Screening Of Complement C3aR Receptor Antagonist Analogues And Preliminary Study Activity

BackgroundMalignant tumor is an important cause of death of the global population,which seriously affects the health and life of hundreds of millions of people.tumor development is a complex biological process.The growth of tumor is constantly controlled by the immune system.The immune system regards cancer cells as a potential threat to the stability of the body environment,leading to innate immunity and adaptive immune effects.tumor growth depends not only on the accumulation of genetic abnormalities in primary cancer cells,but also on the local microenvironment that provides an excellent condition for the survival,growth and migration of cancer cells.The complement system is an important component of the innate immune system,regulating inflammation,promoting immune defense mechanisms,and maintaining tissue homeostasis.Complement is a key component of tumor-associated inflammation.The delicate balance between complement activation and regulation in tumor microenvironment leads to the continuous activation of complement and the continuous production of complement mediators,some of which have the function of supporting tumor growth and metastasis.Malignant tumor cells can use complement activation to produce a range of favorable cancer growth responses,such as chronic inflammation,immunosuppression,angiogenesis,and cancer cell signaling.Among the large number of complement factors potentially associated with tumors,C3a and C5 a are now considered to be key factors in establishing a favorable microenvironment for tumor growth.C3a plays a role in binding to G protein-coupled receptor C3a R.Some studies have determined that C3a-C3a R plays an important role in tumor growth,but so far there are no selective and effective agonists or antagonists for detecting the pharmacological effects of C3a in humans.The subject was screened by 3D-QASR virtual screening and pharmacophore screening in computer-aided drug design（CADD）to study the structure of C3a R receptor antagonists,cytotoxicity to THP-1 cells and RAW264.7 cells,and The Ca²⁺release assay studies its antagonistic activity and is of great significance for the design of more potent C3a R receptor antagonists.ObjectiveIn this study,the C3a receptor active compounds obtained in the literature were used to study the 3D-QASR in CADD and to construct the pharmacophore model based on the structure-activity relationship of the compounds.The compound molecular library was screened,and the cytotoxicity into the cell activity was detectedby CCK8 method and Ca²⁺ release experiment,and the active C3a receptor antagonist was found.Contents1.Three-dimensional quantitative structure-activity relationship of C3a Rreceptor antagonistAmong the 53 C3a receptor active compounds with known structure and activity,17 compounds were randomly selected as the test set to evaluate the prediction ability of the model,and the remaining 36 compounds were the training set to establish Co MFA model and Co MSIA model.2.Screening of pharmacophores and compounds based on structure-activity relationship of compounds100 compounds were screened out of the training set,and the preparation of the training set molecules,the selection of the characteristic elements of the pharmacophore,the pharmacophore model based on the structure-activity relationship of the compounds,and the pharmacophores constructed by the test set compound molecules were used.The optimal pharmacophore was constructed to screen the active compound molecular library after primary screening by ADMET,and the activity of the selected lead compound was predicted by the constructed pharmacophore.3.Cytotoxicity of the compound to thp-1 cells and RAW264.7 cells was detected by CCK-8 assayThe growth curves of THP-1 cells and RAW264.7 cells were tested to determine the optimal culture conditions of the two cells,namely the number of cells cultured and the culture time;the trypan blue exclusion method was used to quickly and easily detect the cell viability;The effect of concentration of DMSO reagent on the proliferation of THP-1 cells and RAW264.7 cells was excluded,and the effect of DMSO reagent on the experiment was excluded.Finally,the cytotoxicity of THP-1cells and RAW264.7 cells was detected by CCK-8 method.4.Ca²⁺ release assay was used to detect the activity of the compoundTo determine whether the 197 and 233 compounds have an antagonistic effect on C3a R,a Ca²⁺ release assay based on C3a-induced THP-1 cells and RAW264.7 cells was employed.Results1.Co MFA model and Co MSIA model with good prediction ability and high credibility were successfully constructed.In addition,according to the three-dimensional potential diagram of Co MFA and Co MSIA models,the active structure of C3a R receptor antagonist should have a stable nucleus structure,as wellas stereoscopic field,hydrophobic field and electrostatic field,which provides effective Suggestions for the later construction of pharmacophors and the screening of C3a R receptor antagonist active compounds.2.Constructed a number of active pharmacophores and screened a higher scored3D-QASR pharmacophore with four pharmacophore elements.Eight compounds with complete matching characteristics of four pharmacophores were screened by pharmacophore and their activities were predicted.The results of pharmacophore activity prediction were consistent with those predicted by Co MFA and Co MSIA models.3.Thp-1 and RAW264.7 cells increased rapidly at 24 h,and the number of thp-1cells was appropriate at 8×103 and the number of RAW264.7 cells was appropriate at6×103.DMSO concentration within 0.25% had no significant effect on cell growth and proliferation.The results of CCK-8 assay indicated that the toxicity of the screened compound on thp-1 cells and RAW264.7 cells gradually increased with the increase of concentration,and the inhibition rate of the highest concentration on cell proliferation was less than 20%,which could be used for the next Ca²⁺ release assay.4.Antagonist SB290157 had certain antagonistic effects on Ca²⁺ release of THP-1 cells and RAW264.7 cells.Two screened compounds 197 and 233 had certain antagonistic effects on C3a R antagonist,while compound 197 had negligible antagonistic effects on C3a R antagonist.ConclusionThrough virtual screening and the construction and screening of pharmacophore,we found that among the lead compounds,compound 233 had the same effect as the antagonist SB290157 in inhibiting the release of Ca²⁺ in cells,and compound 197 had no significant effect on the release of Ca²⁺ in cells.Therefore,agonist or antagonist with better selectivity and effectiveness of C3a receptor can be designed and modified.