Search

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer's disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-β (Aβ) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: The incidence of gestational diabetes mellitus (GDM) is increasing worldwide. GDM patients have a significantly higher rate of cesarean section and postpartum hemorrhage, suggesting changes in uterine contractility. TWIK-1-related potassium channel (TREK1) expressed in the pregnant uterus and its role in uterine contraction. In this study, we examined the expression of HIF-1α and TREK1 proteins in GDM uterine and investigated whether high glucose levels are involved in the regulation of human uterine smooth muscle cells (HUSMCs) contraction through TREK1, and verified the role of HIF-1α in this process., Methods: Compared the uterine contractility between GDM and normal patients undergoing elective lower segment cesarean section. The HUSMCs were divided into normal glucose group, high glucose group, normal glucose with CoCl2 group, CoCl2 with echinomycin/L-Methionine group, and high glucose with echinomycin/L-Methionine group; Compare the cell contractility of each group. Compared the expression of hypoxia-inducible factor-1α (HIF-1α) and TREK1 protein in each group., Results: The contractility of human uterine strips induced by both KCl and oxytocin was significantly lower in patients with GDM compared with that in normal individuals, with increased TREK1 and HIF-1α protein expression. The contractility of cultured HUSMCs was significantly decreased under high glucose levels, which was consistent with increased expression of HIF-1α and TREK1 proteins. The contractility of HUSMCs was decreased when hypoxia was induced by CoCl2 and increased when hypoxia was inhibited by echinomycin. The TREK1 inhibitor L-methionine also recovered the decreased contractility of HUSMCs under high glucose levels or hypoxia., Discussion: The high glucose levels decreased the contractility of the myometrium, and increased expression of HIF-1a and TREK1 proteins play a role in changes in uterus contractility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Fei, Yu, Wang, Bai, Chen, Li and Yin.)

Mangrove actinomycetia have been proven to be one of the promising sources for discovering novel bioactive natural products. Quinomycins K ( 1 ) and L ( 2 ), two rare quinomycin-type octadepsipeptides without intra-peptide disulfide or thioacetal bridges, were investigated from the Maowei Sea mangrove-derived Streptomyces sp. B475. Their chemical structures, including the absolute configurations of their amino acids, were elucidated by a combination of NMR and tandem MS analysis, electronic circular dichroism (ECD) calculation, advanced Marfey's method, and further unequivocally confirmed by the first total synthesis. The two compounds displayed no potent antibacterial activity against 37 bacterial pathogens and had no significant cytotoxic activity against H460 lung cancer cells.

The main objective of this investigation was to characterize a collection of actinomycetes strains isolated from unexplored polluted ecosystems and to evaluate their antimicrobial potential in order to discover interesting bioactive compounds. Based on morphological and culture characters, 32 different strains were isolated: 20 strains from compost heap, seven strains from manure, and five strains from waste water. As expected, the genus Streptomyces was the most prevalent followed by the genus Micromonospora. Analysis of the antimicrobial activities of the isolated strains showed that those from compost heap were more efficient against the tested microorganisms (Candida albicans, Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli). Several bioactive compounds were identified by liquid chromatography (LC) combined with mass spectrometry (MS) and then analyzed by both MEDINA's database, which contains the most common secondary metabolites, and Dictionary of Natural Products Chapman & Hall. Many interesting well-known and unknown biomolecules were identified. Quinomycin A and Daidzein were the most fascinating compounds isolated, respectively, by Streptomyces sp. WW2 and Streptomyces sp. WW4. The most active strain was identified based on 16S rDNA's sequences and it seems to be a new strain. The crude extract of the strain CH12 was analyzed and the UV absorption spectra and mass spectra (MS) of the main active compound were reported. It's an interesting compound (possible purpuromycin) with the molecular formula C26H18O13., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms., Methods: EBs in suspension culture were divided into normoxic (21% O 2 ) and hypoxic (5% O 2 ) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1 α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1 α , β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment., Results: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture ( P < 0.05) with also a greater outgrowth rate of the adherent EBs ( P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA ( P < 0.05) and protein expressions of HIF-1 α , β-catenin and VEGFA ( P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin ( P < 0.05)., Conclusion: Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1 α /β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin., (© 2022 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Nonribosomal peptides (NRPs) are natural products that are biosynthesized by large multi-enzyme assembly lines called nonribosomal peptide synthetases (NRPSs). We have previously discovered that backbone or side chain methylation of NRP residues is carried out by an interrupted adenylation (A) domain that contains an internal methyltransferase (M) domain, while maintaining a monolithic AMA fold of the bifunctional enzyme. A key question that has remained unanswered is at which step of the assembly line mechanism the methylation by these embedded M domains takes place. Does the M domain methylate an amino acid residue tethered to a thiolation (T) domain on same NRPS module (in cis), or does it methylate this residue on a nascent peptide tethered to a T domain on another module (in trans)? In this study, we investigated the kinetics of methylation by wild-type AMAT tridomains from two NRPSs involved in biosynthesis of anticancer depsipeptides thiocoraline and echinomycin, and by mutants of these domains, for which methylation can occur only in trans. The analysis of the methylation kinetics unequivocally demonstrated that the wild-type AMATs methylate overwhelmingly in cis, strongly suggesting that this is also the case in the context of the entire NRPS assembly line process. The mechanistic insight gained in this study will facilitate rational genetic engineering of NRPS to generate unnaturally methylated NRPs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Two new cytotoxic antibiotics designated quinomycins H1 (2) and H2 (3) were isolated from the culture broth of Streptomyces sp. RAL404. The molecular formula of both compounds was established as C 52 H 65 N 11 O 13 S 2 by electrospray ionization mass spectrometry (ESI-MS). Their structures were determined as echinomycin (1) derivatives containing a 3-hydoxyquinaldic acid molecule in place of one of the two quinoxaline-2-carboxylic acid chromophores. Quinomycins H1 (2) and H2 (3) showed selective cytotoxicity against RG-E1-4 cells bearing the adenovirus oncogenes with IC 50 s of 11 nM and 12 nM, respectively.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Noncanonical G-C + and A-T Hoogsteen base pairs can form in duplex DNA and play roles in recognition, damage repair, and replication. Identifying Hoogsteen base pairs in DNA duplexes remains challenging due to difficulties in resolving syn versus antipurine bases with X-ray crystallography; and size limitations and line broadening can make them difficult to characterize by NMR spectroscopy. Here, we show how infrared (IR) spectroscopy can identify G-C + and A-T Hoogsteen base pairs in duplex DNA across a range of different structural contexts. The utility of IR-based detection of Hoogsteen base pairs is demonstrated by characterizing the first example of adjacent A-T and G-C + Hoogsteen base pairs in a DNA duplex where severe broadening complicates detection with NMR., (© 2017 Federation of European Biochemical Societies.)

Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor-1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was -10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR., (© 2014 Society for Laboratory Automation and Screening.)

Two novel quinomycin derivatives, RK-1355A (1) and B (2), and one known quinomycin derivative, UK-63,598 (3), were isolated from a microbial metabolites fraction library of Streptomyces sp. RK88-1355 based on Natural Products Plot screening. The structural elucidation of 1 and 2 was established through two-dimensional NMR and mass spectrometric measurements. They belong to a class of quinomycin antibiotics family having 3-hydroxyquinaldic acid and a sulfoxide moiety. They are the first examples for natural products as a quinoline type quinomycin having a sulfoxide on the intramolecular cross-linkage. They showed potent antiproliferative activities against various cancer cell lines and they were also found to exhibit moderate antibacterial activity.

Echinomycin, a bis-intercalator antitumor cyclic peptide, is biosynthesized by a unique nonribosomal peptide synthetase (NRPS). Successful heterologous expression of the whole gene cluster of echinomycin in Escherichia coli let us to investigate a further application of echinomycin NRPS. To construct a cyclic peptide library, our approach through both chemoenzymatic and rational genetic engineering has been successfully demonstrated. These achievements provided the further support that E. coli-based system can serve as a flexible yet robust platform for producing complex natural products and their analogs.

Excised thioesterase domains are versatile catalysts for macrocyclization. However, thioesterase-catalyzed cyclization is often precluded due to the occurrence of hydrolysis and product inhibition. To circumvent these obstacles, we devised an unprecedented strategy: coincubation with DNA to capture the cyclic products possessing DNA-binding properties. In experiments involving echinomycin thioesterase-catalyzed macrolactonization leading to the cyclic triostin A analog TANDEM, we found that the addition of DNA drastically improved the yield of TANDEM (19% --> 67%), with a complete reversal of the cyclization:hydrolysis ratio (1:2 --> 18:1). Furthermore, the applicability of this protocol was demonstrated for a variety of substrates. The results described herein provide insight into the mechanism of echinomycin thioesterase-catalyzed conversions and also pave the way for chemoenzymatic synthesis of the quinoxaline antibiotics and their analogs.

More than 400 compounds isolated from soil microorganisms, and catalogued in the antibiotic library of the Kitasato Institute for Life Sciences, were screened against African trypanosomes. Ten compounds were found to have selective and potent antitrypanosomal activity in vitro: aureothin, cellocidin, destomycin A, echinomycin, hedamycin, irumamycin, LL-Z 1272beta, O-methylnanaomycin A, venturicidin A and virustomycin A. Results of the in vitro assays using the GUTat 3.1 strain of Trypanosomal brucei brucei and the STIB900 strain of T. b. rhodesiense are presented. Cytotoxicity was determined using a human MRC-5 cell line. This is the first report of antitrypanosomal activities of the 10 microbial metabolites listed above.

Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. In this study, triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de novo production of triostin A by means of high cell density fed-batch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with small-scale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy system could easily provide us with valuable information for maximizing the production titer. Our entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns.

A bis-intercalator echinomycin (ECHI) and a simple intercalator [Co(phen)3]3+ were used as a novel electrochemical redox indicators to detect DNA hybridization at gold electrodes (AuE). In order to minimize the nonspecific adsorption of oligonucleotides (ODN), the thiol-derivatized oligonucleotides were immobilized onto AuE in the first step, and the exposition of AuE to 6-mercapto-1-hexanol (MCH) followed in the second step of this procedure. In this arrangement good reproducibility and discrimination between single-stranded (ss) probe and double-stranded (ds) hybrid DNA were obtained. While both redox indicators showed a good ability to discriminate between the ss probe and ds hybrid DNA, the signals of ECHI were by an order of maginitude higher than those of [Co(phen)3]3+ in a good agreement with stronger DNA binding by the bis-intercalator as compared to the simple intercalator. In addition, DNA single-base mismatch (DNA point mutation) was easily detected by means of ECHI.

A new crystal form was obtained for the complex between (ACGTACGT)2 and echinomycin and X-ray data were collected to 1.6 A. The structure was phased by the SAD method based on a single unexpected anomalous scatterer that could be identified as a mixture of nickel and zinc by measurements of the anomalous scattering at different wavelengths. This cation is coordinated by two guanines from two different duplexes and four water molecules. The structure resembles previously reported crystal structures of DNA-echinomycin complexes, except that three of the eight base pairs flanking the echinomycin bisintercalator sites have the Watson-Crick rather than the Hoogsteen configuration. Hoogsteen binding was found for the corresponding base pairs of the crystallographically independent duplex, indicating that the two configurations are very close in energy.

The major structural determinant of the preference to bind to CpG binding sites on DNA exhibited by the natural quinoxaline bis-intercalators echinomycin and triostin A, or the quinoline echinomycin derivative, 2QN, is the 2-amino group of guanine (G). However, relocation of this group by means of introduction into the DNA molecule of the 2-aminoadenine (=2,6-diaminopurine, D) base in place of adenine (A) has been shown to lead to a drastic redistribution of binding sites, together with ultratight binding of 2QN to the sequence DTDT. Also, the demethylated triostin analogs, TANDEM and CysMeTANDEM, which bind with high affinity to TpA steps in natural DNA, bind much less tightly to CpI steps, despite the fact that both adenosine and the hypoxanthine-containing nucleoside, inosine (I), provide the same hydrogen bonding possibilities in the minor groove. To study both the increased binding affinity of 2QN for DTDT relative to GCGC sites and the remarkable loss of binding energy between CysMeTANDEM and ICIC compared with ATAT, a series of thermodynamic integration free energy simulations involving conversions between DNA base pairs have been performed. Our results demonstrate that the electrostatic component of the stacking interactions between the heteroaromatic rings of these compounds and the bases that make up the intercalation sites plays a very important role in the modulation of their binding affinities.

Atomic force microscopy (AFM) has been used to examine the conformational effects of echinomycin, a DNA bis-intercalating antibiotic, on linear and circular DNA. Four different 398 bp DNA fragments were synthesized, comprising a combination of normal and/or modified bases including 2,6-diaminopurine and inosine (which are the corresponding analogues of adenine and guanosine in which the 2-amino group that is crucial for echinomycin binding has been added or removed, respectively). Analysis of AFM images provided contour lengths, which were used as a direct measure of bis-intercalation. About 66 echinomycin molecules are able to bind to each fragment, corresponding to a site size of six base-pairs. The presence of base-modified nucleotides affects DNA conformation, as determined by the helical rise per base-pair. At the same time, the values obtained for the dissociation constant correlate with the types of preferred binding site available among the different DNA fragments; echinomycin binds to TpD sites much more tightly than to CpG sites. The structural perturbations induced when echinomycin binds to closed circular duplex pBR322 DNA were also investigated and a method for quantification of the structural changes is presented. In the presence of increasing echinomycin concentration, the plasmid can be seen to proceed through a series of transitions in which its supercoiling decreases, relaxes, and then increases.

Differential scanning calorimetry and UV thermal denaturation have been used to determine a complete thermodynamic profile for the bis-intercalative interaction of the peptide antibiotic echinomycin with DNA. The new calorimetric data are consistent with all previously published binding data, and afford the most rigorous and direct determination of the binding enthalpy possible. For the association of echinomycin with DNA, we found DeltaG degrees = -7.6 kcal mol(-1), DeltaH = +3.8 kcal mol(-1) and DeltaS = +38.9 cal mol(-1) K(-1) at 20 degrees C. The binding reaction is clearly entropically driven, a hallmark of a process that is predominantly stabilized by hydrophobic interactions, though a deeper analysis of the free energy contributions suggests that direct molecular recognition between echinomycin and DNA, mediated by hydrogen bonding and van der Waals contacts, also plays an important role in stabilizing the complex.

We have examined the interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments which contain isolated ligand binding sites. A 145 base pair fragment was prepared on the basis of the sequence of tyrT DNA, which contained no CpG or (A/T)(4) binding sites for these ligands. Isolated binding sites were introduced into this fragment at discrete locations where the minor groove is known to face toward or away from the protein core when reconstituted onto nucleosome core particles. The interaction of ligands with target sites on these nucleosomal DNA fragments was assessed by DNase I footprinting. We find that Hoechst 33258 can bind to single nucleosomal sites which face both toward and away from the protein core, without affecting the nucleosome structure. Hoechst binding is also observed on nucleosomal fragments which contain two or more drug binding sites, though in these cases the footprints are accompanied by the presence of new cleavage products in positions which suggest that the ligand has caused a proportion of the DNA molecules to adopt a new rotational positioning on the protein surface. Hoechst 33258 does not affect nucleosome reconstitution with any of these fragments. In contrast, the bifunctional intercalating antibiotic echinomycin is not able to bind to single nucleosomal CpG sites. Echinomycin footprints are observed on nucleosomal fragments containing two or more CpG sites, but there are no changes in the cleavage patterns in the remainder of the fragment. Echinomycin abolishes nucleosome reconstitution when included in the reconstitution mixture.

Full details of the total syntheses of thiocoraline (1) and BE-22179 (2), C(2) symmetric bicyclic octadepsipeptides possessing two pendant 3-hydroxyquinoline chromophores, are described in which their relative and absolute stereochemistry were established. Key elements of the approach include the late-stage introduction of the chromophore, symmetrical tetrapeptide coupling, macrocyclization of the 26-membered octadepsipeptide conducted at the single secondary amide site following disulfide formation, and a convergent assemblage of the tetradepsipeptide with introduction of the labile thiol ester linkage in the final coupling reaction under near racemization free conditions. By virtue of the late-stage introduction of the chromophore and despite the challenges this imposes on the synthesis, this approach provides ready access to a range of key chromophore analogues. Thiocoraline and BE-22179 were shown to bind to DNA by high-affinity bisintercalation analogous to echinomycin, but with little or no perceptible sequence selectivity. Both 1 and 2 were found to exhibit exceptional cytotoxic activity (IC(50) = 200 and 400 pM, respectively, L1210 cell line) comparable to echinomycin and one analogue, which bears the luzopeptin chromophore, was also found to be a potent cytotoxic agent.

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G.C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.

A DNA-multiple drug complex, d(ACGTAGCTACGT)(2):[actinomycin D, (echinomycin)(2)] has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 85.6, b = 72.8, c = 56.6 A, beta = 101.5 degrees at 93 K and Z = 8. The crystal diffracted to 3.0 A resolution along the DNA fiber axis and to 3.5 A resolution in other directions. The Patterson maps indicate that all complexes in the crystal are oriented along their helical axes in the [101;] direction.

Footprinting experiments with DNase I provide a starting-point for investigating the molecular basis of nucleotide sequence recognition by 2QN, a bis-quinoline derivative of the quinoxaline antibiotic echinomycin produced by directed biosynthesis in Streptomyces echinatus. Using tyrT DNA molecules variously substituted with inosine and/or 2,6-diaminopurine residues it is shown that the location of the 2-amino group of purine nucleotides in the minor groove of the double helix exerts a dominant influence in determining where the antibiotic will bind, as it does for echinomycin. However, newly created binding sites in DNA molecules substituted with diaminopurine (D), all located round TpD steps, bind 2QN with so much higher affinity than the canonical CpG steps that the latter fail completely to appear as footprints in D-substituted DNA; indeed CpG sequences appear in regions of enhanced susceptibility to nuclease cleavage as do CpI steps in doubly D + I-substituted DNA. Quantitative footprinting plots confirm that sequences surrounding TpD steps bind 2QN several hundred-fold more tightly than do CpG-containing sequences, with dissociation constants of the order of 25 nM. To test the hypothesis that differences in stacking interactions between the chromophores of the drug and the DNA base pairs could account for the differences in binding affinities, models of 2QN bound to two DNA hexamers containing either a central CpG or a central TpD step were built. Calculation of the molecular electrostatic potential (MEP) of 2QN in solution using a continuum method revealed a distinctive pattern that is considered relevant to DNA binding. When the MEPs calculated for the two DNA hexamers in the complexed state were compared, substantial differences were found in the major groove and in the space between the base pairs that is occupied by the chromophores of the drug upon binding. The modelling data support the notion that electrostatic stacking interactions underlie the considerably preferred binding of echinomycin and 2QN around TpD steps rather than CpG steps.

We have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time-dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37 degrees C.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

We have examined the kinetics of dissociation of echinomycin from CpG sites in several DNA fragments containing synthetic DNA inserts by a variation of the footprinting technique. Complexes of the ligand with radiolabeled DNA fragments were dissociated by adding an excess of unlabeled calf thymus DNA. Samples were removed from this mixture at subsequent time intervals and subjected to DNase I footprinting. The rate of disappearance of the footprints varied considerably between the various CpG sites. At 20 degrees C, echinomycin dissociates more slowly from CpG sites flanked by (AT)n (t1/2 approximately 40 min) and (CA)n.(TG)n (t1/2 approximately 11 min) than by An.Tn (t1/2 < 3 min). In each sequence context the dissociation from ACGT is slower than that from TCGA. (TAA)4CG(TTA)4 also represents a very good binding site (t1/2 approximately 35 min), which is less sensitive to changes in temperature than most other sites. Within sequences (AT)10(G/C)4(AT)10, the dissociation from CGGC is slower than that from CCCG or CCGC.

The crystal structures of three quinoxaline antibiotics-echinomycin 2QN, triostin C and the C222(1) form of triostin A--have been determined, and the structure of the P2(1)2(1)2(1) form of triostin A has been re-refined against our previously reported data. The molecular conformations are compared with those deduced from NMR data and those reported for two complexes of triostin A with oligonucleotides. Although the depsipeptide ring conformations are basically similar, the effective twofold molecular symmetry is violated by the folding of one of the quinoxaline chromophores in echinomycin 2QN and by a rotation of one of the ester planes with the formation of an intramolecular hydrogen bond in triostin C. In the oligonucleotide complexes of triostin A the chirality of the disulfide bridge is inverted. The alanine NH groups are involved in intermolecular hydrogen bonds in all four structures, and (except in echinomycin 2QN) the stacking of the chromophores in the crystal emulates the intercalation involved in DNA complex formation. In echinomycin 2QN, the antibiotic molecules are hydrogen bonded to form a helix along the crystallographic 6(5) screw axes, with a channel of disordered solvent running through the middle of the helix. Crystal data: (1), echinomycin 2QN, C53H66N10O12S2.2.5(C3H6O).2.5(H2O), M(r) = 1289.5, hexagonal, P6(5), a = b = 22.196(15), c = 24.64 (2) A, V = 10,513 (13) A3, Z = 6, Dx = 1.222 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.275 mm-1, T = 193 K, R = 9.0% for 4828 I > 2 sigma (I) and 11.8% for all 7102 unique reflections; (2), triostin C, C54H70N12O12S2.0.67(CHCl3).0.67(H2O), M(r) = 1234.2, orthorhombic, P2(1)2(1)2(1), a = 16.054 (8), b = 17.128 (9), c = 22.706 (12) A, V = 6244 (6) A3, Z = 4, Dx = 1.313 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.239 mm-1, T = 188 K, R = 7.7% for 4678 I > 2 sigma (I) and 14.0% for all 7260 unique reflections; (3), triostin A, C50H62N12O12S2.2(C7H14O2), M(r) = 1347.6, orthorhombic, P2(1)2(1)2(1), a = 20.94 (2), b = 18.53 (2), c = 18.80 (2) A, V = 7292 (13) A3, Z = 4, Dx = 1.228 Mg m-3, lambda (Cu K alpha) = 1.5418 A, mu = 1.245 mm-1, T = 293 K, R = 6.8% for 2116 I > 2 sigma (I) and 9.3% for all 2928 unique reflections; (4), triostin A, C50H62N12O12S2.HCl.2(C3H7NO), M(r) = 1269.9, monoclinic, C222(1), a = 10.622 (10), b = 17.035 (17), c = 35.21 (3) A, V = 6371 (10) A3, Z = 4, Dx = 1.324 Mg m-3, lambda (Mo K alpha) = 0.71073 A, mu = 0.199 mm-1, T = 153 K, R = 7.5% for 2164 I > 2 sigma (I) and 13.2% for all 3402 unique reflections. Extensive use was made of restraints on the geometrical and displacement parameters in the successful anisotropic refinement of these structures against weak data.

The quinomycin antibiotic UK-63052 (designated QN) exhibits a chemical structure related to the antibiotic echinomycin which is known to bisintercalate into DNA. Common features among these antibiotics include two heterocyclic aromatic ring systems propagating from a cross-bridged cyclic octadepsipeptide scaffold. We report on the solution structure of the QN-d(A1-C2-A3-C4-G5-T6-G7-T8) complex (one QN molecule per duplex) based on a combined NMR-molecular dynamics study including intensity-based refinement. The 3-hydroxy quinaldic acid rings bisintercalate into the duplex at (A3-C4).(G5-T6) steps and stack with flanking Watson-Crick A3.T6 and C4.G5 base-pairs. The intercalation sites at (A3-C4).(G5-T6) steps are wedge-shaped and unwound, with significant unwinding also observed at the (C4-C5).(C4-G5) step bracketed between the intercalation sites. The cross-bridged cyclic octadepsipeptide is positioned in the minor groove with the methyl groups on its Ala and NMe-MCp residues directed towards and making van der Waals contacts with the minor groove edge of the duplex. A pair of adjacent intermolecular hydrogen bonds between the Ala backbone atoms and the G5 minor groove edge (Ala-NH to G5-N(3) and G5-NH2e to Ala-CO) account for the sequence specificity associated with complex formation. The solution structure of the QN-DNA oligomer complex, which contains only Watson-Crick base-pairs flanking the bisintercalation site, is compared with the crystal structure of the related echinomycin-DNA oligomer complex, which contains Hoogsteen base-pairs on either side of the bisintercalation site.

In order to assess the utility of different methods of footprinting applied to the study of sequence-selective small molecule-DNA interaction we have performed a homologous series of experiments on the binding of echinomycin, a bis-intercalator, to a 133 base pair DNA restriction fragment containing a small number of discrete binding sites. Two of those sites each contained a pair of closely clustered CpG steps, the cognate dinucleotide sequence which is the common denominator of sites recognised by echinomycin. DNAse I was found to be much the best enzyme for footprinting in terms of sensitivity, accuracy, and ease of handling. DNAase II and micrococcal nuclease were of limited value. Excellent results were recorded with methidiumpropyl-EDTA.FeII which picked up strong binding sites and yielded sharp footprints from which a parsimonious estimate of site size could be determined. Orthophenanthroline.CuI proved to be a very suitable, sensitive chemical nuclease but hydroxyl radical footprinting with EDTA.FeII was only partially successful. Positive footprinting with conformation-sensitive probes diethylpyrocarbonate, osmium tetroxide and potassium permanganate yielded information to complement that afforded by the enzymic and chemical nucleases. Evidence of binding to both CpG steps in the clustered pair was obtained, with indications of possible cooperativity.

Quinomycin antibiotic UK-65,662 binds selectively to the 5'-CpG-binding sites of the DNA duplexes d(ACGT)2 and d(GACGTC)2; the complexes have been studied in detail by 1H-n.m.r. spectroscopy and molecular-modelling techniques employing nuclear Overhauser effect-restrained energy minimization and molecular dynamics. Whereas the terminal A.T base pairs of the tetamer duplex d(ACGT)2 adopt a stable Hoogsteen alignment (characterized by a syn glycosidic conformation of the purine base), when internalized within the hexamer duplex d(GACGTC)2, the A.T base pairs revert to anti glycosidic torsion angles characteristic of the Watson-Crick hydrogen-bonding scheme. The energetics of base-pair stacking at the terminal 5'-GpA steps of the hexamer complex, with base pairs in the Watson-Crick alignment, are concluded to be important determinants of the adopted conformation, whereas an energetic preference for stacking interactions between terminal Hoogsteen A.T base pairs and the drug quinoline chromophores is evident in the tetramer complex. The internal G.C base pairs in both complexes are highly stabilized, as indicated by the very slow exchange rates of the guanine imino protons; in contrast, the flanking A.T base pairs are no more stable than in the ligand-free DNA duplexes. A large number of intermolecular nuclear Overhauser effects are indicative of many van der Waals contacts and hydrogen-bonding between the antibiotic and the minor groove of the central G.C base pairs in both complexes, indicating that interactions with the G.C base pairs in each duplex are very similar providing the essential features for recognition and tight binding. Despite the difference in the conformation of the A.T base pairs, stacking with the quinoline rings occurs primarily with the adenine bases in both complexes. Relative intensities of intranucleotide versus internucleotide nuclear Overhauser effects indicate that both duplexes are substantially unwound by drug binding (particularly at the CpG step) and this is confirmed by the structure calculations. Both duplexes have ladder-like structures that must lead to significant local distortions of the DNA conformation in vivo.

Hoogsteen base pairs have been demonstrated to occur in base pairs adjacent to the CpG binding sites in complexes of triostin A and echinomycin with a variety of DNA oligonucleotides. To understand the relationship of these unusual base pairs to the sequence specificity of these quinoxaline antibiotics, the conformation of the base pairs flanking the YpR binding sites of the 2:1 drug-DNA complexes of triostin A with [d(ACGTACGT)]2 and of the TpA specific [N-MeCys3, N-MeCys7] TANDEM with [d(ATACGTAT)]2 have been studied by 1H NMR spectroscopy. In both the 2:1 triostin A-DNA complex and the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the terminal A.T base pairs are Hoogsteen base paired with the 5' adenine in the syn conformation. This indicates that both TpA specific and CpG specific quinoxaline antibiotics are capable of inducing Hoogsteen base pairs in DNA. However, in both 2:1 complexes, Hoogsteen base pairing is limited to the terminal base pairs. In the 2:1 triostin A complex, the internal adenines are anti and in the 2:1 [N-MeCys3, N-MeCys7] TANDEM-DNA complex, the internal guanines are anti regardless of pH, which indicates that the central base pairs of both complexes form Watson-Crick base pairs. This indicates that the sequence dependent nature of Hoogsteen base pairing is the same in TpA specific and CpG specific quinoxaline antibiotic-DNA complexes. We have calculated a low resolution three-dimensional structure of the 2triostin A-[d(ACGTACGT)]2 complex and compared it with other CpG specific quinoxaline antibiotic-DNA complexes. The role of stacking in the formation of Hoogsteen base pairs in these complexes is discussed.

The expedient of preparing homologous DNA samples substituted with I for G, DAP for A, or both, has been used to investigate the role of the purine 2-amino group in determining the preferred binding sites for antibiotics on DNA. The selectivity of echinomycin for CpG steps, of actinomycin for GpC steps, and of netropsin for A + T-rich tracts, is seen to be radically altered in the substituted DNA molecules.

Experiments are described which probe the role of the 2-amino group of guanine as a critical determinant of the recognition of nucleotide sequences in DNA by specific ligands. Homologous samples of tyrT DNA substituted with inosine or 2,6-diaminopurine residues in place of guanosine or adenine respectively yield characteristically modified footprinting patterns when challenged with sequence-selective antibiotics such as echinomycin, actinomycin or netropsin. The capacity of small molecules to recognise particular DNA sequences is exploited in the 'combilexin' strategy to target small molecules to defined sites in DNA. A composite molecule containing a distamycin moiety linked to an intercalating ellipticine derivative has been synthesised and shown to bind tightly to DNA but without much sequence-selectivity. Refinement of this molecule based on predictions from molecular modelling has led to the synthesis of a second generation derivative bearing an additional positive charge: this new hybrid molecule is strongly selective for binding to AT-rich tracts in DNA.

The binding of echinomycin to DNA hexamers of the form GpApXpZpTpC, where the central XpZ step can be CpG, TpA, GpC, or ApT, has been studied by molecular modeling and molecular mechanics techniques. Interaction energies have also been calculated for the complexation of echinomycin with sequences containing the preferred central CpG step and different flanking base pairs. Besides, two more sets of sequences incorporating either 2,6-diaminopurine (DAP) or hypoxanthine in place of adenine or guanine, respectively, have been examined. The aim of this work was to evaluate the relative importance of hydrogen-bonding and stacking interactions in the association of echinomycin with DNA and further rationalize the experimental evidence. The results of these calculations are in consonance with available data from footprinting experiments and appear to support our previous hypothesis that, in addition to the crucial intermolecular hydrogen bonds in the central region, the stacking interactions involving the quinoxaline-2-carboxamide chromophores of the drug and the DNA base pairs play an important role in modulating the binding specificity of this bisintercalating antitumor antibiotic. This is most clearly seen when sequences with similar minor-groove environments are compared (e.g. CpI vs TpA or CpG vs TpDAP). The dipole moment of N-methylquinoxaline-2-carboxamide has been measured (mu = 4.15 +/- 0.03 D) and compares very well with the calculated value (mu = 4.14 D). The fact that G:C, I:C, A:T, and DAP:T base pairs are shown to be endowed with distinct van der Waals and electrostatic stacking properties with respect to this heteroaromatic ring system could have important implications for the design of novel DNA mono- and bis-intercalating agents.

In order to investigate the possible importance of Hoogsteen base pairing to the DNA-binding ability of echinomycin, quantitative DNase I footprinting has been performed. The substrate was the tyrT DNA restriction fragment, either "native" or substituted with one of the purine analogs 2'-deoxy-7-deazaadenosine and 2'-deoxy-7-deazaguanosine in both strands. The modified DNA species were prepared by PCR and selectively labeled at the 5' terminus of one strand (usually the upper "Watson" strand) with [32P]ATP and polynucleotide kinase. Proper incorporation of the analog nucleotides was verified by Maxam-Gilbert G- and C-sequencing reactions as well as exposure to osmium tetroxide and diethyl pyrocarbonate. OsO4 was found to react strongly with the 7-deaza nucleotides, providing a good check of faithful incorporation. The previously observed echinomycin-induced hyperreactivity of purines toward diethyl pyrocarbonate was eliminated by incorporating the appropriate 7-deazapurine. The DNase I footprinting titration studies greatly refined the existing knowledge of the DNA-binding characteristics of echinomycin, as they revealed five general types of concentration-dependent behavior at single-bond resolution. Estimates of microscopic binding constants at individual DNA binding sites were obtained by measuring the antibiotic concentration which produced a half-maximal effect on the concentration of a given DNase I cleavage product. All binding sites contained one or more CpG steps, and all CpG steps analyzed formed part of a binding site for echinomycin. No consistent differences in the estimated binding constants for these sites were observed by comparing normal and modified DNAs, indicating that the abolition of formal Hoogsteen pairs did not significantly alter the thermodynamics of echinomycin-DNA interaction. The lack of any detectable decrease in binding constants for critical sites in the 7-deazapurine-substituted DNAs argues against any anti-syn conformational transition of purine nucleosides occurring in association with the bis-intercalative complex formation.

We have studied the sequence-specific interaction of mithramycin with nucleosome core particles which have been reconstituted with various DNA fragments. Mithramycin binds to these DNAs without disrupting the integrity of the nucleosome and produces clear DNase I footprints centered around GC-rich regions. In some instances, the footprints produced on free DNA are resolved into two or more smaller sites when the DNA is complexed with the nucleosome core. In a few cases, novel footprints are produced in sequences which did not bind the drug in free DNA samples. The results are explained by suggesting mithramycin binds to GC-rich regions in which the minor groove faces away from the protein core, and which possess a wider than normal narrow groove on account of their location. Hydroxyl radical footprinting and diethyl pyrocarbonate modification confirm that mithramycin does not affect the rotational positioning of the nucleosome-bound DNA. Although distamycin and echinomycin induce novel DNase I digestion products in nucleosomal DNA which are consistent with the proposed change in DNA positioning [Low, C. M. L., Drew, H. R., & Waring, M. J. (1986) Nucleic Acids Res. 14, 6785-6801], hydroxyl radical footprinting and diethyl pyrocarbonate modification suggest these ligands do not change the rotational positioning of the DNA on the nucleosome cores.

The behavior of the complexes of echinomycin with the DNA tetramers d(ACGT)2 and d(TCGA)2, in which the terminal AT base pairs are in either a Hoogsteen or a Watson-Crick conformation, has been explored by molecular dynamics taking into account experimental data from NMR studies (Gao and Patel. Biochemistry 1988, 27, 1744-1751). The DNA binding specificity of echinomycin appears to be the result of a subtle balance between stabilizing and destabilizing forces. Among the former is a number of hydrogen bonds between the alanine residues of echinomycin and both the N3 and 2-amino groups of the guanine bases which decisively determine the strong affinity of the antibiotic for CpG steps. On the other hand, there appears to be an unfavorable dipolar interaction between the chromophores of the antibiotic and the CpG step. This electrostatic component of the stacking interactions also contributes to explaining the conformational preferences of the flanking sequences: upon Hoogsteen pairing, the dipole moment of an AT base pair is found to increase significantly and alter its relative orientation. In the d(ACGT)2:echinomycin complex, this arrangement helps to improve the stacking interactions with the quinoxaline-2-carboxamide system, but would lead to unfavorable dipolar interactions in the d(TCGA)2 complex. The bearing of these findings on the binding of echinomycin to several sequences as well as on the altered binding selectivity of other members of the quinoxaline family of antibiotics is also discussed.

The 2-amino group of guanine is believed to be a critical determinant of potential DNA binding sites for echinomycin and related quinoxaline antibiotics. In order to probe its importance directly we have studied the interaction between echinomycin and DNA species in which guanine N(2) is deleted by virtue of substitution of inosine for guanosine residues. The polymerase chain reaction was used to prepare inosine-substituted DNA. Binding of echinomycin, assessed by DNAse I footprinting, was practically abolished by incorporation of inosine into one or both strands of DNA. We conclude that both the purines in the preferred CpG binding site need to bear a 2-amino group to interact with echinomycin.

The interactions of echinomycin and the DNA decamer [d(ACGTATACGT)]2 were studied by proton NMR. Echinomycin binds cooperatively as a bisintercalator at the CpG steps. The terminal A.T base pairs are Hoogsteen base paired, but none of the four central A.T base pairs are Hoogsteen base paired. However, binding of the drug induces unwinding of the DNA which is propagated to the central ApT step. All four central A.T base pairs are destabilized relative to those in the free DNA. Furthermore, based on these and other results from our laboratory, we conclude that the formation of stable Hoogsteen base pairs may not be the relevant structural change in vivo. The structural changes propagated between adjacent ACGT binding sites are the unwinding of the duplex and destabilization of the base pairing between binding sites.

We have prepared DNA fragments containing the sequences A15CGT15, T15CGA15 and T(AT)8CG(AT)15 cloned within the SmaI site of the pUC19 polylinker. These have been used as substrates in footprinting experiments with DNase I and diethylpyrocarbonate probing the effects of echinomycin, binding to the central CG, on the structure of the surrounding sequences. No clear DNase I footprints are seen with T15CGA15 though alterations in the nuclease susceptibility of surrounding regions suggest that the ligand is binding, albeit weakly at this site. All the other fragments show the expected footprints around the CG site. Regions of An and Tn are rendered much more reactive to DNase I and adenines on the 3'-side of the CG become hyperreactive to diethylpyrocarbonate. Regions of alternating AT show unusual changes in the presence of the ligand. At low concentrations (5 microM) cleavage of TpA is enhanced, whereas at higher concentrations a cleavage pattern with a four base pair repeat is evident. A similar pattern is seen with micrococcal nuclease. Modification by diethylpyrocarbonate is strongest at alternate adenines which are staggered in the 5'-direction across the two strands. We interpret these changes by suggesting secondary drug binding within regions of alternating AT, possibly to the dinucleotide ApT. DNase I footprinting experiments performed at 4 degrees C revealed neither enhancements nor footprints for flanking regions of homopolymeric A and T suggesting that the conformational changes are necessary consequence of drug binding.

We have studied the binding of echinomycin to DNA fragments containing GC-rich regions flanked by blocks of alternating AT by DNase I footprinting and diethylpyrocarbonate modification. Regions of alternating AT flanking the sequences CCCG, CCGC, CGGC and GG show a four base pair DNase I cleavage pattern and reaction of alternate adenines with diethylpyrocarbonate. This pattern is strongest when the AT-block is immediately adjacent to the CpG ligand binding site. We explain these phenomena by suggesting that echinomycin binds to the dinucleotide step ApT in a cooperative fashion. The cooperative effects can be transmitted through the dinucleotide step GC but not CC or AA. No such repetitive patterns are seen with surrounding regions of (ATT).(AAT). Evidence is presented for secondary drug binding sites at CpC and TpG with weaker interaction at the CpG site within the hexanucleotide TTCGAA.

The complexes formed between the cyclic octadepsipeptide antibiotic echinomycin and the two DNA octamers [d(ACGTACGT)]2 and [d(TCGATCGA)]2 have been investigated by using one- and two-dimensional proton NMR spectroscopy techniques. The results obtained for the two complexes are compared to each other, to the crystal structures of related DNA-echinomycin complexes, and to enzymatic and chemical footprinting results. In the saturated complexes, two echinomycin molecules bind to each octamer by bisintercalation of the quinoxaline moieties on either side of each CpG step. Binding of echinomycin to the octamer [d(ACGTACGT)]2 is cooperative so that only the two-drug complex is observed at lower drug-DNA ratios, but binding to [d(TCGATCGA)]2 is not cooperative. At low temperatures, both the internal and terminal A.T base pairs adjacent to the binding site in the [d(ACGTACGT)]2-2 echinomycin complex are Hoogsteen base paired (Gilbert et al., 1989) as observed in related crystal structures. However, as the temperature is raised, the internal A.T Hoogsteen base pairs are destabilized and are observed to be exchanging between the Hoogsteen base-paired and an open (or Watson-Crick base-paired) state. In contrast, in the [d(TCGATCGA)]2-2 echinomycin complex, no A.T Hoogsteen base pairs are observed, the internal A.T base pairs appear to be stabilized by drug binding, and the structure of the complex does not change significantly from 0 to 45 degrees C. Thus, the structure and stability of the DNA in echinomycin-DNA complexes depends on the sequence at and adjacent to the binding site. While we conclude that no single structural change in the DNA can explain all of the footprinting results, unwinding of the DNA helix in the drug-DNA complexes appears to be an important factor while Hoogsteen base pair formation does not.

The interaction of a new group of 'quinomycin-like' antibiotics with the DNA duplexes d(ACGT)2 and d(GACGTC)2 has been investigated in solution by 1H NMR spectroscopy. By monitoring the intensity of intranucleotide base H6/H8 to deoxyribose H1'NOE cross-peaks we conclude that the terminal A-T basepairs flanking the CG bisintercalation site in the d(ACGT)2 complex adopt the Hoogsteen bonding scheme, with the purine base in a syn conformation. By comparison in the d(GACGTC)2 complex all glycosidic bond angles are anti, consistent with a preferred Watson-Crick basepairing scheme. Both DNA duplexes appear to be significantly unwound compared with the ligand-free DNAs. The data illustrate the influence of helical constraints on the stability of the Hoogsteen bonding scheme adjacent to the drug binding sites.

The interaction between UK63052, a novel derivative of the quinomycin group of bifunctional intercalating antibiotics, with DNA has been investigated by footprinting techniques and the results compared with echinomycin. UK63052 binds strongly but reversibly to DNA and decreases the gel mobility of most DNA fragments, although the mobility of bent kinetoplast DNA is increased. The drug binds selectively to the dinucleotide CpG though not all such sequences present good binding sites. Binding is best when CG is surrounded by AT base pairs. UK63052 and echinomycin have different effects on DNA structure as assessed by changes in the sensitivity to modification by diethylpyrocarbonate. The results are interpreted by suggesting that substitutions on the chromophores affect the precise details of DNA recognition.

Echinomycin complexes with polymeric DNAs and model duplex oligonucleotides have been studied by low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoxaline chromophores of the drug used as intrinsic probes. Although not optically resolved, plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of echinomycin, which was ascribed to the presence of two distinct quinoxaline triplet-state environments (referred to as the blue and red triplet states of echinomycin in this report). We think that a likely origin of the two triplet states of echinomycin is the occurrence of two or more distinct conformations of the drug in aqueous solutions. Spectroscopically observed perturbations of the triplet-state properties of echinomycin such as the phosphorescence emission spectrum, phosphorescence lifetime, ODMR spectrum, and zero-field splitting (zfs) energies were investigated upon drug binding to the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) [abbreviated as poly[d(G-C)2]] and poly(dA-dT).poly(dA-dT) [abbreviated as poly[d(A-T)2]], the homopolymer duplexes poly(dG).poly(dC) [abbreviated as poly(dG.dC)] and poly(dA).poly(dT) [abbreviated as poly(dA.dT)], and the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, and calf thymus. Echinomycin bisintercalation complexes with the self-complementary oligonucleotides d(ACGT), d(CGTACG), and d(ACGTACGT), which are thought to model drug binding sites, were also investigated. Phosphorescence and ODMR spectroscopic results indicate that the quinoxaline chromophores of the drug are involved in aromatic stacking interactions in complexes with the natural DNAs as evidenced by red shifts in the phosphorescence 0,0 band of the drug, a small but significant reduction in the phosphorescence lifetime of the red triplet state, and reduction of the zfs D-value of both the blue and red triplet states upon drug complexation. These changes in the triplet-state properties of echinomycin are consistent with stacking interactions that increase the polarizability of the quinoxaline environment. The extent of the reduction of the D parameter for the red triplet state upon complexation with the polymeric DNAs was found to correlate with the binding affinities measured for these targets [Wakelin, L. P. G., & Waring, M. J. (1976) Biochem. J. 157, 721-740], with the single exception of the drug-poly[d(G-C)2] complex, for which an increase in the D-value was noted. In addition, upon drug binding to the natural DNAs, there is a reversal of signal polarity in the ODMR spectra of the red triplet state. Among the synthetic DNA polymers investigated, a reversal of ODMR signal polarity was found only with the echinomycin-poly[d(A-T)2] complex.(ABSTRACT TRUNCATED AT 400 WORDS)

The interaction of echinomycin with a kinetoplast DNA fragment which contains phased runs of adenine residues has been examined by various footprinting techniques. DNAase I footprinting confirms that all drug-binding sites contain the dinucleotide CpG. However, not all such sequences are protected. Three sites, each of which is located between two adenine tracks in the sequence GCGA, are not protected from DNAase I attack. Enhanced cleavage by DNAase I, DNAase II and micrococcal nuclease is observed in regions surrounding drug-binding sites. The results suggest that echinomycin alters the conformation of the AT tracks, making them more like an average DNA structure. Echinomycin renders adenine residues in the sequence CGA hyper-reactive to diethyl pyrocarbonate.