Discovery of catalytic-site-fluorescent probes for tracing phosphodiesterase 5 in living cells

Small molecule fluorescent probes provide a powerful labelling technology to enhance our understanding of particular proteins. However, the discovery of a proper fluorescent probe for detecting PDE5 is still a challenge due to the highly conservative structure of the catalytic domain in the phosphodiesterase (PDE) families. Herein, we identified probes based on the key amino residues in the ligand binding pocket of PDE5 and catalytic-site-fluorescent probes PCO2001–PCO2003 were well designed and synthesized. Among them, PCO2003 exhibited extraordinary fluorescence properties and the ability to be applied to PDE5 visualization in live cells as well as in pulmonary tissue slices, demonstrating the location and expression level of PDE5 proteins. Overall, the environment-sensitive “turn-on” probe is economical, convenient and rapid for PDE5 imaging, implying that the catalytic-site-fluorescent probe will have a variety of future applications in pathological diagnosis as well as drug screening.


Materials and instruments
All reagents and solvents are commercially available and used as received unless otherwise noted. Water used for the fluorescence and biological studies was doubly distilled and further purified with a Mill-Q filtration system. Fluorescence spectra were obtained under the fluorescence spectrograph HORIBA Fluoromax-4. The CCK-8 cytotoxicity assay was conducted on a microplatereader (POLARstar, Omega). Fluorescence imaging was performed by using the fluorescence microscopy (DMI-3000B, OLYMPUS) and the confocal fluorescence microscopy (FV-3000, OLYMPUS). Quantitative Real-time PCR (qRT-PCR) assays were performed by using qRT-PCR system (LightCycler480II, Roche). To a solution of CaCl 2 (2.2 g, 20 mmol), KOH(448 mg,8 mmol) in water (20 mL) was added methyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(benzo[d][1,3]dioxol-5-yl)propanoate(1.78 g, 4 mmol) in THF (8 mL) slowly at 0 °C. Then the reaction mixture was allowed to warm to room temperature and stirred overnight. Upon completion of the reaction, it was acidified by the addition of 2 mol/L aqueous HCl. The resulting solution was extracted with ethyl acetate and washed with water, brine and dried over anhydrous Na2SO4 and was concentrated. Then, it was added the solution of n-hexane: ethyl acetate=20:1 and stirred for 4 h. The mixture was filtered to afford the desired 1 as white solid(1.7 g, 95%).

Experiment section 4.1 Protein expression and purification
The expression and purification of PDE5A were carried out similarly to previously published protocols 6 . Briefly, the catalytic domain coding (535-860) of PDE5A was cloned to vector pET-15b and then the cDNA was transferred to E. coli strain BL21 (CodonPlus, Stratagene) for overexpression. When the cell carrying the plasmid was grown in LB medium at 37°C until OD600=0. 6

Fluorescence properties of PCO2003 with PDE5A recombinant protein
PDE5A recombinant protein was prepared in our group following the protocol published previously. PDE5A protein were diluted to different concentration from 0 to 0.2 mg/mL and incubated with probe PCO2003 (20 μM) in PBS buffer (1×, pH 7.4). After blending the two samples in a quartz cell, the fluorescent spectra were scanned with fluorescence spectrophotometer at 467 nm.

Cell culture
LX-2 cells, A549 cells and HeLa cells were cultured in DMEM medium with 10% FBS(v/v). All the cells were cultivated in an incubator under 5% CO 2 at 37 °C.
All the medium used for cell culture contained 1% penicillin-streptomycin (v/v) unless otherwise noted.

Cytotoxicity evaluation
Cell viability assay was performed to evaluate the cytotoxicity of PCO2001-2003 by CCK8 method. Cells were planted into a 96-well cell culture plate at approximate 6000 cell/well in 100 μL medium. The cells were incubated for 12 h at 37°C under 5% CO 2 in an incubator. Then, a solution of PCO2001-2003 (100 μL/well) at concentrations of 0, 0.39, 0.78, 1.56, 3.14, 6.25, 12.5, 25, 50, 100 μM in DMEM medium was added to the wells, respectively. After 24 h incubation, cell viability was measured by CCK-8 method.

Cell fluorescent imaging
Cells were seeded into a confocal dish at 1 × 10 5 / per dish in 1 mL medium and incubated for 24 h. After the medium was removed, the cells were carefully washed with 37 °C PBS (1×, pH 7.4), and Subsequently the cells were incubated with PCO2003(10 μM) and Hoechst 33342 (Beyotime, 100×) in DMEM for 30 min. They were then washed with DMEM to remove the unbound probes. For the competitive assay, cells were pretreated with sildenafil (25 μM) and dipyridamole (25 μM )for 12h respectively and then washed with the above procedure. For siRNA-transfected assay, the cells were transfected with siRNA at concentration of 100 nM with DMEM with 10% FBS without penicillin and streptomycin for 48 h in advance. Fluorescence imaging was then carried out by confocal fluorescence microscope (FV3000,