Use of compounds capable of inhibiting the interaction between coronavirus spike protein and ace2
A coronavirus, compound technology, applied in the field of pharmaceutical use, can solve the problems of poor oral bioavailability, poor product uniformity, unstable production and storage process, etc.
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Embodiment 1
[0046] Preparation and structure identification of the compounds of the present invention.
[0047] Three compounds will be used as examples (structures are shown below):
[0048]
[0049] experimental method:
[0050] (1) The preparation method of compound 1 is as follows:
[0051] (1) Take the dried herbaceous stems of Ephedra herbaceae, dry them at 50°C, pass through a 65-mesh sieve after crushing, and take the ephedra powder under the sieve for later use;
[0052] (2) Add 50 times its weight of ethanol with a weight percentage concentration of 70% to the ephedra powder for ultrasonic-assisted extraction. The extraction temperature is 25°C, the extraction time is 0.5 h, and the ultrasonic frequency is 40 KHz. Filter and remove the solvent to obtain The extract is ready for use;
[0053] (3) The product in step (2) was dissolved in distilled water, ultrafiltered and centrifuged for 0.5 h, the lower layer solution was taken, concentrated under reduced pressure, the solv...
Embodiment 2
[0067] The compound of the present invention inhibits the interaction between the RBD region of the SARS-CoV-2 novel coronavirus Spike protein and the ACE2 receptor.
[0068] Experimental method: Competitive binding inhibition experiment. Add 100 μl of 0.5 μg / ml SARS-Cov-2 RBD recombinant protein dissolved in coating solution (50 mM carbonate buffer, pH 9.6) to a 96-well microplate, and place at 4°C overnight. The next day, rinse three times with 300 μl washing solution (phosphate buffered saline containing 0.05% Tween 20, pH 7.4) to remove uncoated proteins. Then add 300 μl of blocking solution (washing solution containing 2% bovine serum albumin, pH 7.4) and block at 37°C for 1h. After washing three times, 50 μl of different concentrations of the test compound was mixed with 50 μl of 0.12 μg / ml biotinylated ACE2, added to the 96-well plate, and incubated at 37°C for 1 hour. After washing three times, 100 μl of streptavidin-labeled horseradish peroxidase was added to each w...
Embodiment 3
[0071] The compound of the present invention promotes the dissociation of the RBD region of the SARS-CoV-2 novel coronavirus Spike protein and the ACE2 receptor complex.
[0072]Experimental method: Competitive binding inhibition experiment. Add 100 μl of 0.5 μg / ml SARS-CoV-2 RBD recombinant protein dissolved in coating solution (50 mM carbonate buffer, pH 9.6) to a 96-well microplate, and place at 4°C overnight. The next day, rinse three times with 300 μl washing solution (phosphate buffered saline containing 0.05% Tween 20, pH 7.4) to remove uncoated proteins. Then add 300 μl of blocking solution (washing solution containing 2% bovine serum albumin, pH 7.4) and block at 37°C for 1h. After washing three times, 50 μl of 0.12 μg / ml biotinylated ACE2 was added to the 96-well plate and incubated at 37°C for 1 h. After washing three times, 50 μl of the compound to be tested at a concentration of 25 μM was added to the 96-well plate and incubated at 37°C for 1 h. After washing t...
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