Dipak Dasgupta

Inhibition of Lysine Acetyltransferase KAT3B/p300 Activity by a Naturally Occurring Hydroxynaphthoquinone, Plumbagin

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Mechanism of p300 Specific Histone Acetyltransferase Inhibition by Small Molecules

Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, diabetes, and asthma. Therefore, small molecule inhibitors and activators of HATs are being considered as new generation therapeutics. Here, we report the molecular mechanisms of p300 HAT inhibition by specific and nonspecific HAT inhibitors: garcinol, isogarcinol, and 1 (LTK14). The p300 specific HAT inhibitor 1 behaves as a noncompetitive inhibitor for both acetyl-CoA and histone, unlike nonspecific HAT inhibitors garcinol and isogarcinol. The isothermal calorimetric data suggest that there is a high affinity enthalpy driven single binding site for 1 on p300HAT domain in contrast to two binding sites for garcinol and isogarcinol. Furthermore, the precise nature of molecular interactions was determined by using fluorescence, docking, and mutational studies. On the basis of these observations, we have proposed the mechanisms of specific versus nonspecific HAT inhibition by these small molecule compounds, which may be useful to design therapeutically favorable HAT inhibitors.

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.

Sanguinarine Interacts with Chromatin, Modulates Epigenetic Modifications, and Transcription in the Context of Chromatin

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

Structural basis of DNA recognition by anticancer antibiotics, chromomycin A3, and mithramycin: Roles of minor groove width and ligand flexibility

Anticancer antibiotics, chromomycin A(3) (CHR) and mithramycin (MTR), inhibit cellular processes like transcription and replication, by binding reversibly to double-stranded DNA via minor groove, in the presence of bivalent metal ions like Mg(2+) with GC base specificity. Here, we have attempted to assess the roles of two parameters-namely DNA groove dimension and flexibility of the ligand-in the structural recognition between the ligands, (drug)(2)Mg(2+) and DNA. For the purpose we have employed three synthetic oligonucleotides with minor groove width lying between B- and A-type structures as model DNA sequences: d(GCGCGCGC)(2) in B-form, d(CCGGCGCCGG)(2) in B-form with unusual wide minor groove, and (GGGGCCCC)(2) in A-form. Association of the (drug)(2)Mg(2+) with the oligomers have been probed using spectroscopic techniques like absorbance, fluorescence, and CD. The binding and thermodynamic parameters for the different association processes have also been characterized. Major conclusions from the above studies are as follows. Groove size of the oligomers influences the conformation of the bound ligand. A saccharide dependent variation in structural rigidity of the ligands, (MTR)(2)Mg(2+) and (CHR)(2)Mg(2+), has been observed that leads to differences in the energetics of recognition of the same DNA sequence by the two ligands. In contrast to (CHR)(2)Mg(2+), higher flexibility in (MTR)(2)Mg(2+) makes its conformation in the DNA bound form less sensitive to the groove dimension of DNA.

Association of chromatin with anticancer antibiotics, mithramycin and chromomycin A3

Mithramycin and chromomycin A(3) are two anticancer antibiotics, which inhibit protein biosynthesis via transcription inhibition. They bind reversibly to DNA with (G.C) base specificity. At and above physiological pH in the absence of DNA, they form two types of complexes with Mg(2+), complex I (1:1 in terms of antibiotic: Mg(2+)) and complex II (2:1 in terms of antibiotic: Mg(2+)). These are the DNA binding ligands. In vivo, the antibiotics interact with chromatin, a protein-DNA complex. In order to understand the mode of action of these antibiotics at molecular level, we have carried out spectroscopic, gel electrophoretic and UV melting studies of complex I of these antibiotics with rat liver chromatin, nucleosome core particle and DNA stripped of all chromosomal proteins. Analysis of the results has led us to propose that the antibiotic: Mg(2+) complex binds to both nucleosomal and linker DNA in native chromatin. Histone proteins reduce the binding potential and accessibility of the complexes to the minor groove of (G.C) rich regions of chromosomal DNA. The antibiotic: Mg(2+) complex stabilizes DNA duplex and histone- DNA contacts in chromatin fiber. It also leads to the aggregation of chromatin fibers. From a comparison of the association of the antibiotic: Mg(2+) complexes with different levels of chromatin structure and their effects upon the structure, we suggest that the sugar moieties of the antibiotics play a role in the binding process. Significance of these results to understand the molecular basis of the transcription inhibition potential of the antibiotics in eukaryotes is discussed.

Role of Mg++ in the mithramycin-DNA interaction : evidence for two types of mithramycin-Mg++ complex

Mithramycin(MTR, structure shown in Figure 1) [and the related compound Chromomycin A3(CHRA3)] are antitumor antibiotics which inhibit DNA dependent RNA polymerase activity via reversible interaction with DNA only in the presence of divalent metal ion such as Mg++. In order to understand the role of Mg++ in MTR-DNA interaction, absorbance and CD spectroscopic techniques are employed to study the binding of MTR to Mg++. These studies show: i) the drug alone binds to Mg++ and ii) two different types of drug-Mg++ complexes are formed at low(Complex I) and high(Complex II) ratios of the concentration of Mg++ and MTR. We propose that these two complexes would bind to the same DNA with different affinities and rates. This result suggests that the relative concentration of Mg++ is an important factor to be taken into account to understand the molecular basis of MTR-DNA interaction.

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I. Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Aspergillus niger.

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Aspergillus niger was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34 mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the ortho-position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.

DNA-binding characteristics of a synthetic analogue of distamycin

The interaction between a synthetic analogue (structure shown in fig. 1) of distamycin, and DNA has been studied with a view to understanding the conformational and chemical basis of the sequence specific binding of distamycin with DNA. The complex formation between the trimer and DNA is apparent from the red shift in the UV spectrum and appearance of the induced CD band in 300nm-350nm region. The relevant data suggest: (i) the binding is A-T base specific though the specificity is not as pronounced as in distamycin (1,2) and (ii) it occurs via the minor groove of DNA. The partial loss in A-T base specificity may be due to the replacement of N-methyl pyrrole by benzene or the increase in curvature of the backbone of the ligand as a result of this replacement.

Plant alkaloid chelerythrine induced aggregation of human telomere sequence--a unique mode of association between a small molecule and a quadruplex.

Small molecules that interact with G-quadruplex structures formed by the human telomeric region and stabilize them have the potential to evolve as anticancer therapeutic agents. Herein we report the interaction of a putative anticancer agent from a plant source, chelerythrine, with the human telomeric DNA sequence. It has telomerase inhibitory potential as demonstrated from telomerase repeat amplification assay in cancer cell line extract. We have attributed this to the quadruplex binding potential of the molecule and characterized the molecular details of the interaction by means of optical spectroscopy such as absorbance and circular dichroism and calorimetric techniques such as isothermal titration calorimetry and differential scanning calorimetry. The results show that chelerythrine binds with micromolar dissociation constant and 2:1 binding stoichiometry to the human telomeric DNA sequence. Chelerythrine association stabilizes the G-quadruplex. Nuclear magnetic resonance spectroscopy ((1)H and (31)P) shows that chelerythrine binds to both G-quartet and phosphate backbone of the quadruplex leading to quadruplex aggregation. Molecular dynamics simulation studies support the above inferences and provide further insight into the mechanism of ligand binding. The specificity toward quartet binding for chelerythrine is higher compared to that of groove binding. MM-PBSA calculation mines out the energy penalty for quartet binding to be -4.7 kcal/mol, whereas that of the groove binding is -1.7 kcal/mol. We propose that the first chelerythrine molecule binds to the quartet followed by a second molecule which binds to the groove. This second molecule might bring about aggregation of the quadruplex structure which is evident from the results of nuclear magnetic resonance.