Application of DB-1 in preparation of drug for preventing and treating NLRP3 inflammation correlative diseases and pharmaceutical composition thereof
A technology of inflammasomes and compositions, applied in the field of medicine, can solve problems that have not been fully elucidated
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Embodiment 1
[0072] Validation of the inhibitory effect of DB-1 on NLRP3 inflammasome activation
[0073]DB-1 inhibits the release of pro-inflammatory factors associated with ATP-induced NLRP3 inflammasome activation: three batches of THP-1 cells (THP-Ms cells) after PMA-induced adherence were first treated with 15, 30, and 45 μM DB-1, respectively, Stimulate again with LPS, and finally induce with ATP (see the above-mentioned stimulation of inflammasome for specific experimental process), and perform western blot analysis with IL-1β antibody, Caspase-1 antibody, NLRP3 antibody and ASC antibody (see above for specific experimental process) Western blotting), the content of IL-1β and active caspase-1 in the supernatant of THP-Ms cells was measured by ELISA (see the above enzyme-linked immunosorbent assay for details of the experimental process), and the results are shown in figure 1 A-1C, the results showed that under the joint action of the first signal LPS and the second signal ATP, the i...
Embodiment 2
[0079] To verify whether DB-1 can inhibit the upstream signaling of NLRP3 inflammasome
[0080] THP-Ms cells were treated with DB-1 before and after LPS stimulation, and the expressions of NLRP3 and IL-1β were compared in two ways: three batches of THP-Ms cells were treated with 15, 30, and 45 μM DB-1 before and after LPS stimulation, respectively, Then use ATP to induce (see the above-mentioned stimulation of inflammasome for the specific experimental process), and finally use IL-1β antibody, caspase-1 antibody, and NLRP3 antibody to perform western blot analysis on the cell lysates of THP-Ms cells (see the specific experimental process for details). See Western blot above). see results figure 2 A and 2B, the results show that the expressions of NLRP3 and IL-1β were slightly suppressed in THP-Ms cells treated with DB-1 before LPS stimulation compared with DB-1 after LPS stimulation.
[0081] The effect of DB-1 on ATP-induced NLRP3 inflammasome activation-related protein mR...
Embodiment 3
[0085] DB-1 targets the NACHT domain of NLRP3 and inhibits ATPase activity
[0086] Using computer drug-aided design, the binding mode of DB-1 and NLRP3 NACHT domain was simulated, and the docking site was derived from the position of co-crystallized ADP at the binding site of NLRP3 (PDB 6NPY), see image 3 A-3B. In the figure, hydrogen bonds are represented by green dotted lines, Pi-Pi stacking interactions are represented by Li red dotted lines, and electrostatic interactions are represented by yellow dotted lines. The carbon atoms of the ligand and the NLRP3 NACHT residues are colored light green and dashed gray. From the binding mode in the figure, the key residues for the interaction between DB-1 and the active pocket of NLRP3 NACHT domain are 165Arg, 232Ile, 410Pro, 506Phe, 519Ile and 520His. DB-1 interacts with these key amino acid residues of the NACHT domain in the NLRP3 protein through hydrogen bond interactions and Pi-Pi interactions.
[0087] The direct interact...
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