Laurence Hurley

PMID: 2979741;Abstract:

The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds. © 1988 American Chemical Society.

PMID: 7853333;Abstract:

The quinobenzoxazine compounds A-62176 and A-85226 belong to a novel class of antineoplastic agents that are catalytic inhibitors of topoisomerase II and also structural analogs of the antibacterial DNA gyrase inhibitor Norfloxacin. In vitro studies have shown that their antineoplastic activity is dependent upon the presence of divalent metal ions such as Mg²⁺ and Mn²⁺, although the precise role of these ions in the mechanism of action is unknown. In this study we have investigated the structures of the binary complex between the quinobenzoxazines and Mg²⁺ and the ternary complex between quinobenzoxazine-Mg²⁺ and DNA. The stoichiometry of the binary and ternary complexes and the biophysical studies suggest that a 2:2 drug:Mg²⁺ complex forms a "heterodimer complex" with respect to DNA in which one drug molecule is intercalated into DNA and the second drug molecule is externally bound, held to the first molecule by two Mg²⁺ bridges, which themselves are chelated to phosphates on DNA. There is a cooperativity in binding of the quinobenzoxazines to DNA, and a 4:4 drug:Mg²⁺ complex is proposed in which the two externally bound molecules from two different 2:2 dimers interact via π-π interactions. The externally bound quinobenzoxazine molecules can be replaced by the quinolone antibacterial compound Norfloxacin to form mixed-structure dimers on DNA. Based upon the proposed model for the 2:2 quinobenzoxazine:Mg²⁺ complex on DNA, a parallel model for the antibacterial quinolone-Mg²⁺-DNA gyrase complex is proposed that relies upon the ATP-fueled unwinding of DNA by gyrase downstream of the cleavable complex site. These models, which have analogies to leucine zippers, represent a new paradigm for the structure of drug-DNA complexes. In addition, these models have important implications for the design of new gyrase and topoisomerase II inhibitors, in that optimization for structure-activity relationships should be carried out on two different quinolone molecules rather than a single molecule. © 1995 American Chemical Society.

PMID: 15599;Abstract:

Anthramycin, tomaymycin and sibiromycin are pyrrolo(1,4)benzodiazepine antitumor antibiotics. These compounds react with DNA and other guanine-containing polydeoxynucleotides to form covalently bound antibiotic · polydeoxynucleotide complexes. Experiments utilizing radiolabelled antibiotics have led to the following conclusions: 1. 1. Sibiromycin reacts much faster than either anthramycin or tomaymycin with DNA. 2. 2. At saturation binding the final antibiotic to base ratios for sibiromycin, anthramycin and tomaymycin are 1 : 8.8, 1 : 12.9, and 1 : 18.2 respectively. 3. 3. No reaction with RNA or protein occurs with the pyrrolo(1,4)benzodiazepine antibiotics. 4. 4. Sibiromycin effectively competes for the same DNA binding sites as anthramycin and tomaymycin; however, there is only partial overlap for the same binding sites between anthramycin and tomaymycin. 5. 5. Whereas all three pyrrolo(1,4)benzodiazepine antibiotic · DNA complexes are relatively stable to alkaline conditions, their stability under acidic conditions increases in the order tomaymycin, anthramycin and sibiromycin. 6. 6. No loss of non-exchangeable hydrogens in either the pyrrol ring or the side chains of these antibiotics occurs upon formation of their complexes with DNA. 7. 7. Unchanged antibiotic has been demonstrated to be released upon acid treatment of the anthramycin · DNA and tomaymycin · DNA complexes. 8. 8. A Schiffbase linkage between the antibiotics and DNA has been eliminated. The comparative reactivity of the three antibiotics towards DNA and the stability of their DNA complexes is discussed in relation to their structures. A working hypothesis for the formation of the antibiotic · DNA covalent complexes is proposed based upon the available information. © 1977.

PMID: 455297;Abstract:

Anthramycin-DNA adducts, produced in vitro by reaction of anthramycin with calf thymus DNA, have been shown to be stable only as long as the secondary structure of DNA is maintained. Denaturation either by heat or enzymatic degradation of the DNA adduct, with DNase I and snake venom phosphodiesterase, leads to the release of significant amounts of the bound drug as unchanged anthramycin. These observations led us to suspect that the DNA adduct might be a suitable prodrug system for anthramycin, which might be more efficacious and less toxic than the administration of the free drug. In order to test this hypothesis, the ability of the adduct versus free drug to inhibit DNA synthesis and induce unshceduled DNA synthesis in a human cell line was evaluated. The results demonstrated that the anthramycin-DNA adduct was less potent than the free drug in these systems in both respects. The anthramycin and anthramycin-DNA conjugate were compared in mice for lethality, tisue levels, alteration of hexobarbital sleeping times, and efficacy against a mouse ascites tumor model. These results showed that the DNA adduct was three times more lethal and produced similar increases in sleeping times at equitoxic doses. The increase in lethality of the anthramycin-DNA adduct could be explained by elevated and more prolonged blood and tissue levels following administration of the DNA conjugate as compared to free anthramycin. When tested for efficacy against a mouse ascites tumor line, the anthramycin-DNA adduct was found to be less efficacious than the free drug.