ABSTRACT
Lysine methyltransferases are important regulators of epigenetic signaling and are emerging as a novel drug target for drug discovery. This work demonstrates the positioning of novel 1,5-oxaza spiroquinone scaffold into selective SET and MYND domain-containing proteins 2 methyltransferases inhibitors. Selectivity of the scaffold was identified by epigenetic target screening followed by SAR study for the scaffold. The optimization was performed iteratively by two-step optimization consisting of iterative synthesis and computational studies (docking, metadynamics simulations). Computational binding studies guided the important interactions of the spiro[5.5]undeca scaffold in pocket 1 and Lysine channel and suggested extension of tail length for the improvement of potency (IC50: up to 399 nM). The effective performance of cell proliferation assay for chosen compounds (IC50: up to 11.9 nM) led to further evaluation in xenograft assay. The potent compound 24 demonstrated desirable in vivo efficacy with growth inhibition rate of 77.7% (4 fold decrease of tumor weight and 3 fold decrease of tumor volume). Moreover, mirosomal assay and pharmacokinetic profile suggested further developability of this scaffold through the identification of major metabolites (dealkylation at silyl group, reversible hydration product, the absence of toxic quinone fragments) and enough exposure of the testing compound 24 in plasma. Such spiro[5.5]undeca framework or ring system was neither been reported nor suggested as a modulator of methyltransferases. The chemo-centric target positioning and structural novelty can lead to potential pharmacological benefit.
Subject(s)
Aza Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Epigenesis, Genetic/drug effects , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Quinones/pharmacology , Spiro Compounds/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Aza Compounds/chemical synthesis , Aza Compounds/chemistry , Cell Survival/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Epigenesis, Genetic/genetics , Female , HEK293 Cells , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Male , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Mice, Nude , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Docking Simulation , Molecular Structure , Quinones/chemical synthesis , Quinones/chemistry , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Structure-Activity RelationshipABSTRACT
DNA methyltransferase is the key enzyme in the process of DNA methylation, playing an important role in regulation of gene expression in vivo. According to the Ganoderma lucidum transcriptome data, a full-length cDNA sequence of MET1 from G. lucidum was cloned for the first time, the GenBank registration number is KU239998, and we conducted a comprehensive bioinformatics analysis of the genetic characteristics and spatial structure. The prokaryotic expression analysis showed that E.coli[pET28a(+)-GlMET1] in BL21(DE3) could induce objective protein, shaking the culture at 16 â until the host bacterium(A600) was approximately 0.8, and added IPTG to finally concentration of 0.2 mmolâ¢L⻹, and then the optimal expression of GlMET1 recombinant protein was accumulated for the induction time of 20 h. The real-time PCR results showed that the expression levels of GlMET1 had obvious differences among varieties of G. lucidum. During the maturity stage, the expression levels of GlMET1 were lower than that in juvenile stage, the results showed that with the growth of G. lucidum, the expression levels of GlMET1 were on the decline. The research provided an important basis for studying the mechanism of DNA methyltransferase thoroughly.
Subject(s)
Cloning, Molecular , DNA (Cytosine-5-)-Methyltransferase 1/genetics , Fungal Proteins/genetics , Reishi/enzymology , DNA (Cytosine-5-)-Methyltransferase 1/chemistry , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reishi/chemistry , Reishi/classification , Reishi/genetics , TranscriptomeABSTRACT
DNA methyltransferase is the key enzyme in the process of DNA methylation, playing an important role in regulation of gene expression in vivo. According to the Ganoderma lucidum transcriptome data, a full-length cDNA sequence of MET1 from G. lucidum was cloned for the first time, the GenBank registration number is KU239998, and we conducted a comprehensive bioinformatics analysis of the genetic characteristics and spatial structure. The prokaryotic expression analysis showed that E.coli[pET28a(+)-GlMET1] in BL21(DE3) could induce objective protein, shaking the culture at 16 ℃ until the host bacterium(A₆₀₀) was approximately 0.8, and added IPTG to finally concentration of 0.2 mmol•L⁻¹, and then the optimal expression of GlMET1 recombinant protein was accumulated for the induction time of 20 h. The real-time PCR results showed that the expression levels of GlMET1 had obvious differences among varieties of G. lucidum. During the maturity stage, the expression levels of GlMET1 were lower than that in juvenile stage, the results showed that with the growth of G. lucidum, the expression levels of GlMET1 were on the decline. The research provided an important basis for studying the mechanism of DNA methyltransferase thoroughly.
