Suman Kalyan Pradhan

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.

Molecular basis of recognition of quadruplexes human telomere and c-myc promoter by the putative anticancer agent sanguinarine.

BACKGROUND Interaction of putative anticancer agent sanguinarine with two quadruplex forming sequences, human telomeric DNA (H24) and NHE III1 upstream of the P1 promoter of c-myc (Pu27), has been studied to understand the structural basis of the recognition. METHODS Absorption, fluorescence and circular dichroism spectroscopy have been employed to characterize the association. Energetics of the interaction was studied by isothermal titration and differential scanning calorimetry. TRAP assay was done to assess the inhibitory potential of sanguinarine. RESULTS Absorption and fluorescence studies show that sanguinarine has high binding affinity of ~10(5)M(-1) for both sequences. Binding stoichiometry is 2:1 for H24 and 3:1 for Pu27. Results suggest stacking interaction between planar sanguinarine moiety and G-quartets. Circular dichroism spectra show that sanguinarine does not cause structural perturbation in the all-parallel Pu27 but causes a structural transition from mixed hybrid to basket form at higher sanguinarine concentration in case of H24. The interaction is characterized by total enthalpy-entropy compensation and high heat capacity values. Differential scanning calorimetry studies suggest that sanguinarine binding increases the melting temperature and also the total enthalpy of transition of both quadruplexes. TRAP results show that sanguinarine effectively blocks telomerase activity in a concentration dependent manner in cell extracts from MDAMB-231 breast cancer cell lines. CONCLUSION These results suggest that there is a difference in the structural modes of association of sanguinarine to the quadruplexes. GENERAL SIGNIFICANCE It helps to understand the role of quadruplex structures as a target of small molecule inhibitors of telomerase.

Human telomere d[(TTAGGG)4] undergoes a conformational transition to the Na+-form upon binding with sanguinarine in presence of K+.

Guanine-rich telomeric sequences fold into G-quadruplex conformation and are known to bind a variety of ligands including potential drug candidates. By means of CD spectroscopy and fluorescence lifetime measurements we demonstrate that putative anticancer therapeutic sanguinarine (SGR) exhibits two distinct interactions with human telomere d[(TTAGGG)(4)] (H24) in presence of K(+). Up to about 1:2 M ratio of H24:SGR (10 μM H24), two molecules of SGR bind H24. Above this molar ratio, SGR induces a conformational transition in H24 from the K(+)-form to the Na(+)-form. The demonstration of SGR-induced conformational transition in a G-quadruplex formed by a human telomeric sequence could provide new insights into interaction of drugs with quadruplex DNA structure.

Mechanism of interaction of small transcription inhibitors with DNA in the context of chromatin and telomere

Small molecules from natural and synthetic sources have long been employed as human drugs. The transcription inhibitory potential of one class of these molecules has paved their use as anticancer drugs. The principal mode of action of these molecules is via reversible interaction with genomic DNA, double and multiple stranded. In this article we have revisited the mechanism of the interaction in the context of chromatin and telomere. The established modes of association of these molecules with double helical DNA provide a preliminary mechanism of their transcription inhibitory potential, but the scenario assumes a different dimension when the genomic DNA is associated with proteins in the transcription apparatus of both prokaryotic and eukaryotic organisms. We have discussed this altered scenario as a prelude to understand the chemical biology of their action in the cell. For the telomeric quadruplex DNA, we have reviewed the mechanism of their association with the quadruplex and resultant cellular consequence.

Chromatin as a Target for the DNA-Binding Anticancer Drugs

Chemotherapy has been a major approach to treat cancer. Both constituents of chromatin, chromosomal DNA and the associated chromosomal histone proteins are the molecular targets of the anticancer drugs. Small DNA binding ligands, which inhibit enzymatic processes with DNA substrate, are well known in cancer chemotherapy. These drugs inhibit the polymerase and topoisomerase activity. With the advent in the knowledge of chromatin chemistry and biology, attempts have shifted from studies of the structural basis of the association of these drugs or small ligands (with the potential of drugs) with DNA to their association with chromatin and nucleosome. These drugs often inhibit the expression of specific genes leading to a series of biochemical events. An overview will be given about the latest understanding of the molecular basis of their action. We shall restrict to those drugs, synthetic or natural, whose prime cellular targets are so far known to be chromosomal DNA