Biological characterization of cyclothialidine, a new DNA gyrase inhibitor

Abstract

Cyclothialidine is a new DNA gyrase inhibitor isolated from Streptomycesfilipinensis NR0484. Structurally, it belongs to a new class of natural products containing a unique 12-membered lactone ring that is partly integrated into a pentapeptide chain When ATP is present, DNA gyrase catalyzes the introduction of negative supercoils into DNA; in the absence of ATP, it removes negative supercoils, albeit inefficiently. The mammalian topoisomerase, however, can only relax supercoiled DNA. DNA gyrase has proved to be essential for bacterial growth. These factors provide the basis for DNA gyrase inhibitors to exert their antibacterial activities with selective toxicities. DNA gyrase is known to be the target of two classes of antibiotics. One comprises the synthetic quinolones, typified by nalidixic acid and oxolinic acid, which target the A subunit of the enzyme and probably act by interfering with the DNA rejoining step of the gyrasemediated DNA strand-passing reaction (14,27,30). The other class consists of the natural coumarin-type compounds, such as novobiocin and coumermycin Al, that inhibit gyrase probably by competing with ATP for binding to the B subunit of the enzyme (7,8,15,29). Although synthetic quinolones are commercially successful antibacterial agents, there is still room for improvement in their toxicities (33) and activities against resistant strains (6, 26). The natural novobiocin derivatives are also toxic. These situations led us to initiate microbial broth screening for DNA gyrase inhibitors with the aim of identifying new lead compounds. From the screening of natural products for DNA gyrase inhibitors, we discovered a novel DNA gyrase inhibitor, cyclothialidine (Ro 09-1437) (Fig. 1) (1). It was isolated from the fermentation products of Streptomyces * Corresponding author. filipinensis NR0484. Cyclothialidine contains a unique 12-member lactone ring which is partly integrated into a pentapeptide chain. In this report, we describe the biological activity of cyclothialidine as a lead structure for a new class of antibacterial agents. MATERIALS AND METHODSChemicals. Cyclothialidine (molecular weight, 641; >98% pure) was isolated from the culture broth of S. filipinensis NR0484. Nalidixic acid, oxolinic acid, novobiocin, coumermycin Al, and adriamycin were purchased from Sigma Chemical Company. Ofloxacin (Daiichi), norfloxacin (Kyorin), ciprofloxacin (Bayer), and enoxacin (Dainippon) were >98% pure by high-pressure liquid chromatographic analysis. All other chemicals were at least analytical grade. DNA

Mechanism of inhibition of DNA gyrase by cyclothialidine, a novel DNA gyrase inhibitor

“…Therefore, cyclothialidine is a B-subunit inhibitor, similar to novobiocin and coumermycin Al. The efficacies of cyclothialidine, novobiocin, and coumermycin Al as inhibitors of the ATPase activity closely correlated with their potencies in the DNA-supercoiling assay (17), implying that the inhibition of DNA supercoiling, which includes the whole DNA gyrase reaction process, resulted from the inhibition of the ATPase by the B-subunit inhibitors.…”

“…However, two important differences were observed between cyclothialidine and the coumarin-type antibiotics. First, cyclothialidine was much more selective than novobiocin and coumermycin Al for the DNA gyrase (17). Cyclothialidine inhibited E. coli DNA gyrase potently, with an IC50 of 0.03 ,ug/ml; furthermore, cyclothialidine showed high selectivity toward DNA gyrase in comparison with its inhibition of DNA topoisomerases I and II of calf thymus, with IC50s of 1,700 and 1,900 ,ug/ml, respectively.…”

“…DNA replication directed from the oriC origin of E. coli was inhibited by cyclothialidine (17). However, the activity of cyclothialidine in the DNA replication assay was weaker than its inhibitory activity against DNA gyrase (IC50 replication/ IC50 gyrase = 11.7).…”

“…4) (10). Cyclothialidine potently inhibited DNA gyrases from several bacterial species, including E. coli and Staphylococcus aureus, with a high degree of selectivity (17). In the study described here we investigated the mechanism by which cyclothialidine inhibits DNA gyrase and compared its mode of action with those of known DNA gyrase inhibitors, i.e., quinolones and coumarin antibiotics.…”

Total Synthesis of Cyclothialidine

“…The metaposition of the two OH groups on the phenyl ring differs from the typical ortholpara-substitution pattern of the orsellinic acid type macrolides produced by the polyketide biosynthetic pathway [4]. It is noteworthy that 1 was discovered in a DNA-supercoiling-assay-guided screening program aiming at new DNA gyrase inhibitors as potential antibacterials [5]. Its mode of action was shown to be inhibition of the ATPase activity conferred by the B subunit of DNA gyrase [6], the target of the coumarin antibiotics novobiocin and coumermycin [7].…”