Purification,Activity And Inhibitors Of The Phosphodiesterase 5

Phosphodiesterase5 (PDE5) can hydrolyze cGMP specially, to reduce its concentration. Male erectile dysfunction (ED) is closely related to a reduction in the level of cGMP. We can enhance the level of cGMP by inhibiting PDE5 activity to achieve the treatment of ED. ED seriously affected the physical and mental health of men. Therefore, research and development of specific PDE5 inhibitor is particularly important. The activity and the inhibitors of PDE5 were investigated.1. Isolation of Human Platelet and Purification of PDE5Fresh apheresis platelets from a healthy donor added ice-cold Hepes buffer, then were crushed by ultrasonic grinder, centrifuged and the supernatant was purified to get PDEs by Pharmacia FPLC system Acta Primer and Pharmacia Mono Q anion exchange column HR 5 / 5.2. PDE activity assayTo analyze the activity of PDE, we dissolved the step-by-step collection of fluid (2 - 5 uL), ³H-cGMP (2 mCi / mL), MgCl₂ (5 mmol / L) in Tris-HCl buffer solution. ³H-cGMP activated PDE5 hydrolysis reaction in water bath incubator, boiling water bath to terminate the response. And each tube was added venom (1mg/ml), 30℃heated, then added Bio-Rad AG1-X2 anion exchange resin, continuous mixed, placed after 30min ,instantaneous centrifugation. We took out of 0.5ml from the reaction system, then added 5ml scintillation fluid and used sigma LS1801 liquid scintillation to measure the cpm value of the end product. Comparison of each tube, s hydrolysis activity of 3H-cGMP, we know PDE5 Concentrated in the fifth and sixth tube. Further determine the optimal reaction conditions were as follows: the optimum reaction time was 15min, the optimal amount of enzyme was 2ul (PDE5 stock solution) and the optimum substrate concentration was 0.2pmol/ul. Activity unit of PDE5enzyme is 8.89×10-5nkat.3.Phosphodiesterase type 5 inhibitor screeningOptimum reaction conditions for the analysis of CYâ… ,CYII, CYIII,CYIV, CYâ…¤and CYâ…¥effect of PDE5 inhibition kinetics, the concentration of six new compounds are 10^-6mol / L ,10^-7mol / L, 10^-8mol / L ,10^-9mol / L ,10^-10mol / L and 10^-11mol / L respectively. We used sigma LS1801 liquid scintillation detection instrument to measure the cpm value of reaction system with Samples or not separately, and calculated their rate of PDE5 inhibition (with SDL as a control), using linear regression Eagle Ver2.5 software to get PDE5 inhibitory effect on the regression analysis. We obtained the IC50 value of CYâ… , CYII, CYâ…¤, CYâ…¥and SDL were0.0200mmol / L, 0.0032 mmol / L, 0.0027 mmol / L,0.0044mmol/L and 0.0031 mmol / L. When the concentration of CYIII and CYâ…£10-6 mol / L, they did not inhibit PDE5 activity. The results showed that, CYâ…¤on the degree of PDE5 inhibition is stronger than the SDL; CYII and SDL for the PDE5 were similar; In contrast, CYâ…¥was relatively weaker; CYâ… was the weakest. So it was concluded that CYâ…¤has strong inhibitory effect on PDE5 in this six new compounds, which is helpful for further investigation. This compound is expected to be effective drug treatment for ED.