Destabilization of the non-pathogenic, cellular prion-protein by a small molecular drug.

The presence of the normal cellular prion-protein (PrPc) is a prerequisite for the development of fatal, neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). We discovered a new biological activity of the well-known coumarin antibiotic novobiocin; the treatment of eukaryotic cells with novobiocin induces the rapid depletion of PrPc. This activity is shared by coumermycin A1, another coumarin with a related molecular structure. Novobiocin's effects on the prion-protein are time- and dose-dependent. No permanent damage to the treated cells was observed, which continue to proliferate after cessation of drug exposure. Most of the cellular proteins are unaffected by novobiocin treatment. Pretreatment with geldanamycin, an inhibitor of the aminoterminal ATPase of heat-shock protein 90 (Hsp90) partially antagonizes novobiocin's depletory activity. Concurrent treatment with the protease inhibitor chymostatin completely prevents PrPc loss. Here we show that the stability of the normal cellular prion-protein may be targeted pharmacologically. These findings open up a hitherto unknown avenue to the study of TSEs in general and may have therapeutic implications.

DNA intercalating drugs inhibit positive supercoiling induced by novobiocin in halophilic archaea.

The two DNA intercalators, actinomycin D and 2-methyl-9-hydroxy-ellipticine, and the DNA minor groove ligant DAPI inhibited the growth of the haloarchaeon Halobacterium sp. GRB and bind to its plasmid pGRB-1. In contrast to specific DNA topoisomerase II inhibitors, they produced neither double-stranded breaks nor relaxation of plasmidic DNA. The two DNA intercalators inhibited positive supercoiling induced by novobiocin, suggesting that positive supercoiling in haloarchaea is due to transcription, as in the domain Bacteria. Plasmids from haloarchaea could thus be used to prescreen for DNA intercalators and to discriminate between different drug families via their mode of action.

Na transport stimulation by novobiocin: transepithelial parameters and evaluation of ENa.

The action of the antibiotic novobiocin on transepithelial Na transport was studied in isolated skins obtained from two different frog species. In Rana esculenta addition of novobiocin to the outer bath (1 mM) resulted in a sustained and reversible stimulation of the short-circuit current, transepithelial potential, and transepithelial conductance. Similar, though more variable and much less pronounced changes were observed in Rana temporaria. In the presence of amiloride (0.1 mM) novobiocin had no effect on any of the investigated transport parameters and all novobiocin induced changes were fully reversed when amiloride was given subsequently. At reduced external Na concentration or low pH the action of novobiocin was found to be greatly attenuated. In the presence of novobiocin an increased affinity to amiloride and a linearization of the transepithelial current-voltage relationship was observed. The results are consistent with the view that novobiocin increases the Na permeability of the outer membrane, possibly by an attenuation of an Na self-inhibition mechanism. In addition, the driving force of transepithelial Na transport was estimated by means of novobiocin. Several different methods were employed, providing varying results. As shown in an Appendix, for the most part the discrepancies can be explained by changes in the intracellular Na and K concentration. In some cases, novobiocin induced large secondary increases in the skin conductance which can be referred to an increased Cl permeability.

Curing of plasmid pE194 with novobiocin and coumermycin A1 in Bacillus subtilis and Staphylococcus aureus.

Plasmid pE194 determining macrolide-lincosamid-streptogramin B resistance could be eliminated with novobiocin and coumermycin A1 in Bacillus subtilis and Staphylococcus aureus. In both organisms the curing effect depended on the temperature. In the case of Staphylococcus aureus it increased very much at high temperatures, while in the case of Bacillus subtilis only a moderate increase could be observed. The curing activity of coumermycin was substantially weaker than that of novobiocin in both bacteria. This difference decreased at higher temperatures only in Staphylococcus aureus. In Bacillus subtilis the curing with novobiocin could be antagonized with coumermycin in accordance with the growth experiments reported previously.

[Nature of the phenotypic expression of novobiocin resistance in staphylococci]. / Priroda fenotipicheskogo vyrazheniia novobiotsinoustoichivosti u stafilokokkov.

The phenotypic features of novobiocin-sensitive and novobiocin-resistant staphylococci were studied comparatively and it was shown that mutations of staphylococci to phenotype Novr resulted in alteration of their pathogenicity, phage lysing and antigenic properties. Loss of phage receptors and a number of antigens as well as changes in multiplication activity of novobiocin resistant staphylococci were defined by alteration of the functional properties and antigenic specificity to teichoic acids. Possible transformation of pathogenic staphylococci into nonpathogenic ones on mutation to Novr is discussed.

[The membrane apparatus of Staphylococci in relation to acquired novobiocin resistance]. / Membrannyi apparat stafilokokkov v sviazi s priobreteniem ustoichivosti k novobiotsinu.

The properties of the membrane respiration apparatus of three clinical staphylococcal strains and their novobiocin resistant variants were studied comparatively. Changes in the specific activity of the respiration enzymes were shown: when the dehydrogenase activity of the membrane preparations of the sensitive and resistant variants was equal and the specific quantity of cytochromes in the novobiocin resistant staphylococci was lower, the oxidase activity of their respiration chains was increased by 60-70 per cent. Disintegration of the cells of the novobiocin resistant staphylococci resulted in a higher yield of the membrane protein, the membrane fraction being characterized by a activity of phospholipase A.

Effect of novobiocin and its combination with tetracycline, chloramphenicol, erythromycin, and lincomycin on the microbial generation of Escherichia coli.

Inhibition of the steady-state generation of Escherichia coli by the bacteriostatic antibiotic novobiocin is linearly related to drug concentration in the range of 0 to 30 mug/ml. Increased cell sizes result because the drug inhibits cell division. The generation rate dependence on drug concentration depends on the nonionized fraction of novobiocin and is invariant with inoculum size or medium composition. However, the antibacterial activity of novobiocin decreases as the concentration of nutrients and Mg(2+) increases, although the inhibitory action of novobiocin on generation rate remains unchanged for concentrations of Mg(2+) above 8.1 x 10(-4) M. Novobiocin is synergistic in combinations with tetracycline in broth, but not when the Mg(2+) was maintained at 4.05 x 10(-3) M. Combinations of novobiocin with the 50S ribosomal subunit inhibitors chloramphenicol, erythromycin, or lincomycin are antagonistic, and the degree of growth inhibition is determined only by that component of the binary combination that would have the greater potency if it were acting alone.