Discovery Of A Natural BRD4 Inhibitor 3',4',7,8-Tetrahydroxyflavone And Its Inhibitory Mechanism Against Acute Myeloid Leukemia

In recent years,with the continuous improvement of the technology of gene sequencing and structure analysis,the related mechanism research on malignant tumors has made great progress.Many studies have shown that the single central principle can no longer explain the complex biological behavior of malignant tumors,while the development of epigenetics has enabled us to have more understanding of the pathogenesis of malignant tumors.Epigenetic inheritance refers to changes in phenotype that are not rooted in DNA sequence.This phenomenon has largely been studied in the context of chromatin modification,including histone modification after translation,DNA and non-coding RNA in the form of regulation.At present,with the research on the key epigenetic events related to cancer is getting more and more in-depth and new epigenetic key targets are constantly discovered,the discovery of new generation drugs targeting these targets is the main strategy and direction of epigenetic targeted therapeutics.Bromodomain-containing protein 4(BRD4)is one of the most studied members of bromodomain and extra terminal protein(BET)which has many family members.It identifies and binds acetylated lysine residues on the N-terminal tails of histones through two bromodomains(BD1 and BD2)to participate in the transmission of histone acetylation signals and regulate gene transcription.Inhibiting one or both of bromodomains resulted in different phenotypes,suggesting BD1 and BD2 may have different functions.Although some BRD4 inhibitors have already entered the clinical research stage,similar structural skeletons may bring the risk of drug resistance for future clinical application,it is still of great significance to find novel BRD4 inhibitors with new structural skeletons.Meanwhile,natural compounds,as the product of natural evolution which form a 'natural molecular library',have a variety of chemical skeletons,a wide range of biological activity,rich resources and other advantages.They have been an important source of drugs for a long time.Therefore,based on the scientific problems caused by the important biological functions of BRD4 protein,this paper gives full play to the advantages of natural compounds,and starts the research on BRD4 natural inhibitors from the perspective of screening novel natural inhibitors and exploring the mechanism of action of budding compounds.This paper mainly includes the following aspects of work:1.AlpHaScreen assay was used to screen out a novel BRD4 inhibitor 3',4',7,8-tetrahydroxy flavonoids(3478)from natural flavonoids,and Counter assay was also used to analyze the affinity of 3478 to BRD4 bromodomain 1(BD1)and 2(BD2),respectively.The results showed that the 3478 had good affinity to the BRD4 protein,and other similar flavonoid compounds and derivatives has no obvious such affinity activity,suggesting that the specific structure skeleton of 3478 had very important significance to the affinity to the BRD4 bromodomain,which isn't the general activity of flavonoid.Meanwhile,the affinity(IC50=204 nM)of 3478 was?100-fold selective for BD2(IC50=204 nM)than BD1(IC50=17.9 ?M),which exhibited the selectivity of BRD4-BD2.2.In this study,the human acute myelomonocytic leukemia cell line(MV4-11)was used to research the activity of 3478 and its possible molecular regulation mechanism on cell proliferation and apoptosis,and protein expression of c-myc and BRD4.The results showed that 3478 can inhibit the proliferation of MV4-11 cells,promote the apoptosis of MV4-11 cells,simultaneously down-regulate the mRNA transcription level and its expression of c-Myc.3.AML xenograft mice model was constructed to study the suppression effect of 3478 on the subcutaneous transplanted tumor in vivo and its potential molecular regulation mechanism,which may provide an objective experimental basis for the possible intervention of 3478 in AML.The results showed that 3478 can suppress the growth of tumor volume and weight,simultaneously having no effect on body weight and organ index of mice.Meanwhile,3478 can also down-regulate the transcription level of c-Myc mRNA and reduce its expression in the cell nucleus and cytoplasm.4.To better understand the mechanism of different affinity activities of 3478 to BD1 and BD2,we performed X-ray structures of 3478 in complex with BD1 and BD2 which were determined at resolution of 1.30 A and 1.73 A,respectively.3478 binds very similarly to BD1 and BD2 in the binding pocket.In both structures,the carbonyl group of 3478 forms strong H-bonds with an asparagine residue(BD1:N144,BD2:N433),and a water molecule near the tyrosine residue(BD1:Y97,BD2:Y390).7-hydroxy of 3478 forms a third H-bond with the main chain carbonyl group of a proline residue(BD1:P82,BD2:P375).These 3 H-bonds fix the 2,3-dihydro-1-benzopyran-4-one ring tightly in the binding pocket.In addition,hydrophobic residues V87,1146,and 194(BD1)or V380,V439,and L387(BD2)form strong hydrophobic interaction with 3478.These binding characteristics reveal the mechanism that 3478 has promising affinity to both BD1 and BD2 at the molecular level.However,in the crystal of BD1-3478 complex,the weak electron density of the phenyl group of 3478 suggests a weak interaction of this phenyl group with BD1.In contrast,its density is well defined in BD2-3478 structure,suggesting a fixed conformation and stronger interactions in BD2.3478 forms one additional H-bond with the water molecule near N433,and likely establishes hydrophobic interaction with H437,as NH of H437 points to two carbon atoms in its phenyl ring.The corresponding position of H433 in BD1 is an aspartic acid(D144)whose side chain is too distant from 3478 to establish effective interaction.In a recently published paper,H437 of BD2 is exploited to design inhibitors with selectivity for BD2.These structural characteristics reveal the molecular mechanism by which the activity of 3478 against BD2 is significantly higher than that of BD1.The chemical skeleton of 3478 and its binding pattern to the BRD4 are tremendously different from the reported BRD4 inhibitors before.Considering its high potency,selectivity for BD2,its small size(molecular weight=286.24),large space for optimization,and novel chemical scaffbold and binding mode,it is worth to be explored as a lead compound to inspire anti-cancer drug design and to treat other BRD4-related diseases.