Application of dipeptide as ace enzyme activity inhibitor

Abstract

An application of dipeptide as an ACE enzyme activity inhibitor. Virtual screening is performed on 400 types of dipeptide based on ACE inhibiting effects thereof according to a detected ACE enzyme crystal structure by using self-developed software and adopting a molecular docking method, experiments are conducted to verify the ACE inhibitory activity of the dipeptide obtained by virtual screening, and it finds out that the dipeptide with the N terminal as cysteine has better ACE inhibitory activity.

Claims

1. A method of inhibiting an ACE enzyme by exposing the ACE enzyme to an ACE enzyme activity inhibitor, wherein the ACE enzyme activity inhibitor is a dipeptide, wherein said dipeptide is selected from the group consisting of CA, CH, CI, or CK; the N-terminal of the dipeptide is a cysteine, the amino acid in the dipeptide is in L-form or D-form, and at least one amino acid in the dipeptide is optionally modified with a group which can improve the stability of the dipeptide in vivo; wherein the C-terminal amino acid of the dipeptide is a basic amino acid or an aliphatic amino acid; wherein the basic amino acid is H or K; and wherein the aliphatic amino acid is A or I.

2. The method according to claim 1, wherein the ACE enzyme activity inhibitor is administered to a patient to lower blood pressure.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is the kinetic curves with different amounts of ACE enzyme;

(2) FIG. 2 is a relation curve of slope of linear segment of the kinetic curve vs enzyme activity; and

(3) FIG. 3 is an ACE inhibition curve with Captopril.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) The technical solution of the present invention is further described hereinafter with reference to experiments.

(5) Determination of ACE Enzyme Activity

(6) Kinetic Curves with Different Amounts of ACE Enzyme

(7) Preparation for the reaction is made according to the following table:

(8) TABLE-US-00001 No. 1 2 3 4 5 6 FAPGG 200 200 200 200 200 200 reagent (l) 115 u/L 20 16 12 8 4 0 ACE (l) Ultrapure water 20 34 28 32 36 40 (l) Note: enzyme 115 u/L 92 u/L 69 u/L 43 u/L 23 u/L 0 u/L activity

(9) The substances are mixed and placed in a SpectraMax microplate reader to determine change of absorbance value with 340 nm as a main wavelength and 405 nm as a reference wavelength at 37 C., and continuously monitored for 1 hour.

(10) The kinetic curves with different amounts of ACE enzyme are shown in FIG. 1, and the relation curve of slope of linear segment of the kinetic curve vs the enzyme activity is shown in FIG. 2. The results show that, slope of linear segment of the kinetic curve and the ACE enzyme activity are in a linear relation, with a regression equation y=7*10.sup.6x+5*10.sup.5, R.sup.2=0.995.

(11) Inhibitory Effect of Different Concentrations of Captopril on ACE

(12) Captopril is prepared with ultrapure water in 2 mg/mL, and then diluted with ultrapure water in 10-time proportion (till 10.sup.8).

(13) Preparation for the reaction system is shown in the following table:

(14) TABLE-US-00002 No. 1 2 3 4 5 6 FAPGG reagent 200 200 200 200 200 200 (l) 115 u/L ACE (l) 20 20 20 20 20 20 2 g/L 20 16 12 8 4 0 Captopril (l) Ultrapure water 0 4 8 12 16 20 (l) FAPGG reagent 200 200 200 200 200 200 (l) 115 u/L 20 20 20 20 20 20 ACE (l) 0.2 g/L 20 16 12 8 4 0 Captopril (l) Ultrapure water 0 4 8 12 16 20 (l)

(15) The substances are mixed and placed in a SpectraMax microplate reader to determine change of absorbance value with 340 nm as a main wavelength and 405 nm as a reference wavelength at 37 C., and continuously monitored for 1 hour.

(16) The inhibition curve with Captopril is shown in FIG. 3. It can be seen from FIG. 3 that, viewed from the overall trend, the inhibition rate is decreased as the concentration of Captopril decreases. Since the concentrations are in linear in the experiment, the variation range of the inhibition rate is reduced.

(17) Inhibitory Effect of Dipeptides on ACE

(18) 20 g/mL stock solution is prepared by dissolving synthetic dipeptide samples in ultrapure water. The stock solution is then diluted to a 20 g/mL sample as test sample.

(19) TABLE-US-00003 No. 1 2 3 4 5 6 FAPGG reagent (l) 200 200 200 200 200 200 115 u/L ACE (l) 20 20 20 20 20 20 g/mL dipeptide (l) 20 Captopril (l) 4 20 20 Ultrapure water (l) 0 16 0 0 20 40

(20) The substances are mixed and placed in a SpectraMax microplate reader to determine change of absorbance value with 340 nm as a main wavelength and 405 nm as a reference wavelength at 37 C., and continuously monitored for 1 hour. The slopes of linear segment of the enzymatic kinetic curve are calculated, and the inhibition rates of the samples are calculated according to formula (1);

(21) $\mathrm{Inhibition}\mathrm{rate}\left(\%\right)=\frac{115-E}{115}100$$\mathrm{Wherein},E=\frac{S_s-S_b}{S_p-S_b}115$ E-ACE enzyme activity in a sample well S.sub.s-slope of linear segment of ACE kinetic curve for a sample well S.sub.p-slope of linear segment of ACE kinetic curve for a well without inhibitor S.sub.b-slope of linear segment of ACE kinetic curve for a blank well

(22) The experimental results are shown in the following table:

(23) TABLE-US-00004 No. Dipeptide Inhibition rate (%) 3 FE 6.18 5 KW 16.42 9 IF 18.35 11 KY 11.54 13 AY 8.18 15 KP 7.88 19 WL 7.27 20 KA 6.45 22 AG 3.08 25 CA 84.38 28 CH 70.79 29 CI 67.52 30 CK 38.39 38 DE 15.16 47 EV 14.26 48 EW 9.91 49 FD 5.05 50 FH 10.06 51 FI 9.58 55 FQ 10.96 57 FT 1.92 58 FW 3.16 59 GC 0.12 64 GT 3.20 65 HC 5.06 66 HD 10.98 67 HE 9.95 Cap1 86.95 Cap2 72.84 Cap3 35.84

(24) It can be seen from the table that when the N-terminal of a dipeptide is a cysteine, the ACE inhibition rate of the dipeptide is significantly higher than those of other dipeptides; and more particularly, when the dipeptide is CA, CH, CI or CK, the inhibition rate is even higher.