The Discovery And Research Of Novel BTK Inhibitors Against Rheumatoid Arthritis

Bruton's tyrosine kinase(BTK)is a non-receptor tyrosine kinase that plays an important role in the development of B cells and is highly expressed in a variety of B-cell malignant hematological tumors.It is one of the popular targets of anti-tumor drugs..Currently,Ibrutinib and ACP-196 have been approved for marketing,and several drug candidates are in clinical trials.Ibrutinib has been approved by the FDA for the clinical treatment of B cell malignancy and anti-transplantation host disease(GCHD);the second generation of BTK small molecule inhibitor ACP-196 was also approved by the FDA in 2017 for mantle cell lymphoma(MCL)treatment,while its treatment in autoimmune is also in the second phase of clinical.In this study,structural optimization was performed using Ibrutinib and ACP-196 as positive drugs,and activity evaluation and mechanism studies were performed on more than 300 modified compounds from the molecular,cellular and animal levels,and then combined with the overall animal-level drug evaluation to obtain independent knowledge.Property innovation candidate drug.First,the inhibitory activity of the engineered compounds on BTK kinases was tested at the molecular level,of which 42 compounds had an IC50 of less than 10 nM;for 42 molecules with activity less than 10 nM,cell proliferation inhibition experiments(IC50 less than 10 nM)were performed at the cellular level,and 12 compounds were obtained.10 nM of the compound,and calcium flow experiments were performed on these compounds to obtain 5 active compounds having an IC50 of less than 100 nM.Using chemical biology methods to carry out cell-level mechanism studies,by synthesizing a fluorescent probe with a similar structure and positive activity of the compound Ibrutinib,and selecting the same compound as the calcium flow assay.Using the occupancy-competitive assay to test the fluorescence intensity proves that the compound is indeed related to BTK Target-specific binding and activity screening(complete occupancy activity less than 100 nM),combined with data from the above molecular and cellular levels,data comparisons to obtain candidate compounds SPI-SIMM-519 and SPI-SIMM-718(complete occupancy activity 25 n M and 50nM).After the compound was subjected to an acute occupancy test in animals,the total occupancy of SPI-SIMM-519 in vivo was 5 mg/kg,which was comparable to that of Ibrutinib and superior to ACP-196.Taken together above data,SPI-SIMM-519 and SPI-SIMM-718 can indeed bind to BTK and has high activity.The indication for the candidate compound is arthritis,an autoimmune disease that requires long-term administration of the compound and low toxicity.Therefore,it is particularly important to consider its drug resistance and safety.Since Ibrutinib is currently undergoing clinical therapy,patients with drug resistance appear to have BTKC481 S and BTKT361 A mutations,and Ibrutinib and ACP-196,which are irreversible inhibitors,mainly bind to the C481 position of the BTK kinase through covalent bonds,and this site is mutated.This will lead to a decrease in compound inhibitory activity.Therefore,in this dissertation,the construction of BTKC481 S and the expression of eukaryotic protein in BTKC481 S were performed at the molecular level.The kinase activity assay system was established.The experimental system was optimized at the cellular level.The positive compounds were Ibrutinib and ACP-196 and the reversible BTK inhibitor GDC-0853.The experimental conditions for occupying the reversibility of the compound were optimized.The results showed that the candidate compounds SPI-SIMM-519 and SPI-SIMM-718 were all irreversible BTK inhibitors,and their activity was dependent on the position of BTK C481.In order to detect the toxic side effects of the selected compounds on normal B cells,a cell proliferation inhibition experiment was performed on normal human PBMCs.The screened candidate drugs SPI-SIMM-519 and SPI-SIMM-718 were evaluated on normal human PBMCs.According to the experimental results,the side effects of SPI-SIMM-519 were small and comparable to that of ACP-196.In summary,SPI-SIMM-519 shows good drug-making properties and has the potential for further research.Further comprehensive evaluation of SPI-SIMM-519 drug-property,SPI-SIMM-519 mouse pharmacokinetic evaluation,its Cmax 2254ng/mL,AUClas up to 4872h*ng/mL,and oral bioavailability up to 84.2%,far superior to Ibrutinib and ACP-196;further evaluation of chronic pharmacological effects on CIA rat rheumatoid arthritis model found that it has a good effect of relieving arthritis and can be significantly reduced after 29 days of administration The arthritis index and the degree of effective relieving the swelling of the athlete's foot;Finally,acute toxicity and 14-day sub-toxicity evaluation of Babl/c mice in SPI-SIMM-519 was evaluated.The results of acute toxicity test showed no significant toxic effect when administered orally at 1000 mg/kg.The 14-day sub-toxicity test showed no significant toxic response dose(NOAEL)of 125 mg/kg for SPI-SIMM-519,and at least 30 times the therapeutic window compared to the dose of 4 mg/kg.Based on the above data,SPI-SIMM-519 exhibits good drug-making properties and has the potential for further development.