Masaru Uyeda

Inhibitory Properties of Antitumor Prostaglandins against Topoisomerases

Prostaglandins (PGs) having antitumor activity such as Δ12,14-PGJ2, Δ12-PGJ2, PGA2 and PGA1 strongly inhibited topoisomerase II (topo II) from human placenta, the potential order of inhibitory activity of the PGs resembling that of the antitumor activity. PGs having no antitumor activity did not inhibit topo II. Δ12,14-PGJ2 to be a potent inhibitor showed inhibitions to some extent against topo I from wheat germ, NIH3T3 and calf thymus gland, and showed no inhibition against the enzymes from Vero, A549, HeLa and COLO 201 cells. Δ12,14-PGJ2 differentially inhibited topo I from different sources. Δ12,14-PGJ2 was a topo inhibitor of the cleavable complex-nonforming type without DNA intercalation.

Inhibition of Topoisomerases by Fatty Acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure-activity relationships and mechanism of action were studied. Saturated fatty acids (C6:0 to C22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C16:1 to C22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.

Inhibition of Hyaluronidases and Chondroitinases by Fatty Acids

The inhibitory effects of various fatty acids on three hyaluronidases (h-ST, h-SH and h-SD) and four chondroitinases (c-ABC, c-B, c-ACI and c-ACII) were examined, and their structure-activity relationships and mechanism of action were studied. The fatty acids used in this experiment showed various inhibitory activities against the enzymes. None of the fatty acids did not inhibit h-ST and h-SH. The saturated fatty acids (C 10:0 to C 22:0) showed very weak or no inhibition against h-SD, c-ABC, c-B, c-ACI and c-ACII but the unsaturated fatty acids (C 14:1 to C 24:1) with one double bond strongly inhibited the enzymes, and the inhibitory potency increased with increase in carbon chain length of the fatty acids. In contrast, the increase in number of double bonds caused a decrease in inhibitory potency against the enzymes. The position of the double bond and the stereochemistry of the cis - trans form of oleic acid (C 18:1) did not influence the inhibitory potency against the enzymes. Carboxyl and hydroxyl groups in the fatty acid molecule were concerned in the inhibition of c-ACI. Among the fatty acids, eicosatrienoic acid (C 20:3) generally inhibited h-SD, c-ABC, c-B and c-ACI, and nervonic acid (C 24:1) was a potent inhibitor of c-ACII, and the fatty acids inhibited the enzymes in a noncompetitive manner.

Evaluation of the antimicrobial activity and materials compatibility of orthophthalaldehyde as a high-level disinfectant

We tested the antimicrobial activity of orthophthalaldehyde (OPA) against 21 strains (16 species) of pathogenic microorganisms that cause hospital-associated infections. Changes in hepatitis B surface antigen (HBs-Ag) resulting from the addition of OPA to HBs-Ag-positive serum were measured using a radio-immunoassay. We also examined the effect of immersing medical instruments in OPA (0.55%) for 168 h at room temperature. OPA (0.5%, 0.37% and 0.25%) killed 11 strains of vegetative bacteria within 15 s, and it killed the test microorganisms faster than 3.0% glutaraldehyde (GTA). Incubation with OPA or GTA caused levels of HBs-Ag to fall below a cut-off value within 30 s. OPA did not adversely affect instruments made from various materials. OPA demonstrated more effective antimicrobial activity than GTA against a range of microorganisms. We conclude that OPA should replace GTA as the first-choice high-level disinfectant for endoscopes, considering its antimicrobial efficacy and low inhalation toxicity.

Inhibition of Dna Topoisomerases by Microbial Inhibitors

New inhibitors of DNA topoisomerase named 2280-DTI and 2890-DTI have been discovered in the culture filtrates of Micromonospora sp. strain No. 2280 and Streptomyces sp. strain No. 2890, respectively. Both inhibitors were purified from each culture filtrate by column chromatography on Diaion, Dowex and gel filtration. Both inhibitors were thermostable acidic substances with high molecular weight and inhibited topoisomerase I in a non-competitive manner. They differed from well-known inhibitors of topoisomerases such as camptothecin and doxorubicin, which inhibit the DNA rejoining reaction of the enzyme by intercalation into DNA strands or stabilizing the cleavable complex (enzyme-DNA reaction intermediate). 2280-DTI and 2890-DTI did not intercalate into DNA strands and also had no ability to stabilize the cleavable complex. It is suggested that 2280-DTI and 2890-DTI inhibit the DNA breaking and rejoining reactions of topoisomerase by direct action on the enzyme molecule.

Macrostatin, A Novel Macromolecular Inhibitor of Topoisomerases Produced by Streptomyces a Vermitilis No. C-127

A novel inhibitor of topoisomerases designated as Macrostatin has been isolated from the culture filtrate of Streptomyces avermitilis strain No. C-127 and purified by successive chromatography on Dowex, activated charcoal, gel filtration and cellulose. It is an acidic macromolecule having 45 kD molecular weight as determined by gel filtration. Macrostatin inhibited topoisomerase I and II in a noncompetitive manner with Ki = 3.7 and 1.3 nM respectively. Macrostatin differed from well-known inhibitors of topoisomerase such as camptothecin, etoposide and doxorubicin which induce topoisomerase-mediated DNA cleavage by stabilizing the cleavable complex or intercalation into DNA strands. Macrostatin had neither ability to stabilize the cleavable complex nor ability to intercalate into DNA strands. It was suggested that Macrostatin inhibits topoisomerase by a direct action on the enzyme.

Inhibition of DNA Methyltransferase by Microbial Inhibitors and Fatty Acids

Streptomyces sp. strain No. 560 produces four kinds of DNA methyltransferase inhibitors in the culture filtrate. One of them, DMI-4 was distinguished from DMI-1, -2 and -3 previously reported with respect to certain properties, DMI-4 is considered to be a triglyceride consisting of the fatty acids anteisopentadecanoic acid (C15:0), isopalmitic acid (C16:0) and isostearic acid (C18:0) from the results of gas chromatography analysis. Since DMI-4 contains three molecules of fatty acid, and the previously reported DMI-1, 8-methylpentadecanoic acid, is analogous to a fatty acid, the inhibitory activity has been examined of various fatty acids and their methyl esters against Eco RI DNA methyltransferase (M. Eco RI). Oleic acid (C18:1) was found to be a potent inhibiton of M. Eco RI. The inhibitory activity of oleic acid was shown to be pH- and temperature-dependent and inhibited M. Eco RI in a noncompetitive manner with respect to DNA or S-adenosylmethionine (SAM). The number of carbon atoms and double bonds in the fatty acid molecule affected the inhibitory activity, but their methyl esters were not inhibitors. Our results suggest that the length of the carbon chain, the number of double bonds and the presence of a carboxyl group and branched methyl group in the fatty acid molecule may play an important role in the inhibition of DNA methyltransferase.

2070-DTI, A Topoisomerase Inhibitor Produced by Streptomyces sp. Strain No. 2070

A novel inhibitor of topoisomerase II designated as 2070-DTI was isolated from the culture filtrate of Streptomyces sp. strain No. 2070. The structure was determined to be that of the known soyasaponin I on the basis of spectroscopic methods (NMR and MS). 2070-DTI strongly inhibited the decatenation activity of human placenta topoisomerase II in a noncompetitive manner, and weakly inhibited or was inert towards the relaxation activities of various topoisomerase Is and DNA-related enzymes. 2070-DTI is an inhibitor belonging to the cleavable complex-nonforming type without DNA intercalation.

1513-DMIA AND 1513-DMIB, DNA METHYLTRANSFERASE INHIBITORS PRODUCED BY STREPTOMYCES SP. STRAIN NO. 1513

Two new methyltransferase inhibitors were isolated from the culture filtrate of Streptomyces sp. strain No. 1513 and named 1513-DMIa and 1513-DMIb. 1513-DMIa and 1513-DMIb were distinguished in certain properties from DMI-1, DMI-2, DMI-3 and DMI-4 previously reported. The molecular weight of 1513-DMIa and 1513-DMIb were estimated to be 576 and 8400 from the results of FAB-MS and gel filtration, respectively. The inhibitory activities of 1513-DMIa and 1513-DMIb were shown to be pH- and temperature-dependent and both inhibited M. EcoRI in an uncompetitive manner with respect to DNA or S-adenosylmethionine (SAM).

DMI-1, A NEW DNA METHYLTRANSFERASE INHIBITOR PRODUCED BY STREPTOMYCES SP. STRAIN NO. 560

A new inhibitor of DNA methyltransferase named DMI-1 has been discovered in the culture filtrate of Streptomyces sp. strain No. 560. DMI-1 was purified by extraction with ethyl acetate followed by Diaion HP-20SS and silica gel column chromatography. The structure of DMI-1 was determined to be 8-methylpentadecanoic acid (C16H32O2). DMI-1 is a novel inhibitor of methyltransferase isolated from microorganisms and is structurally different from sinefungin and A9145C which are structural analogs of S-adenosylmethionine (methyl donor). DMI-1 was a strong inhibitor of N6-methyladenine-DNA methyltransferase (M. Eco RI, EC 2.1.1.72) in a noncompetitive manner and its inhibition depended on the pH and temperature in the assay media.