Application of db-1 in the preparation of medicines for preventing and treating nlrp3 inflammasome-related diseases and its pharmaceutical composition
A technology for inflammasomes and diseases, applied in the field of medicine, can solve problems that have not been fully elucidated, and achieve significant prevention and treatment effects and good prevention and treatment effects.
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Embodiment 1
[0072] Validation of the inhibitory effect of DB-1 on NLRP3 inflammasome activation
[0073]DB-1 inhibits ATP-induced release of pro-inflammatory factors related to NLRP3 inflammasome activation: three batches of PMA-induced adherent THP-1 cells (THP-Ms cells) were first treated with 15, 30, and 45 μM DB-1, respectively. Then stimulated with LPS, and finally induced with ATP (for the specific experimental process, please refer to the stimulation of the inflammasome above), and use IL-1β antibody, Caspase-1 antibody, NLRP3 antibody and ASC antibody for western blot analysis (for the specific experimental process, please refer to the above for details. Western blotting), the content of IL-1β and active caspase-1 in the supernatant of THP-Ms cells was determined by ELISA method (for details of the experimental process, please refer to the above enzyme-linked immunosorbent assay), and the results are shown in figure 1 A-1C, the results show that under the combined action of the fi...
Embodiment 2
[0079] Validating whether DB-1 can inhibit upstream signaling of the 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, 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 lysate of THP-Ms cells (the specific experimental process is detailed in See Western blot above). see the results figure 2 A and 2B, the results show that the expression of NLRP3 and IL-1β was slightly inhibited in THP-Ms cells treated with DB-1 before LPS stimulation compared with DB-1 treatment after LPS stimulation.
[0081] The effect of DB-1 on ATP-induced mRNA levels of NLRP3 inflammasome activation...
Embodiment 3
[0085] DB-1 targets the NACHT domain of NLRP3 and inhibits ATPase activity
[0086] Using computer-aided drug design, the binding mode of DB-1 to the NACHT domain of NLRP3 was simulated, and the docking site was derived from the position of ADP co-crystallized at the binding site of NLRP3 (PDB 6NPY), see image 3 A-3B. In the figure, hydrogen bonds are represented by green dashed lines, Pi-Pi stacking interactions are represented by Li red dashed lines, and electrostatic interactions are represented by yellow dashed lines. The carbon atoms of the ligands 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 the 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 bonding interactions and Pi-Pi interactions.
[0087] The d...
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