We have investigated the binding ability to DNA of compounds belonging to the 2-azaanthraquinone-type structure and have examined the effect on the activity of DNA gyrase as well as on mammalian topoisomerases in vitro. Using different biophysical techniques it was found that one of these ligands, 9-((2-dimethylamino)ethyl)amino)-6-hydroxy-7-methoxy-5, 10-dihydroxybenzo[g]isoquinoline-5,10-dione (TPL-I), is an intercalating DNA binding agent, whereas the parent compound tolypocladin (TPL) and a derivative (TPL-II) showed almost no similar affinity to DNA. CD measurements demonstrated a significant and selective binding tendency of TPL-I to alternating purine/pyrimidine sequences with some preference for poly(dA-dT). poly(dA-dT). Tm values were increased of the ligand complex with the alternating AT-containing duplex polymer. The binding to various DNAs was characterized by CD and visible absorption spectral changes. From the latter, different binding constants of 6.2 x 10(5) and 1.5 x 10(5) M-1 were obtained for poly(dA-dT).poly(dA-dT) and poly(dA). poly(dT), respectively. Sedimentation measurements with supercoiled pBR322 plasmid DNA clearly indicated an intercalative binding mechanism associated with an unwinding angle of about 18 degrees. These results suggest that the intercalative binding of TPL-I is promoted by the 2-(dimethylamino)ethylamino group substituted on carbon 9 of the anthraquinone system. The cytotoxic compound TPL-I, but not TPL or TPL-II, effectively inhibited the DNA supercoiling reaction of DNA gyrase and the activity of mammalian topoisomerases I and II as measured by the relaxation assay. TPL-I affects the cleavage reaction of topoisomerases on a single site located in alternating purine-pyrimidine sequence regions. The inhibitory potency of TPL-I can be ascribed to a blocking of cleavage sites on the DNA substrate, which correlates with the sequence preference of the ligand.

Non-intercalating DNA minor-groove binders may effectively inhibit the supercoiling activity of gyrases by influencing the enzyme recognition and cleavage site on DNA. For gyrase from Streptomyces noursei a wide range of inhibitory potency for different classes of ligands is observed. This can be explained by a number of structural and binding factors of the ligands competing with the gyrase on the target site of DNA, the mechanism of which is different from the classical gyrase inhibitors.

Supercoiling of bacterial DNA is regulated by topoisomerases and influences most of the metabolic processes involving DNA. The present review is devoted to a brief outline of the supercoiled state of DNA in bacteria and to all microbial topoisomerases hitherto described. Recent studies on topoisomerases of archaebacteria led to the discovery of a so-called reverse gyrase, the properties of which are also discussed. Special emphasis is given to a selective treatment of the effects of those antibiotics which act as gyrase inhibitors.