Discovery And Bioactivity Evaluation Of Small Molecule Inhibitors Targeting Epigenetic Regulator Proteins KDM4D And JMJD6

Epigenetic refer to heritable changes in gene expression without alteration in DNA sequences.Epigenetics is regulated by a variety of proteins.Aberrant epigenetic modifications can lead to changes in gene function and cause many diseases,such as autoimmune diseases,metabolic diseases,neurological disorders,and cancer.Therefore,epigenetic regulatory proteins have become potential targets for the treatment of these diseases.However,compared with kinases,G protein-coupled receptors(GPCR),drugs targeting epigenetic regulatory protein targets are still very few.For most epigenetic regulatory proteins,small molecule inhibitors are still lacking.During my Ph.D.study,I mainly focused on two epigenetic regulatory proteins,namely histone demethylase KDM4D and hydroxylase JMJD6,and carried out the design and screening of small molecule inhibitors,as well as structural optimization and bioactivity evaluation.The details are introduced as follows:Part ?.Discovery and Bioactivity Evaluation of Inhibitors Against Histone Demethylase KDM4DKDM4D(also known as JMJD2D)is a histone demethylase containing the Jmj C domain and belongs to the KDM4(JMJD2)subfamily.Its main function is to catalyze the removal of methylation modifications on H3K9 with the participation of?-ketoglutarate(2-OG).Abnormal expressin of KDM4D is closely associated with the genesis,progression and drug tolerance of malignant tumors.Thus,small molecule inhibitors targeting KDM4D can be used as potential drugs for the therapy of cancer.Prior to this study,there is no selective inhibitors of KDM4D have been reported.To this end,drug design and inhibitor screening was carried out targeting the 2-OG binding site and the H3K9 substrate binding site respectively.1.1 Discovery and Complex Crystal Structure of KDM4D Inhibitors Targeting the Catalytic SitesIn this part of study,molecular docking and substructure search was performed nd two hit compounds were retrived.Among them,5-hydroxy-9-nitropyrazolo[1,5-a]quinazoline-3-carbonitrile(2)showed a better inhibitory efficiency against KDM4D.The inhibition rate of compound 2 at 10?M is 73.33%and the potency of this compound need further improvement.Structural optimization was carried out to compound 2 and a total number of 32 compounds were synthesized.Compound 21was the most active one with an IC₅₀ value of 0.41?M.The results of Differential Scanning Fluorimetry(DSF),Isothermal Titration Calorimetry(ITC)and Orthogonal assays of Microscale Thermophoresis(MST)showed that compound 21 could bind to KDM4D.This compound showed nearly no inhibition towards KDM2,KDM3 and KDM5(IC₅₀>10?M),implying that compound 21 was a selective KDM4D inhibitor.Finally,in order to explore the interaction mode between compound 21 and KDM4D,we used BL21(DE3)to express and purify human KDM4D protein.By optimizing the crystallization conditions,we successfully solved the structure of KDM4D in complex with compound 21.It revealed the pyridyl nitrogen atom of 21formed a coordination interaction with the metal ion Fe(II).The hydroxyl group of21 formed two hydrogen bonds with residue ASN202 and LYS210.Besides,?-?interactions formed between the tri-aromatic-ring of 21 and the two active-site aromatic residues PHE189 and TRP212.The crystal structure has been uploaded to the PDB database(PDB:7DYQ).In summary,we successfully obtained the first KDM4D inhibitor which had highly activity and selectivity.Compound 21 laid a solid foundation for the development of small molecule drugs for KDM4D and deserves further optimization and research.1.2 Discovery and Bioactivity Evaluation of KDM4D Inhibitors Targeting the Substrate Binding PocketIn this part of study,molecular docking was performed and the substrate binding pocket of KDM4D was defined as the active site for virtual screening.One hit compound were retrived with an IC₅₀ value of 1.751?M.Structure optimization and structure-activity relationship(SAR)analyze was carried out and about 60compounds were synthesized.Among them,compound 24s was the most active one(IC₅₀=0.023?M).The binding ability of compound 24s to KDM4D was confirmed y experimental results of ITC,DSF and CETSA.Kinetic analysis showed that compound 24s is not a 2-OG competitive inhibitor,and the results of molecular docking showed that good interactions formed between 24s and the substrate binding pocket of KDM4D.Compound 24s exhibited more than 1500-fold selectivity towards KDM4D versus other tested KDM family members(including KDM4 subfamily).In in vitro assays,the results of MTT and clone formation showed that compound 24s had exellent anti-viability and antiproliferative activities against various colorectal cancer cells(HCT116,SW480 and SW620).Wound healing assay and transwell assay showed that compound 24s significantly inhibited the migration of HCT116cells.In conclusion,we discovered the first KDM4D inhibitor which had exhibited selectivity within the KDM4 subfamily.Compound 24s has potential to be used as a chemical probe for KDM4D biological function studies.Part ?.Discovery and Bioactivity Evaluation of Selective Inhibitors Against Hydroxylase JMJD6Jumonji-domain-containing protein 6(JMJD6)is a Fe(II)and 2-oxogluterate(2OG)dependent oxygenase.Previously it was reported to function as a histone demethylase.Recent investigations revealed that it was more likely a protein hydroxylase.Numerous studies have shown that dysregulation of JMJD6 is associated with pathologies of various diseases,particularly cancer,including colon carcinoma,neuroglioma,melanoma,breast cancer and oral cancer.Thus,JMJD6inhibitors are thought as potential agents for treating these diseases.Currently only few JMJD6 inhibitors have been reported and these compounds showed poor selectivity.To this end,drug design and inhibitor screening was carried out against this target.Molecular docking was performed to screen small molecule database and 30compounds were selected to test their inhibitory activity against JMJD6.Finally three hit compounds were obtained.Among them,compound J2 was the most active one(IC₅₀=1.99?M).However,compound J2 showed inhibitory efficiency against other histone demethylases(KDM3B,KDM4D,KDM5A)as well.Based on these results,the selectivity of this compound were poor and required further structural optimization.The co-crystal structure of compound J2 in complex with homologous protein KDM4D was solved,and the amino acids involved in JMJD6 binding pocket were compared with the corresponding residues of KDM3B/4D/5A via structure alignment.These structural information provided direction for optimization.We designed and synthesised compound 1 which showed a better selectivity.Then structure optimization was further carried out upon this compound and a total of 32compounds were synthesised.Among them,the most active compound against JMJD6 was compound 7p with an IC₅₀ value of 0.681?M.Both DSF and CETSA assay showed that compound 7p could bind to JMJD6.More importantly,compound7p displayed no activity against other tested Jmj C domain-containing protein family members,indicating good selectivity(>100 fold).Collectively,this investigation offers a JMJD6 inhibitor with the best selectivity among the JMJD6 small molecule inhibitors reported so far.This compound can be used as a probe to explore the biological functions of JMJD6 and it can also be taken as a lead compound for subsequent drug discovery targeting JMJD6.In summary,during my Ph.D.studying,computer-aided drug design,chemical synthesis,bioactivity evaluation,structure-activity relationship analysis,and crystallographic study have been conducted to rationally design novel,potent and selective small molecule inhibitors for histone demethylase KDM4D and hydroxylase JMJD6.Multiple active compounds were obtained for these potential targets.Among them,the first selective and potent inhibitor 21 was obtained for KDM4D catalytic site;the inhibitor 24s which had selectivity within the KDM4 subfamily was discovered for KDM4D substrate binding site and the most selective inhibitor 7p was found for JMJD6.In conclusion,our studies laid a solid foundation for the development of small molecule drugs for KDM4D and JMJD6.