As a methyltransferase,NSD2 can transfer methyl groups to the lysine side chain of H3,thus affecting gene transcription,DNA replication and repair.It plays an important role in maintaining the stability of chromatin.In addition,the abnormal expression of NSD2 is closely related to a variety of cancers,including multiple myeloma,acute lymphoblastic leukemia and breast cancer.Therefore,NSD2 is a potential target for cancer treatment.However,due to the lack of small molecule binding detection methods and structural information of SET domain,the development of effective selective inhibitors has been hindered.In this paper,the SET domain of NSD2 is used as a target,and computer-aided drug design is used to screen "hit" compounds at substrate and coenzyme sites and verify their inhibitory activity.Finally,we try to analyze the eutectic structure of NSD2 and "hit" compounds,design derivatives and analyze the synthesis route according to the binding mode of NSD2-SET.In order to obtain the key binding information of NSD2-SET and potential inhibitors targeting binding sites,we designed a new combination method of homology modeling,virtual screening,pharmacophore construction and molecular dynamics simulation.Based on this method,we first constructed a high-quality model through homology modeling,model evaluation,and molecular dynamics simulation.The pharmacophore models F1,F2,F3 and F4 were constructed through multiple docking analysis of small molecule fragment compounds to screen a database containing 120 million compounds.The screened compounds were docked again and the binding modes of the first 100 compounds were comprehensively analyzed.The combination pattern at the coenzyme site is that the nitrogen on the pyrimidine or pyrazole ring forms hydrogen bonds with His1142 and Arg1192,forms a salt bridge with Lys1069 and forms hydrophobic interactions with Thr1189,which tightly bind to the coenzyme sites.Correspondingly,compounds at the substrate site bind to hydrophobic pocket through hydrogen bonds with amino acids such as Tyr1092,Phe1117,Met1119,Leu1184,Asn1186,or through π-cation and π-π stacking.Through the analysis of the medicinal properties of the compounds,it was found that the top ten compounds have good ADMET properties and comply with the Lipinski rule.Finally,molecular dynamics simulations were carried out to verify the stability of the combination of NSD2 with compounds by analyzing RMSD,RMSF,Rg and binding energy.To verify whether the screened compounds can inhibit the activity of NSD2,we tested the inhibitory effect of the compounds on NSD2 in vitro.Four successfully constructed recombinant plasmids were introduced into BL21(DE3)receptive cells for induction of expression using IPTG.It was found that the conditions of 15℃,induction time of 17 hours,and IPTG concentration of 0.2 m M were more suitable for this enzyme.SDS-PAGE and WB validation showed that the protein purified from the nickel column was indeed the target protein.Finally,by detecting the activity of NSD2,it was proved that the selected "hit" compound had NSD2 inhibitory activity,and the IC50 values of compounds were between 50 and 500 μM.The optimal IC50 value of ZINC257312485 is 59.5 μM,which indicates that ZINC257312485 has a better inhibitory effect on the activity of NSD2.By observing the crystal formation state,we have found some good crystal formation conditions.After multiple optimization of the conditions,we have been able to obtain small crystals,but further optimization of the conditions is still needed.At the same time,in order to further optimize the activity of "hit" compounds,we comprehensively used bioelectronic rearrangement,skeleton transition,and molecular hybridization methods to conduct diverse structural modifications.We designed 3000 derivatives and minimized the energy of these compounds before molecular docking.By comprehensively analyzing docking score,binding energy,and spatial complementarity,we selected compounds with excellent performance in various aspects to design a synthesis route,laying a foundation for future activity determination and related experimental research.In summary,in view of the lack of structural information and effective inhibitors of NSD2,this paper obtained a number of “hit” compounds with development prospects by comprehensive use of virtual screening based on structure and pharmacophore model,computer-aided drug design and in vitro activity verification,which laid a foundation for more rational design of NSD2 inhibitors. |