Mithramycin is an anticancer drug that blocks macromolecular synthesis via reversible interaction with the DNA template in the presence of bivalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not clear. We approached the problem in two steps via studies on the interactions between (i) mithramycin and Mg2+ and (ii) mithramycin-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for the purpose. From equilibrium and kinetic studies, we earlier reported that MTR forms two different types of complexes with Mg2+ [Aich, P., & Dasgupta, D. (1990) Biochem. Biophys. Res. Commun. 173, 689]. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of mithramycin: Mg2+) and complex II (with 2:1 stoichiometry in terms of mithramycin: Mg2+). In this report, we have further characterized these complexes by fluorescence spectroscopy. Interactions of these complexes with calf thymus DNA were examined to elucidate their binding. Evaluation of binding parameters (intrinsic binding constant and stoichiometry) from spectrophotometric and fluorimetric titrations suggests that the complexes bind differently to the same DNA. Measurement of van't Hoff enthalpies for the interaction of the two ligands and DNA shows that the complex I-DNA interaction is exothermic, in contrast to the endothermic nature of the complex II-DNA interaction. This could originate from a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. Our studies to detect the nature of the groove via which these complexes bind to DNA suggest that both complexes approach via the minor groove of the DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.
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.
The interaction of the two anticancer antibiotics, chromomycin A3 and mithramycin, with the polynucleotides poly(dG-dC) x poly(dC-dG), representative of B-DNA, and poly(dG) x poly(dC), representative of A-DNA, in the presence of Mg2+ is studied by spectroscopic techniques such as absorbance, fluorescence, and dircular dichroism (CD). The studies were done with both drug x Mg2+ complexes, I and II, having 1:1 and 2:1 stoichiometries with respect to drug and Mg2+, respectively [Aich, P., Sen, R., & Dasgupta, D. (1992) Biochemistry 31, 2988-2997]. The objective of the present work is 2-fold. First, an attempt is made to understand the structural basis of the ligand-DNA interaction, particularly the role of DNA backbone conformation with its groove size and the accessibility of the 2-amino group in the minor groove of guanosine. Second, the role of the antibiotic saccharide moieties in the association with DNA was studied. For this purpose, the spectroscopic characterization of the binding was done followed by the evaluation of binding parameters and associated thermodynamics. Analysis of the observed thermodynamics for the ligand-DNA interactions in terms of the different structures of the polynucleotides was done. The salient results are as follows. Complex I does not discriminate significantly among the A- and B-forms of DNA when it binds to them in an entropy-driven process. On the other hand, complex II for both drugs recognizes B- and A-forms of DNA in different ways. This observation implies that the sequence specificity shown by this complex is a sequel to the difference in the parameters such as groove size and accessibility of the guanosine amino group. Another important finding is that binding with the same polynucleotide is not comparable for the complex II of the two drugs. It emphasizes the involvement of the sugar moieties, when the drug x Mg2+ complex binds to DNA. The presence of an acetoxy group in the sugars of chromomycin A3 imparts some distinctive specific features of the association of the chromomycin dimer x Mg2+ complex with DNA. Finally, the results are compared with those available from NMR studies of different drug-oligonucleotide complexes under conditions where complex II is the ligand.
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