were prepared in DMSO, and the final volume of DMSO added to cells did not exceed 0.5% (vol/vol). The presence of DMSO had no effect on cell viability or initial reaction velocities. Conventional analysis of the kinetics of inhibition followed the protocol described in the previous section on measuring the kinetics of natural substrate metabolism. In short, cellular metabolism experiments were quenched at selected time points (<30% substrate conversion) with ethyl acetate, extracted, chromatographed on TLC plates, and analyzed using an automatic TLC-analyzer. TLC analysis provided the ratio of substrate DHT and reaction products 3a-diol and metabolites after selected time points. The ratios were used to estimate substrate conversion and analyzed by GraFit to provide the initial rate of reaction, which was then fit as described above.

Real-Time PCR. Relative expression of AKR1C2 in differently transfected COS-1 cells was determined by real-time RT-PCR normalized to GAPDH and PBDG as described (3). TriZOL agent (Invitrogen) was used to extract the total RNA from the cell lysates, and 1 mg of total RNA was reverse-transcribed using GeneAmp RNA PCR Kit (Applied Biosystems). Then, 50 ng of cDNA was added to each real-time PCR experiment and performed in triplicate. Real-time PCR was set up by addition of 12.5 ml of the PCR master mix (QuantiTect SYBR Green PCR Kit; Qiagen, Inc.) supplemented with 10 pmol of the respective forward/reverse oligonucleotide primers and either an external standard (0.025-2.5) ´ 10⁶ fg) or cDNA sample to give a final volume of 25 ml. A DNA Engine Opticon 2 (MJ Research) was used for plate analysis. At the end of the PCR, melting curves were performed to ensure the specific amplification of the desired product. The RT-PCR method was linear (>r = 0.995) over a dynamic range (10⁹) as determined by plotting the log₁₀ fluorescence intensity versus the number of cycles. Primer specificity was determined by separating the PCR product on a 3% gel and by sequencing to ensure only the amplification of the desired gene. The conditions for the real-time PCR using SYBR Green were as follows: 95°C for 15 min followed by 40 cycles of 94°C for 15 s, X°C for 30 s, and 72°C for 30 s (where X = 58°C for GAPDH and PBGD and 61° for AKR1C2). Full length standards (2,500,000-0.025 fg) were generated for AKR1C1, AKR1C2, AKR1C3, and AKR1C4 from their appropriate cDNA plasmids (pcDNA3-AKR1C1, -AKR1C2, -AKR1C3, and -AKR1C4). PCR product standards (2,500,000-0.025 fg) were generated using GeneAmp RNA PCR Kit for GAPDH and PBGD by performing reverse transcription on the total mRNA isolated from liver (BD Bioscience). The product was isolated by gel purification and used as real-time PCR standards with correction factors for GADPH (3.30) and PBGD (7.48) due to the difference in molecular weight between full-length and PCR product standards. Each 96-well plate contained nine standards in duplicate and four no-template controls to ensure the absence of contamination and/or primer dimer products. Samples (fg) were divided by the total cDNA in each reaction (ng) and subsequently normalized to the relative amount of PBGD and GAPDH, which was calculated to be the individual sample divided by the sample set average.

1. Yee, D. J., Balsanek, V., & Sames, D. (2004) J. Am. Chem. Soc. 126, 2282-2283.

2. Cheng, Y.-C. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108.

3. Bauman, D. R., Steckelbroeck, S., Williams, M. V., Peehl, D. M., & Penning, T. M. (2006) Mol. Endocrinol. 20, 444-458.

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