Differential scanning calorimetric study of the effect of intercalators and other kinds of DNA-binding drugs on the stepwise melting of plasmid DNA.

The effect of intercalating drugs (the anthracycline group of antibiotics, ethidium bromide, actinomycin D) on stepwise melting of DNA was studied by differential scanning calorimetry (DSC). The DSC DNA melting profile of plasmid pJL3-TB5 DNA (5277 base-pairs in length) consists of seven peaks, and all the intercalators caused shifting of these peaks, particularly those formed at the high temperature ranges, to the higher temperature ranges in a characteristic manner depending upon the binding strength of the drug. The analysis of the anthracycline group of antibiotics, such as aclacinomycin A, daunomycin, adriamycin and pyrarubicin, indicates that the difference in binding is due to the sugar moiety at position O-7 of the chromophore in these antibiotics. Analysis on the basis of the helix-coil transition theory suggests that the anthracycline group of antibiotics interact preferentially with the 5'-CG-3' sequences. The effect of various DNA-binding drugs other than intercalators on stepwise melting of DNA was then studied by DSC. The representative drugs examined were distamycin A, peplomycin, cis-dichlorodiamine-platinum(II) (cis-DDP or cis-Platin) and mitomycin C, which differ in their mode of interaction with DNA; namely, minor groove binding, strand cleavage and intrastrand or interstrand cross-linking. Distamycin A caused shifting of the DSC peaks at the low temperature ranges to a higher temperature range, whereas peplomycin and cis-DDP caused shifting of all the DSC peaks to form a broad peak at a lower temperature range, suggesting that the DSC DNA melting profiles are affected in a characteristic manner depending upon the interaction mode of the drug.     

Synthesis, DNA binding, and cleavage studies of novel PNA binding cyclen complexes

A novel coumarin‐appended PNA binding cyclen derivative ligand, C1 , and its copper(II) complex, C2 , have been synthesized and characterized. The interaction of these compounds with DNA was systematically investigated by absorption, fluorescence, and viscometric titration, and DNA‐melting and gel‐electrophoresis experiments. DNA Melting and viscometric titration experiments indicate that the binding mode of C1 is a groove binding, and C2 is a multiple binding mode that involves groove binding and electrostatic binding. From the absorption‐titration data, we can state that the primary interaction between CT DNA and the two compounds may be H‐bonds between nucleobases. Fluorescence studies indicate that the binding ability of C1 to d(A)₉ is as twice or thrice as that of other oligodeoxynucleotides. Agarose gel‐electrophoresis experiments demonstrate that C2 is an excellent chemical nuclease, which can cleave plasmid DNA completely within 24 h.     

Direct interaction between the N- and C-terminal portions of the herpes simplex virus type 1 origin binding protein UL9 implies the formation of a head-to-tail dimer

UL9, a superfamily II helicase, is a multifunctional protein required for herpes simplex virus type 1 replication in vivo. Although the C-terminal 317-amino-acid DNA binding domain of UL9 exists as a monomer, the full-length protein behaves as a dimer in solution. Thus, it has been assumed that the N-terminal 534 residues contain a region necessary for efficient dimerization and that UL9 dimers are in a head-to-head configuration. We recently showed, however, that residues in the N terminus could modulate the inhibitory properties of UL9 by decreasing the DNA binding ability of the C terminus (S. Chattopadhyay and S. K. Weller, J. Virol. 80:4491-4500, 2006). We suggested that a direct interaction between the N- and C-terminal portions of UL9 might exist and serve to modulate the DNA binding activities of the C terminus. In this study, we used a coimmunoprecipitation assay to show that the N-terminal portion of UL9 can indeed directly interact with the C terminus. A series of truncation mutant proteins were used to show that a region in the N terminus between residues 293 and 321 is necessary for efficient interaction. Similarly, a region in the C terminus between residues 600 and 800 is required for this interaction. The simplest model to explain these data is that UL9 dimers are oriented in a head-to-tail arrangement in which the N terminus is in contact with the C terminus.     

Structural forms of phenprocoumon and warfarin that are metabolized at the active site of CYP2C9

Possible reasons for the observed differences in metabolic behavior and drug interaction liability between the structurally similar oral anticoagulants warfarin and phenprocoumon were explored. Incubating (S)-phenprocoumon with human liver microsomes and cDNA-expressed CYP2C9 and determining its metabolism both in the absence and presence of the CYP2C9 inhibitor, sulfaphenazole, confirmed that phenprocoumon is a substrate for CYP2C9. Comparing the metabolic behavior of (S)- and (R)-warfarin, (S)- and (R)-phenprocoumon, and fixed structural mimics of the various tautomeric forms [(S)- and (R)-4-methoxyphenprocoumon, (S)- and (R)-2-methoxyphenprocoumon, (S)- and (R)-4-methoxywarfarin, (S)- and (R)-2-methoxywarfarin, and 9(S)- and 9(R)-cyclocoumarol] available to these two drugs with expressed CYP2C9 provides compelling evidence indicating that the ring closed form of (S)-warfarin and the ring opened anionic form of (S)-phenprocoumon are the major and specific structural forms of the two drugs that interact with the active site of CYP2C9. The conclusion that (S)-warfarin and (S)-phenprocoumon interact with CYP2C9 in very different structural states provides a clear basis for the significant differences observed in their metabolic profiles. Moreover, in accord with a previously established CoMFA model these results are consistent with the hypothesis that the active site of CYP2C9 possesses at least two major substrate binding sites, a pi-stacking site for aromatic rings and an ionic binding site for organic anions. An additional electrostatic binding site also appears to contribute to the orientation of coumarin analogs in the CYP2C9 active site by interacting with the C2-carbonyl group of the coumarin nucleus.     

A Surfactant-Induced Functional Modulation of a Global Virulence Regulator from Staphylococcus aureus

Triton X-100 (TX-100), a useful non-ionic surfactant, reduced the methicillin resistance in Staphylococcus aureus significantly. Many S. aureus proteins were expressed in the presence of TX-100. SarA, one of the TX-100-induced proteins, acts as a global virulence regulator in S. aureus. To understand the effects of TX-100 on the structure, and function of SarA, a recombinant S. aureus SarA (rSarA) and its derivative (C9W) have been investigated in the presence of varying concentrations of this surfactant using various probes. Our data have revealed that both rSarA and C9W bind to the cognate DNA with nearly similar affinity in the absence of TX-100. Interestingly, their DNA binding activities have been significantly increased in the presence of pre-micellar concentration of TX-100. The increase of TX-100 concentrations to micellar or post-micellar concentration did not greatly enhance their activities further. TX-100 molecules have altered the secondary and tertiary structures of both proteins to some extents. Size of the rSarA-TX-100 complex appears to be intermediate to those of rSarA and TX-100. Additional analyses show a relatively moderate interaction between C9W and TX-100. Binding of TX-100 to C9W has, however, occurred by a cooperative pathway particularly at micellar and higher concentrations of this surfactant. Taken together, TX-100-induced structural alteration of rSarA and C9W might be responsible for their increased DNA binding activity. As TX-100 has stabilized the somewhat weaker SarA-DNA complex effectively, it could be used to study its structure in the future.     

The interaction of adriamycin and adriamycin analogues with nucleic acids in the B and A conformations.

The reinforced intercalative binding to DNA typical of adriamycin and daunomycin can still occur if there is epimerisation at C4' or if the O-methyl group is lost or if the 9-substituents are deleted or if the 4'-hydroxyl group is lost. In the latter two cases however, there is a reduction in affinity for the DNA, supporting the suggested role of the 9-hydroxyl and 4'-hydroxyl groups in secondary stabilization of the complex. Epimerisation at C-1' or at C-3' alters but does not abolish the intercalative mode of binding to DNA whereas epimerisation at C-7 precludes intercalation of the chromophore into the helix of DNA. In contrast to the interaction with the B-form found in DNA, the parent drugs do not intercalate into nucleic acids possessing the A-conformation and none of the above-mentioned structural changes will allow intercalation into A-form nucleic acids.     

Exploration of the binding of proton pump inhibitors to human P450 2C9 based on docking and molecular dynamics simulation

Human P450 protein CYP2C9 is one of the major drug-metabolizing isomers, contributing to the oxidation of 16% of the drugs currently in clinical use. To examine the interaction mechanisms between CYP2C9 and proton pump inhibitions (PPIs), we used molecular docking and molecular dynamics (MD) simulation methods to investigate the conformations and interactions around the binding sites of PPIs/CYPP2C9. Results from molecular docking and MD simulations demonstrate that nine PPIs adopt two different conformations (extended and U-bend structures) at the binding sites and position themselves far above the heme of 2C9. The presence of PPIs changes the secondary structures and residue flexibilities of 2C9. Interestingly, at the binding sites of all PPI–CYP2C9 complexes except for Lan/CYP2C9, there are hydrogen-bonding networks made of PPIs, water molecules, and some residues of 2C9. Moreover, there are strong hydrophobic interactions at all binding sites for PPIs/2C9, which indicate that electrostatic interactions and hydrophobic interactions appear to be important for stabilizing the binding sites of most PPIs/2C9. However, in the case of Lan/2C9, the hydrophobic interactions are more important than the electrostatic interactions for stabilizing the binding site. In addition, an interesting conformational conversion from extended to U-bend structures was observed for pantoprazole, which is attributed to an H-bond interaction in the binding pocket, an internal π–π stacking interaction, and an internal electrostatic interaction of pantoprazole.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Influence of substituent modifications on DNA binding energetics of acridine-based anticancer agents

The DNA binding energetics of a series of analogues derived from the anticancer agent N-[2-(dimethylamino)ethyl]-9-aminoacridine-4-carboxamide (AAC) are investigated. The effects of substituent modification at the C5 position of the acridine chromophore on the interaction of AAC with DNA are determined using spectrophotometry and isothermal titration calorimetry (ITC). The binding affinity and binding free energy associated with the interaction of AAC with DNA are significantly enhanced upon substitution at the C5 position. Energetic profiles describing ligand-DNA complex formation obtained from ITC indicate that C5 substitution significantly enhances binding enthalpy relative to the parent AAC. In many cases, the enhanced binding enthalpies of the C5-substituted analogues correlate with anticancer activity. Because of the cationic character of AAC and its analogues, the DNA binding properties of these compounds are dependent on ionic strength. To quantitate the ionic contributions to complex formation, the observed binding free energy of each compound is parsed into its polyelectrolyte and nonelectrostatic components. Enhanced nonelectrostatic contributions to the overall binding free energies observed with C5-substituted analogues relative to the parent AAC suggest that C5 substituents play a critical role in directing both thermodynamic mechanisms associated with complex formation and molecular interactions between the ligand and its DNA binding site. These studies have demonstrated that substitution of AAC at the C5 position results in enhanced DNA binding affinity and energetics.     

Chemotherapeutic potential of 9-phenyl acridine: biophysical studies on its binding to DNA

Acridines and their derivatives are well-known probes for nucleic acids as well as being relevant in the field of drug development to establish new chemotherapeutic agents. We have shown from molecular modelling studies that 9-phenyl acridine and some of its derivatives can act as inhibitors of topoisomerase I and thus have potential to act as anticancer agents. Rational design of new compounds for therapeutics requires knowledge about their structural stability and interactions with various cellular macromolecules. In this regard it is important to know how these molecules would interact with DNA. Here we report the interaction of 9-phenyl acridine (ACPH) with calf thymus DNA (CT-DNA) based on various biophysical and molecular modelling studies. Spectrophotometric studies indicated that ACPH binds to CT-DNA. DNA melting studies revealed that binding of ACPH to CT-DNA resulted in a small increase in melting temperature, which is unlikely in case of classical intercalator; rather, it indicates external binding. Viscosity measurements show that ACPH exhibits groove binding. Competitive binding of ACPH to CT-DNA pre-bound to ethidium bromide (EB) showed slow quenching. Measurement of the binding constant of ACPH by fluorescent intercalator displacement (FID) assay corroborated the notion that there was groove binding. Molecular modelling studies also supported this finding. Results indicate that binding of ACPH is through partial intercalation in the minor groove of DNA.     

Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9^ΔC-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9^ΔC-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.     

Interaction between second generation anthracyclines and DNA in the nucleosomal structure.

The interaction between three anthracycline antibiotics of second generation (9-deoxydoxorubicin, 9-DAM, 4-demethoxydaunorubicin, 4-DDM, 4'-deoxydoxorubicin, 4'-DAM) and DNA in the nucleosomal structure was investigated using fluorescence and circular dichroism techniques. The thermodynamic parameters of the binding process were obtained at different ionic strength and temperature conditions, thus allowing the calculation of the electrostatic contribution to the free energy and the enthalpy of the process. The same measurements were performed on linear double stranded DNA for comparison. The parent compounds adriamycin and daunomycin were additionally considered. Although the examined drugs greatly vary in biological activity, their binding parameters are only slightly different. Like the parent compounds, 9-DAM, 4-DDM and 4'-DAM exhibit preference for isolated regions of the polynucleotide rather than for nucleosomes. This fact suggests a non-homogeneous distribution of the antibiotics in vivo. The enthalpy values are remarkably lower than the ones characterizing the interaction of adriamycin and daunomycin to DNA. According to CD spectra, all derivatives, but 4-DDM, intercalate into nucleosomal or free DNA in a manner similar to the first generation compounds, namely with the chromophore perpendicular to the hydrogen bonds between the bases. The demethoxy compound, on the other hand, seems to be able to insert its planar moiety in different orientations, which are related to the structure of the nucleic acid being examined. The lack of the methoxy group in the intercalating part of the molecule appears to be responsible for this behaviour. As far as biological activity is concerned, our findings indicate a qualitative correlation between cell cytotoxicity and ability of interaction with nucleosomes at physiological conditions, rather than with free DNA. The modified binding stereochemistry of 4-DDM could play an additive role in modulating the pharmacological effectiveness of the above compounds.     

The effects of local DNA sequence on the interaction of ligands with their preferred binding sites

We have examined the effects of local DNA sequence on the interaction of distamycin, Hoechst 33258, echinomycin, actinomycin and mithramycin with their preferred binding sites using a series of DNA fragments that contain every symmetrical hexanucleotide sequence. In several instances we find that the affinity for the ligands' preferred binding sites is affected by the hexanucleotide context in which they are located. The AT-selective minor groove binding ligand Hoechst 33258 shows a 200-fold difference in binding to the 16 different X(A/T)(4)Y sites; the strongest binding is to AAATTT and the weakest is to (G/C)TTAA(C/G). Although TTAA is generally a poor binding site, ATTAAT is better than TTTAAA and they are both much better than GTTAAC and CTTAAG. Similarly, TTATAA and ATATAT are better binding sites than GTATAC and CTATAG. In contrast, distamycin shows less discrimination between the various X(A/T)(4)Y sites, with a 20-fold difference between the best [(A/T)AATT(T/A)] and worst [GATATC and (G/C)TTAA(C/G)] sites. Although actinomycin binds to GpC it shows little or no interaction with any of the GGCC sites, yet shows only a six-fold variation in affinities for the other XYGCXY sites. Echinomycin binds to CpG yet shows no binding to TTCGAA, TGCGCA and AGCGCT, while the best binding is to AACGTT. The tetranucleotides CCGG and ACGT produce consistently good binding sites, irrespective of the surrounding sequences, while the interaction with TCGA and GCGC is sensitive to the hexanucleotide context. Hexanucleotides with a central GCGC, flanked by A and T are weaker echinomycin sites than those flanked by G and C, especially CGCGCG. The best X(G/C)(4)Y binding sites for mithramycin were located at AGCGCT and GGGCCC, and the worst at CCCGGG and TCCGGA. These footprinting fragments are valuable tools for comparing the binding of ligands to all the potential symmetrical hexanucleotides and provide insights into the effects of local DNA sequence on ligand-DNA interactions.     

Study of DNA interactions with steroidal and nonsteroidal estrogen-platinum (II)-based anticancer drugs

The interactions of the steroidal and nonsteroidal estrogen-platinum (Pt) (II)-based anticancer drugs 16beta-hydroxymethyl-16alpha-[8-(2-pyridin-2-yl-ethylamino)-3,6-dioxaoctyl]-1,3,5(10)-estratrien-3,17betadiol dichloroplatinum (II) (JPM-39), 4-[6-(2'-pyridylethylamino)-butyloxy)-phenyl]-7-methoxy-2,2-dimethyl-3-phenyl-chroman dichloroplatinum (II) (ATG-99), and 1-[(2-aminoethyl)amino]-9,10,10-tris(4-hydroxyphenyl)-9-decene dichloroplatinum (II) (GEB-28) with calf-thymus DNA in vitro using constant DNA concentration and various drug levels were studied. Fourier transform infrared (FTIR) and circular dichroism (CD) were studied with calf-thymus DNA in vitro using constant DNA concentration and various drug levels. FTIR, UV-visible, and CD spectroscopic methods were used to characterize the drug binding mode, the binding constant, and structural variations of DNA in aqueous solution. Spectroscopic evidence showed that the various Pt-based drugs bind indirectly to the major and minor grooves of DNA duplex with some degree of drug-phosphate interaction. The overall binding constants for JPM-39, GEB-28, and ATG-99 are K(JPM-39) = 4.2 (+/-0.75) x 10(3) M(-1), K(GEB-28) = 3.4 (+/-0.65) x 10(3) M(-1), and K(ATG-99) = 2.1 (+/-0.45) x 10(3) M(-1). DNA aggregation occurs at high drug concentration, while DNA remains in the B-family structure.