Mithramycin Research Articles

Solution Structure of Mithramycin Dimers Bound to Partially Overlapping Sites on DNA

Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg 2+-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C)·(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C)·(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A)·(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.

A DNA-Binding Antitumor Antibiotic Binds to Spectrin

Aureolic acid group of antibiotics inhibit transcription by reversible binding to DNA in presence of divalent magnesium. We for the first time report binding of the one of such antitumor antibiotic, mithramycin (MTR), to the major protein component of erythrocyte cytoskeleton, spectrin. A reasonably high apparent dissociation constant was estimated to be 1.5 μM. The binding of mithramycin in the absence of any divalent cation to the large cytoskeletal protein led to quenching in the tryptophan fluorescence of the protein. Stern-Volmer quenching of the tryptophan residues by acrylamide revealed conformational change in the MTR-bound spectrin. This preliminary study might be useful in understanding other possible sites of actions after translocation.

DNA stainability with base-specific fluorochromes: dependence on the DNA topology in situ

The influence of DNA topology on stainability with the externally binding fluorochromes Hoechst 33258 (HO) and mithramycin (MI) was investigated in HeLa nuclei in comparison with the intercalating dye propidium iodide (PI). Changes in DNA topology were induced with a mild DNAse I treatment. Stainability properties of untreated and nuclease-treated nuclei were compared with those of the supercoiled-circular and the relaxed-linear forms of the plasmid pBR322. DNAse-treated nuclei stained with HO showed a higher fluorescence intensity than control samples, independently of the dye concentration, in contrast with the findings obtained with PI. Similar behaviour was observed with the relaxed-linear form of pBR322, compared with the supercoiled-circular molecule. With MI, the stainability of HeLa nuclei did not depend on the DNA topology, whereas the stainability of the plasmid was similar to that of HO. In order to assess whether this discrepancy depended on differences in the availability of DNAse-sensitive sites to the fluorochromes, fluorescence resonance energy transfer (FRET) studies were performed in nuclei stained with HO+PI, or with HO+MI dye pairs. After DNAse I digestion, the relative FRET efficiency between donor (HO) and acceptor molecules (PI or MI) was reduced significantly only when MI was the acceptor. This result may be due to greater stainability of DNAse-sensitive sites with HO than with MI. These findings indicate that DNA stainability with base-specific fluorochromes may be affected by the topology of chromatin regions.

Mithramycin: a very strong metal chelating agent

The interaction of mithramycin (MTR) with Ca 2+, Cd 2+, Tb 3+, Li +, Na + and K + ions has been studied by circular dichroism and absorption spectroscopy. Mithramycin binds strongly to Ca 2+, Cd 2+, Tb 3+ and Gd 3+ forming a 1:4 Ca 2+ : MTR entities with a left-handed screw conformation. The concentration of Ca 2+ present in water currently used being about 10 μM, this leads to the conclusion that, in most of the experiments reported in the literature, about 40 μM mithramycin were actually bound to Ca 2+. Mithramycin also binds to Na +m forming entities with left-handed screw conformation, but not to K + and Li +. None of these cations were able to promote the mithramycin-DNA interaction.

Mithramycin cannot bind to left-handed poly(dG-m 5dC) in the presence of Mg 2+ ion

Mithramycin (MTR) is an antitumor compound that inhibits RNA and DNA polymerase action by forming a non covalent complex with double strand DNA, in the presence of divalent cations. We have shown that in the presence of Mg 2+, MTR binds to right-handed poly(dG-m 5dC) as a dimer in the right-handed screwness conformation but cannot bind to left-handed poly(dG-m 5dC).

Binding of mithramycin to DNA in the presence of second drugs

Comparative DNA equilibrium binding studies with mithramycin (MTR) and ethidium bromide in the presence and in the absence of second drugs were investigated by spectral titrations. Unusual curvatures (in contrast to those due to neighbor exclusion or anticooperativity) are found in the Scatchard plots of MTR-DNA titrations in the presence of netropsin, a minor-groove binder. Parallel studies with ethidium bromide indicate that although the presence of netropsin significantly reduces the binding ability of ethidium, no unusually curved Scatchard plots are obtained. The unusual curvature exhibited by the Scatchard plots of MTR titrations in the presence of netropsin indicates that the binding of netropsin greatly affects the MTR binding to DNA and can be simulated by an explicit incorporation of the second drug-DNA interaction in the binding formalism. Since netropsin is a minor-groove binder, its interference with the binding of MTR is in accord with the notion that MTR also binds at this groove. The observation of negligible effects on the DNA binding ability of MTR in the presence of either a major-groove or a phosphate group binder lends further support to this conclusion. Consistent with its guanine specificity, studies with synthetic polynucleotides suggest that MTR exhibits negligible affinity for poly(dA-dT).poly(dA-dT) or poly(dA).poly(dT).(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent skin ulcers in mice treated with DNA binding antitumor antibiotics.

The DNA-binding agents daunomycin (DAU-NO), mithramycin (MITH), dactinomycin (ACT-D), amsacrine (mAMSA) and esorubicin (ESO) were tested for local vesicant potential in a quantitative intradermal mouse skin model. Only MITH, which adlineates but doses not intercalate DNA, did not produce dose-dependent skin ulcerations in the mouse. The anthracycline antibiotics DAUNO and ESO produced the largest skin ulcers when administered intradermally at clinically relevant doses (adjusted on the basis of comparable body surface areas). Numerous local pharmacologic adjuvants were tested for activity to decrease skin ulceration patterns in mice given one of the DNA intercalators. Inactive local adjuvants included heat, cold, saline, hyaluronidase, glucorticosteroids and isoproternol. Only one adjuvant, topical dimethylsulfoxide (DMSO), was found to reduce DAUNO skin lesions. A single topical DMSO application significantly decreased ulceration size to almost half of control levels. However, it was ineffective for the other intercalating agents. These results show that the DNA intercalators DAUNO, ESO and ACT-D are potent vesicants in a mammalian skin model. These vesicant agents must be administered cautiously to prevent extravasation. No single local adjuvant treatment can be recommended for extravasation of these drugs in the clinic. One significant exception is DAUNO, where topical DMSO may reduce clinical toxicities.

Bone destruction and hypercalcemia in plasma cell myeloma.

M YELOMA is almost always associated with a marked increase in destructive osteolytic bone lesions. Bone destruction in myeloma is responsible for the most distressing clinical features of this disease, including intractable bone pain, fractures occurring either spontaneously or following trivial injury, and hypercalcemia with its attendant symptoms and signs. Eighty percent of patients present with bone pain as a predominant symptom.’ The bone lesions occur in several patterns. Occasionally, patients develop single osteolytic lesions that are associated with solitary plasmacytomas. Some patients have diffuse osteopenia, which mimics the appearance of osteoporosis, and is due to the myeloma cells being spread diffusely throughout the axial skeleton. In most patients there are multiple discrete lytic lesions occurring at the site of deposits or nests of myeloma cells. Bone scans in myeloma show very little abnormality. Isotopic bone scans are performed with labeled bisphosphonates that are taken up at sites of mineral deposition and reflect increased activity of bone forming cells. Myeloma is characterized by discrete lytic lesions with little increase in formation and this is probably the explanation for the difference in appearance between the bone scan in myeloma and the bone scan in breast cancer, where osteoblast activity is frequently increased. Similarly, serum alkaline phosphatase, which also reflects osteoblast activity, is usually not increased in patients with myeloma, unless they have coexistent pathological fractures. Most available evidence suggests that the mechanism of bone destruction in myeloma is primarily osteoclastic. This was first suggested in a study in which morphologic examination was performed on autopsy and biopsy samples of bone from a series of 37 patients with myeloma.’ In this series, active osteoclastic bone resorption was found predominantly in specimens that contained more than 20% marrow myeloma cell infiltration. In specimens of bone from myeloma patients that contained few or no myeloma cells, little osteoclast activity or evidence of active bone resorption was observed. This suggested that the myeloma cells caused the osteoclast activity that led to increased bone resorption. The association between increased osteoclasts on bone resorbing surfaces and areas of heavy myeloma cell infiltration was confirmed in a later study using the technique of quantitative bone histomorphometry on undecalcified transiliac bone biopsies from 118 patients with myeloma.3 Surprisingly, no decrease in mean trabecular bone volume was observed, but abnormalities were observed in bone formation. Bone forming surfaces were increased away from the areas of heavy myeloma cell infiltration but the osteoid seams were reduced in thickness and had a lowered calcification rate. The authors suggested that the activity of individual osteoblasts may be reduced in many patients with myeloma, accounting for these latter findings. Further evidence that the mechanism of bone resorption in myeloma is primarily osteoclastic is provided by data that show drugs that are specific inhibitors of osteoclast activity such as the bisphosphonates, mithramycin, and corticosteroids are usually very effective in lowering the serum calcium in patients with myeloma.4,“16 Not all workers have found increased osteoelastic resorption in myeloma. Schechter et al’ saw little evidence of osteoclastic activity in the patients they studied. MacDonald et al” suggested that murine myeloma cells had the capacity to destroy bone directly in vitro independent of osteoclasts, in a similar manner described earlier for human breast cancer cells.’ Whether this phenomenon has an in vivo significance is not currently known, In some patients with myeloma, osteosclerosis

Rapid nuclear staining method for Saccharomyces cerevisiae.

Mithramycin was used to stain nuclei in mitotically dividing and sporulating yeast.

Reverse fluorescent chromosome banding with chromomycin and DAPI.

Two DNA binding guanine-specific antibiotics, chromomycin A3 (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI.--In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI.--Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The cma-banding pattern appears to be similar to the pattern found by R-banding procedures.

Letter: Mithramycin for hypercalcaemia associated with myeloma and other malignancies.

Letter: Mithramycin for hypercalcaemia associated with myeloma and other malignancies.