The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Macrophage-organoid co-culture model for identifying treatment strategies against macrophage-related gemcitabine resistance.

BACKGROUND: Gemcitabine resistance (GR) is a significant clinical challenge in pancreatic adenocarcinoma (PAAD) treatment. Macrophages in the tumor immune-microenvironment are closely related to GR. Uncovering the macrophage-induced GR mechanism could help devise a novel strategy to improve gemcitabine treatment outcomes in PAAD. Therefore, preclinical models accurately replicating patient tumor properties are essential for cancer research and drug development. Patient-derived organoids (PDOs) represent a promising in vitro model for investigating tumor targets, accelerating drug development, and enabling personalized treatment strategies to improve patient outcomes. METHODS: To investigate the effects of macrophage stimulation on GR, co-cultures were set up using PDOs from three PAAD patients with macrophages. To identify signaling factors between macrophages and pancreatic cancer cells (PCCs), a 97-target cytokine array and the TCGA-GTEx database were utilized. The analysis revealed CCL5 and AREG as potential candidates. The role of CCL5 in inducing GR was further investigated using clinical data and tumor sections obtained from 48 PAAD patients over three years, inhibitors, and short hairpin RNA (shRNA). Furthermore, single-cell sequencing data from the GEO database were analyzed to explore the crosstalk between PCCs and macrophages. To overcome GR, inhibitors targeting the macrophage-CCL5-Sp1-AREG feedback loop were evaluated in cell lines, PDOs, and orthotopic mouse models of pancreatic carcinoma. RESULTS: The macrophage-CCL5-Sp1-AREG feedback loop between macrophages and PCCs is responsible for GR. Macrophage-derived CCL5 activates the CCR5/AKT/Sp1/CD44 axis to confer stemness and chemoresistance to PCCs. PCC-derived AREG promotes CCL5 secretion in macrophages through the Hippo-YAP pathway. By targeting the feedback loop, mithramycin improves the outcome of gemcitabine treatment in PAAD. The results from the PDO model were corroborated with cell lines, mouse models, and clinical data. CONCLUSIONS: Our study highlights that the PDO model is a superior choice for preclinical research and precision medicine. The macrophage-CCL5-Sp1-AREG feedback loop confers stemness to PCCs to facilitate gemcitabine resistance by activating the CCR5/AKT/SP1/CD44 pathway. The combination of gemcitabine and mithramycin shows potential as a therapeutic strategy for treating PAAD in cell lines, PDOs, and mouse models.

Combinatorial biosynthesis yields novel hybrid argimycin P alkaloids with diverse scaffolds in Streptomyces argillaceus.

Coelimycin P1 and argimycins P are two types of polyketide alkaloids produced by Streptomyces coelicolor and Streptomyces argillaceus, respectively. Their biosynthesis pathways share some early steps that render very similar aminated polyketide chains, diverging the pathways afterwards. By expressing the putative isomerase cpkE and/or the putative epoxidase/dehydrogenase cpkD from the coelimycin P1 gene cluster into S. argillaceus wild type and in argimycin mutant strains, five novel hybrid argimycins were generated. Chemical characterization of those compounds revealed that four of them show unprecedented scaffolds (quinolizidine and pyranopyridine) never found before in the argimycin family of compounds. One of these compounds (argimycin DM104) shows improved antibiotic activity. Noticeable, biosynthesis of these quinolizidine argimycins results from a hybrid pathway created by combining enzymes from two different pathways, which utilizes an aminated polyketide chain as precursor instead of lysine as it occurs for other quinolizidines.

Thermodynamics of the Interaction between the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus-2 and the Receptor of Human Angiotensin-Converting Enzyme 2. Effects of Possible Ligands.

Since the end of 2019, the coronavirus SARS-CoV-2 has caused more than 1000000 deaths all over the world and still lacks a medical treatment despite the attention of the whole scientific community. Human angiotensin-converting enzyme 2 (ACE2) was recently recognized as the transmembrane protein that serves as the point of entry of SARS-CoV-2 into cells, thus constituting the first biomolecular event leading to COVID-19 disease. Here, by means of a state-of-the-art computational approach, we propose a rational evaluation of the molecular mechanisms behind the formation of the protein complex. Moreover, the free energy of binding between ACE2 and the active receptor binding domain of the SARS-CoV-2 spike protein is evaluated quantitatively, providing for the first time the thermodynamics of virus-receptor recognition. Furthermore, the action of different ACE2 ligands is also examined in particular in their capacity to disrupt SARS-CoV-2 recognition, also providing via a free energy profile the quantification of the ligand-induced decreased affinity. These results improve our knowledge on molecular grounds of the SARS-CoV-2 infection and allow us to suggest rationales that could be useful for the subsequent wise molecular design for the treatment of COVID-19 cases.

Structures of mithramycin analogues bound to DNA and implications for targeting transcription factor FLI1.

Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.

PLC-beta 1 regulates the expression of miR-210 during mithramycin-mediated erythroid differentiation in K562 cells.

PLC-beta 1 (PLCß1) inhibits in human K562 cells erythroid differentiation induced by mithramycin (MTH) by targeting miR-210 expression. Inhibition of miR-210 affects the erythroid differentiation pathway and it occurs to a greater extent in MTH-treated cells. Overexpression of PLCß1 suppresses the differentiation of K562 elicited by MTH as demonstrated by the absence of γ-globin expression. Inhibition of PLCß1 expression is capable to promote the differentiation process leading to a recovery of γ-globin gene even in the absence of MTH. Our experimental evidences suggest that PLCß1 signaling regulates erythropoiesis through miR-210. Indeed overexpression of PLCß1 leads to a decrease of miR-210 expression after MTH treatment. Moreover miR-210 is up-regulated when PLCß1 expression is down-regulated. When we silenced PKCα by RNAi technique, we found a decrease in miR-210 and γ-globin expression levels, which led to a severe slowdown of cell differentiation in K562 cells and these effects were the same encountered in cells overexpressing PLCß1. Therefore we suggest a novel role for PLCß1 in regulating miR-210 and our data hint at the fact that, in human K562 erythroleukemia cells, the modulation of PLCß1 expression is able to exert an impairment of normal erythropoiesis as assessed by γ-globin expression.

Apoptotic effect of methanol extract of Picrasma quassioides by regulating specificity protein 1 in human cervical cancer cells.

In the present study, we examined the effects of methanol extracts of Picrasma quassioides (MEPQ) on apoptosis in human cervical cancer cells. The results showed that MEPQ decreased the viability and induced caspase-dependent apoptosis in HEp-2 cells. MEPQ decreased specificity protein 1 (Sp1) in HEp-2 cells, whereas Sp1 mRNA was not changed. We found that MEPQ reduced Sp1 protein through proteasome-dependent protein degradation, but not the inhibition of protein synthesis. Also, MEPQ increased the expressions of Bad and truncated Bid (t-Bid) but did not alter other Bcl-2 family members. The knock-down of Sp1 by both Sp1 interfering RNA and Mithramycin A, Sp1 specific inhibitor clearly increased Bad and t-Bid expression to decrease cell viability and induce apoptosis. In addition, MEPQ inhibited cell viability and induced apoptotic cell death through the modulation of Sp1 in KB cells. These results suggest that MEPQ may be a potential anticancer agent for human cervical cancer.

Aureolic acids from a marine-derived Streptomyces sp. WBF16.

A marine-derived actinomycete (Streptomyces sp. WBF16) exhibiting antitumor activities was investigated. The strain was identified using morphological, biochemical and genetic techniques. 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces coelicolor A3 (2). Furthermore, a new aureolic acid (Chromomycin B, 1), along with Chromomycin A(2) (2) and Chromomycin A(3) (3) were isolated from its secondary metabolites. Their structures were determined by chemical and spectroscopic methods including 1D, 2D NMR and HRMS. Compounds 1-3 showed strong cytotoxicity against SGC7901, HepG2, A549, HCT116 and COC1 and HUVEC.

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic.

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu(2+). CHR forms a high affinity 2:1 (CHR:Cu(2+)) complex with dissociation constant of 0.08 × 10(-10) M(2) at 25°C, pH 8.0. The affinity of CHR for Cu(2+) is higher than those for Mg(2+) and Zn(2+) reported earlier from our laboratory. CHR binds preferentially to Cu(2+) in presence of equimolar amount of Zn(2+). Complex formation between CHR and Cu(2+) is an entropy driven endothermic process. Difference between calorimetric and van't Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)(2):Cu(2+)] complex assumes a structure different from either of the Mg(2+) and Zn(2+) complex reported earlier. Both [(CHR)(2):Mg(2+)] and [(CHR)(2):Zn(2+)] complexes are known to bind DNA. In contrast, [(CHR)(2):Cu(2+)] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5'-CCGGCGCCGG-3'). In order to interact with double helical DNA, the (antibiotic)(2) : metal (Mg(2+) and Zn(2+)) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu(2+) complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg(2+) and Zn(2+). The results also indicate that CHR has a potential for chelation therapy in Cu(2+) accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.

The chromomycin CmmA acetyltransferase: a membrane-bound enzyme as a tool for increasing structural diversity of the antitumour mithramycin.

Mithramycin and chromomycin A(3) are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.

DNA binding characteristics of mithramycin and chromomycin analogues obtained by combinatorial biosynthesis.

The antitumor antibiotics mithramycin A and chromomycin A(3) bind reversibly to the minor groove of G/C-rich regions in DNA in the presence of dications such as Mg(2+), and their antiproliferative activity has been associated with their ability to block the binding of certain transcription factors to gene promoters. Despite their biological activity, their use as anticancer agents is limited by severe side effects. Therefore, in our pursuit of new structurally related molecules showing both lower toxicity and higher biological activity, we have examined the binding to DNA of six analogues that we have obtained by combinatorial biosynthetic procedures in the producing organisms. All these molecules bear a variety of changes in the side chain attached to C-3 of the chromophore. The spectroscopic characterization of their binding to DNA followed by the evaluation of binding parameters and associated thermodynamics revealed differences in their binding affinity. DNA binding was entropically driven, dominated by the hydrophobic transfer of every compound from solution into the minor groove of DNA. Among the analogues, mithramycin SDK and chromomycin SDK possessed the higher DNA binding affinities.

Glucose enhances protein tyrosine phosphatase 1B gene transcription in hepatocytes.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin receptor signal transduction pathway. We investigated the effects of glucose on PTP1B transcription in the human hepatocyte cell line Huh7. Using a reporter gene assay, we found that D-glucose dose-dependently enhanced the PTP1B promoter activity. Real-time PCR demonstrated that D-glucose also increased PTP1B mRNA expression. Protein kinase C (PKC) inhibitors partially but significantly inhibited the transactivation by D-glucose. Mithramycin, a Sp1 inhibitor, completely abrogated this transactivation. The deletion of three possible Sp1 sites in the promoter region of PTP1B significantly reduced the basal promoter activity and transactivation by D-glucose. Sp1 activation by PKC is one of the key mechanisms in the regulation of several gene expressions. Our data suggested that glucose enhanced PTP1B transcription through Sp1 activation by PKC. Increased hepatic PTP1B expression may partly explain glucose toxicity in diabetes.

Novel GC-rich DNA-binding compound produced by a genetically engineered mutant of the mithramycin producer Streptomyces argillaceus exhibits improved transcriptional repressor activity: implications for cancer therapy.

The aureolic acid antibiotic mithramycin (MTM) binds selectively to GC-rich DNA sequences and blocks preferentially binding of proteins, like Sp1 transcription factors, to GC-rich elements in gene promoters. Genetic approaches can be applied to alter the MTM biosynthetic pathway in the producing microorganism and obtain new products with improved pharmacological properties. Here, we report on a new analog, MTM SDK, obtained by targeted gene inactivation of the ketoreductase MtmW catalyzing the last step in MTM biosynthesis. SDK exhibited greater activity as transcriptional inhibitor compared to MTM. SDK was a potent inhibitor of Sp1-dependent reporter activity and interfered minimally with reporters of other transcription factors, indicating that it retained a high degree of selectivity toward GC-rich DNA-binding transcription factors. RT-PCR and microarray analysis showed that SDK repressed transcription of multiple genes implicated in critical aspects of cancer development and progression, including cell cycle, apoptosis, migration, invasion and angiogenesis, consistent with the pleiotropic role of Sp1 family transcription factors. SDK inhibited proliferation and was a potent inducer of apoptosis in ovarian cancer cells while it had minimal effects on viability of normal cells. The new MTM derivative SDK could be an effective agent for treatment of cancer and other diseases with abnormal expression or activity of GC-rich DNA-binding transcription factors.

Mithramycin forms a stable dimeric complex by chelating with Fe(II): DNA-interacting characteristics, cellular permeation and cytotoxicity.

Mith (mithramycin) forms a 2:1 stoichiometry drug-metal complex through the chelation with Fe(II) ion as studied using circular dichroism spectroscopy. The binding affinity between Mith and Fe(II) is much greater than other divalent metal ions, including Mg(II), Zn(II), Co(II), Ni(II) and Mn(II). The [(Mith)2-Fe(II)] complex binds to DNA and induces a conformational change of DNA. Kinetic analysis of surface plasmon resonance studies revealed that the [(Mith)2-Fe(II)] complex binds to DNA duplex with higher affinity compared with the [(Mith)2-Mg(II)] complex. A molecular model of the Mith-DNA-Metal(II) complex is presented. DNA-break assay showed that the [(Mith)2-Fe(II)] complex was capable of promoting the one-strand cleavage of plasmid DNA in the presence of hydrogen peroxide. Intracellular Fe(II) assays and fluorescence microscopy studies using K562 indicated that this dimer complex maintains its structural integrity and permeates into the inside of K562 cells, respectively. The [(Mith)2-Fe(II)] complex exhibited higher cytotoxicity than the drug alone in some cancer cell lines, probably related to its higher DNA-binding and cleavage activity. Evidences obtained in this study suggest that the biological effects caused by the [(Mith)2-Fe(II)] complex may be further explored in the future.

Crystallization and X-ray diffraction properties of Baeyer-Villiger monooxygenase MtmOIV from the mithramycin biosynthetic pathway in Streptomyces argillaceus.

The Baeyer-Villiger monooxygenase MtmOIV from Streptomyces argillaceus is a 56 kDa FAD-dependent and NADPH-dependent enzyme that is responsible for the key frame-modifying step in the biosynthesis of the natural product mithramycin. Crystals of MtmOIV were flash-cooled and diffracted to 2.69 A resolution using synchrotron radiation on beamline SER-CAT 22-ID at the Advanced Photon Source. Crystals of MtmOIV are monoclinic and light-scattering data reveal that the enzyme forms dimers in solution. The rotation function suggests the presence of two dimers in the asymmetric unit. L-Selenomethionine-incorporated MtmOIV has been obtained. Structural solution combining molecular-replacement phases and anomalous phases from selenium is in progress.

N-terminal tail domains of core histones in nucleosome block the access of anticancer drugs, mithramycin and daunomycin, to the nucleosomal DNA.

Mithramycin (MTR) and daunomycin are two anticancer drugs that bind reversibly to double stranded DNA with (G.C) base specificity leading to inhibition of transcription. MTR is a groove binder of DNA in the presence of a divalent cation such as Mg(2+), while daunomycin intercalates in the double stranded DNA structure. In order to understand the mechanism of action of the two types of transcription inhibitor, namely, groove binder and intercalator, we have studied the effect of N-terminal tail domains in histone proteins of the nucleosome upon the association of both MTR and daunomycin with the nucleosome core particle, because the tails modulate the accessibility to nucleosome during gene expression. Using a combination of spectroscopic, thermodynamic and biochemical studies, we have shown that N-terminal intact and chopped core particles interact differently with the same ligand and the N-terminal tail domains of core histones in the nucleosome stand in the way of free access of these ligands to the nucleosomal DNA. Tryptic removal of N-terminal tail domains of core histones enhances the binding potential and accessibility of both MTR and daunomycin to nucleosomal DNA. They disassemble the nucleosome structure leading to a release of DNA, N-terminal chopped nucleosomes being more susceptible for disruption compared to N-terminal intact nucleosomes. The extent of these effects is more pronounced in case of the intercalator daunomycin. Thus, N-terminal tail domains protect the eukaryotic genome from external agents, such as anticancer drugs, and the degree of protection is dependent upon the mode of binding to DNA.

MtmMII-mediated C-methylation during biosynthesis of the antitumor drug mithramycin is essential for biological activity and DNA-drug interaction.

The antitumor drug mithramycin consists of a polyketide chromophore glycosylated with a trisaccharide and a disaccharide. Two post-polyketide methylations take place during mithramycin biosynthesis. One of these methylations has been shown to be very relevant for biological activity, that is the introduction of a methyl group at aromatic C-7. We have purified to 282- fold the MtmMII methyltransferase involved in this reaction. The protein is a monomer, and results from kinetic studies were consistent with a model for the enzyme acting via a compulsory order mechanism. The enzyme showed high substrate specificity and was unable to operate on structurally closely related molecules. Structural predictions suggest that the molecule is integrated by two domains, an essentially all alpha-amino domain and an alpha/beta-carboxyl domain displaying a variation of a Rossmann-fold containing the cofactor binding site. Although 7-demethyl-mithramycin did not show any biological activity, it was able to reach the nucleus of eukaryotic cells, with subsequent binding to DNA. Mithramycin and 7-demethylmithramycin were able to form similar complexes with Mg(2+), although their respective DNA binding isotherms were very different. The dinucleotide binding model fit well the isotherms recorded for both compounds, predicting that the C-7 methyl group was essential for high affinity binding to specific GC and CG sequences. Considering previous structural studies, we propose that this effect is performed by positioning the group in the floor of the minor groove, allowing the interaction with the third sugar moiety of the trisaccharide, d-mycarose, which is involved in sequence selectivity.

Association of anticancer drug mithramycin with H1-depleted chromatin: a comparison with native chromatin.

Depletion of histone H1 after covalent modification from chromatin is a key step in eukaryotic transcription initiation. We have studied the effect of depletion of linker histone H1 upon the association of transcription inhibitor, (mithramycin)(2):Mg(2+) complex, with chromatin. We have compared the binding characteristics of the above complex with native, H1-depleted chromatin and naked DNA. Binding site size (number of bases per ligand molecule) of the above complex to the chromosomal DNA increases upon removal of histone H1. It implies an increase in the accessibility of the ligand for the linker DNA. Spectroscopic data, and associated enthalpy and entropy values of the interaction of the complex with H1-depleted chromatin are similar to naked DNA rather than native chromatin. These results suggest that under in vivo conditions, depletion of histone H1 from transcriptionally inert native chromatin during gene activation would lead to an enhanced accessibility of linker DNA to the small ligands with the potential to inhibit transcription.

Evaluation of complexation of metal-mediated DNA-binding drugs to oligonucleotides via electrospray ionization mass spectrometry.

The interactions of self-complementary oligonucleotides with a group of metal-mediated DNA-binding drugs, including chromomycin A(3), mithramycin and the novel compound UK-1, were examined via electrospray ionization quadrupole ion trap mass spectrometry. Both chromomycin and mithramycin were shown to bind preferentially to GC-rich oligonucleotide duplexes in a 2:1 drug:metal ratio, while UK-1 was shown to bind in a 1:1 drug:metal stoichiometric ratio without a strong sequence preference. These trends were observed in the presence of Co(2+), Ni(2+) and Zn(2+), with the exception that chromomycin-Zn(2+) complexes were not readily observed. The binding stoichiometries as well as the sequence specificities are in agreement with literature reports for solution studies. Binding selectivities and stabilities of the complexes were also probed using electrospray ionization mass spectrometry. Both of the GC-rich oligomers 5'-GCGCGC-3' and 5'-GCGCATGCGC-3' exhibited a binding preference for chromomycin over mithramycin in the presence of Co(2+) and Ni(2+). Energy-variable collisionally activated dissociation of the complexes was employed to determine the stabilities of the complexes. The relative metal-dependent binding energies were Ni(2+) > Zn(2+) > Co(2+) for UK-1-oligomer complexes and Ni(2+) > Co(2+) for both mithramycin and chromomycin complexes.

Interactions of chromomycin A3 and mithramycin with the sequence d(TAGCTAGCTA)2.

Anti-cancer antibiotics, chromomycin A3 (CHR) and mithramycin (MTR) inhibit DNA directed RNA synthesis in vivo by binding reversibly to template DNA in the minor groove with GC base specificity, in the presence of divalent cations like Mg2+. Under physiological conditions, (drug)2Mg2+ complexes formed by the antibiotics are the potential DNA binding ligands. Structures of CHR and MTR differ in their saccharide residues. Scrutiny of the DNA binding properties reveal significant differences in their sequence selectivity, orientation and stoichiometry of binding. Here, we have analyzed binding and thermodynamic parameters for the interaction of the antibiotics with a model oligonucleotide sequence, d(TAGCTAGCTA)2 to understand the role of sugars. The oligomer contains two potential binding sites (GpC) for the ligands. The study illustrates that the drugs bind differently to the sequence. (MTR)2Mg2+ binds to both sites whereas (CHR)2Mg2+ binds to a single site. UV melting profiles for the decanucleotide saturated with the ligands show that MTR bound oligomer is highly stabilized and melts symmetrically. In contrast, with CHR, loss of symmetry in the oligomer following its association with a single (CHR)2Mg2+ complex molecule leads to a biphasic melting curve. Results have been interpreted in the light of saccharide dependent differences in ligand flexibility between the two antibiotics.