The next bleomycin news ought to be about the new generation of synthetic derivatives. "There's reason to expect improvements," says Hecht, because "there's no reason to think the activity has been optimized." The natural bleomycins often are toxic, and that toxicity has been reduced only unsystematically so far by modifying the DNA-binding portion of the molecule. The natural bleomycins can be broken down by enzymes in target tissues, and thus enzyme-resistant bleomycins eventually may be fashioned. The bleo-
mycins also tend to destroy themselves and, again, that futile tendency begs for chemical modification. Moreover, bleomycin shows some specificity towards particular DNA sequences, a specificity that varies from one derivative to the next. This opens the possibility of redesigning the molecule so that it goes after only certain DNAs, such as those in specific tumor-promoting genes. "There are lots of reasons to want to change bleomycin," Hecht says, "but it's so complex, you don't want to make just random changes." D
Researchers study how drugs bind to DNA Taking some hints from the structure and activity of bleomycin, chemists at California Institute of Technology, Pasadena, have designed a molecule that binds to and cleaves doublehelical DNA. The researchers have used the compound to study how certain drugs bind to DNA. Bleomycin binds to and cleaves DNA in a sequence-specific fashion; that is, it recognizes either a guanine-cytosine or a guanine-thymine dinucleotide and cuts the DNA double helix symmetrically adjacent to the guanine. The reaction depends on the presence of ferrous ion and molecular oxygen. In work supported by the National Institutes of Health, Caltech chemistry professor Peter B. Dervan and graduate students Robert P. Hertzberg and Michael W. Van Dyke built what Dervan calls a primitive model of bleomycin. "Without understanding how bleomycin binds to DNA and without understanding the cleavage reaction itself," Dervan says, "we followed chemical intuition in designing the molecule." Essentially, the researchers tied together two molecules, each with a property gleaned from bleomycin as important in that molecule's activity. To bind DNA, they chose methidium bromide, a molecule with a planar portion that intercalates DNA. To complex ferrous ion, they chose ethylenediaminetetraacetic acid (EDTA), a well-known iron chelator. They linked the two together with a short hydrocarbon chain. The result is methidiumpropyl-EDTA or MPE. In the presence of ferrous ion and oxygen, 10"6M MPE cleaves plasmid DNA. If dithiothreitol (DTT) also is present, 10~8M MPE cleaves DNA about as efficiently as bleomycin. Dervan speculates that the DTT acts as a reducing agent that regenerates
DNA intercalator
Tether
Iron chelator
(Methidiumpropyl-EDTA)iron(ll)
ferrous ion from ferric ion (which is inactive) to provide a continuous source of active metal ion. According to Dervan, because the portion of MPE that binds to DNA—methidium—does so with no preference for any particular DNA base, MPE cleaves DNA nonsequence specifically. In other words, MPE cuts DNA anywhere along its length. In its activity, therefore, MPE mimics not bleomycin, but a class of enzymes called DNases. Because MPE mimics the action of DNases, the researchers realized that they could use it to study how compounds bind to DNA. Previous workers have studied how proteins bind to DNA by generating what are called DNA cleavage-inhibition patterns, or footprints, on Maxam-Gilbert DNA sequencing gels. The idea is to allow a protein to bind to radioactively labeled DNA and then expose the DNA-protein complex to DNase long enough for the enzyme to make statistically only one cut per strand of DNA in solution. Any posi-
tion on the DNA molecules can be cut except those protected by the bound protein. On a sequencing gel, therefore, spots corresponding to each possible DNA fragment length will appear, except for those fragments that would correspond to cuts at the protected region. The blanks on the gel are the binding protein's footprint. That works fine for some proteins that bind to DNA, but until recently, it had not been done for smaller molecules. Numerous compounds, many of them drugs having antibiotic, antiviral, or antitumor activity, bind to DNA but do not cleave it. Their pharmacological activity results, presumably, from their ability to interfere with some aspect of DNA's function. Equilibrium studies with drugs and synthetic DNA have provided information on the types of DNA regions—for example, a region rich in a particular base—that such molecules bind to preferentially. However, pinpointing the specific binding sites has proved difficult. Dervan's group initially studied four drugs: daunomycin, actinomycin D, distamycin A, and netropsin. Each binds to DNA via an equilibrium reaction. The researchers use a restriction fragment of well-characterized, heterogeneous, plasmid DNA, and label the end of one side of the double helix with radioactive phosphorus. Then, the DNA is exposed first to one of the drugs under study and then to MPE only long enough to allow limited digestion of the DNA. The resulting DNA fragments are sequenced on a Maxam-Gilbert gel and a photographic plate is made of the radioactivity on the gel. That plate is scanned by a microdensitometer. The researchers find that specific protected sites show up clearly on the sequences for distamycin A, netropsin, and actinomycin D. Daunomycin does not produce a pattern. Distamycin A and netropsin, which are similar molecules with similar modes of binding, produce identical patterns. They bind to regions rich in adenine and thymine and protect regions of at least four base pairs. Significantly, Dervan says, the researchers are able to observe new sites becoming protected as the concentration of each of the two drugs is increased, indicating a hierarchy of preferred binding sites. Actinomycin D gives a completely different pattern, with regions four to 16 base pairs protected. The regions center on one or more guanine-cytosine base pairs. The research is described in the September issue of the Proceedings Oct. 18, 1982 C&EN
27
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Control of Isothermal Polystyrene Reactors · Kinetics of Styrene Polymerization · Free Radical Polymerization Kinetics with Chain Length Dependent Termination · Molecular Weight Distribution Using Multicomponent Models · CSMP m Simulating Polymerization, Polycondensation, and Polymer Modification Reactions · Modeling and Control of Copolymenzation Reactors · Semibatch Solution Copolymenzation of Styrene with Methyl Acrylate · Programs for Calculating Structural Features of Terpolymers · UV Spectrophotometers as Detectors for Size Exclusion Chromatography · Computerized Quantitative Analysis of Copolymers by IR Spectroscopy · Modeling Equilibrium Swelling of Latex Particles with Monomers · Poly (vinyl Acetate) Emulsion Polymerization Reactors · Automated Ferranti-Shirley Viscometer · Automated Capillary Rheometer Measurement and Analysis · Computer Simulation of Nip Flow in Roll Coating · Finite Element Modeling of Nomsothermal Polymer Flows · Polarization in Thermoplastic Elastomers with Application to Morphology Studies · Automated Thermal Analysis System for Reaction Kinetics · Analysis of Epoxy Curing Reaction · Automated Torsion Pendulum Control and Data Collection/Reduction Using Desktop Computer · ATHAS Polymer Heat Capacity Data Bank · Viscoelastic Master Plots for Vibration Damping Applications · Degradation Kinetics of Poly (vinyl Hahdes) · Prediction of Polymer Solution Properties from Chain Conformations and Interactions Model · Emulsification of Benzene and Styrene in Aqueous Hexadecyltrimethylammonium Bromide— Cetyl Alcohol Mixtures · Computer Methods for Finding Solvent Blend Replacements and for Predicting Water/Cosolvent Evaporation at Any Humidity · ABS Pipe Compound Experiment 469 pages (1982) Clothbound US & Canada $49.95 Export $59.95 LC 82-13735 ISBN 0-8412-0733-X Order from: Distribution Office — 7 American Chemical Society 1155 Sixteenth Street, N.W. Washington, D.C. 20036 or CALL TOLL FREE 800-424-6747 and use your credit card.
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