beta-Lapachone: synthesis of derivatives and activities in tumor models.

In order to find a 3,4-dihydro-2H-naphtho[1,2-b]pyran-5,6-dione more potent than the naturally occurring 2,2-dimethyl derivative [beta-lapachone (10a)], we synthesized a series of analogous compounds with modifications at position 2 of the pyran ring or at positions 8 and 9 of the benzene ring. Of the compounds tested in vitro for inhibition of RNA-dependent DNA polymerase and in mice infected with Rauscher leukemia, all retained good enzyme activity. Inhibition of the reverse transcriptase activity of the 2,2-substituted derivatives 10b-e was as strong as 10a. However, only the 2-methyl-2-phenyl derivative 10e proved to be about as potent as the 2,2-dimethyl reference compound 10a in prolonging the mean survival time of mice with Rauscher leukemia virus induced leukemia.

Papulacandins--the relationship between chemical structure and effect on glucan synthesis in yeast.

Papulacandin B inhibits glucan biosynthesis in cells of Saccharomyces cerevisiae and Candida albicans. Biological studies with a series of papulacandin derivatives showed that the short fatty acid chain and the galactose residue are not required for activity at the target site, but that they can affect penetration. On the other hand, the long fatty acid residue is essential for biological activity.

CGP 4832, a new semisynthetic rifamycin derivative highly active against some gram-negative bacteria.

CGP 4832 (5) is a new derivative of rifamycin S, showing a very high degree of activity against certain Gram-negative bacteria, with MICs as much as 400 times lower than those of rifampicin. CGP 4832 and rifampicin inhibit DNA-dependent transcription in vitro to a similar extent, which excludes any difference in their effect on the target enzyme. The most plausible explanation for the potent activity of CGP 4832 is that it penetrates into bacterial cells by way of a specific mechanism. This hypothesis is corroborated by the high rate of mutations leading to bacterial strains resistant against CGP 4832.

Resistance of Escherichia coli to rifampicin and sorangicin A--a comparison.

Sorangicin A, a macrolide polyether antibiotic and the ansamycin antibiotic rifampicin inhibit DNA-dependent RNA polymerase to a similar extent. Resistance to sorangicin A is due to a mutation in the RNA polymerase which renders the enzyme less sensitive. Parallel investigations with rifampicin revealed partial cross-resistance, which was more marked in sorangicin A-resistant mutants than in rifampicin-resistant mutants.

Rifampin: mechanisms of action and resistance.

Rifampin specifically inhibits bacterial RNA polymerase, the enzyme responsible for DNA transcription, by forming a stable drug-enzyme complex with a binding constant of 10(-9) M at 37 C. The corresponding mammalian enzymes are not affected by rifampin. Bacterial resistance to rifampin is caused by mutations leading to a change in the structure of the beta subunit of RNA polymerase. Such resistance is not an all-or-nothing phenomenon; rather, a large number of RNA polymerases with various degrees of sensitivity to rifampin have been found. No strict correlation exists between enzyme sensitivity and MIC values, since inhibition of RNA synthesis does not always show up to the same extent in the two different test systems used for the determination of these values.

beta-Lapachone, an inhibitor of oncornavirus reverse transcriptase and eukaryotic DNA polymerase-alpha. Inhibitory effect, thiol dependence and specificity.

beta-Lapachone is a naturally occuring compound that can be isolated from a number of tropical trees. It is shown to be a potent inhibitor of reverse transcriptase activity from both avian myeloblastosis virus and Rauscher murine leukaemia virus. In addition, it affects eukaryotic DNA-dependent DNA polymerase-alpha activity: 50% inhibition is reached in 60-min incubation time by about 8 micron beta-lapachone. Enzyme activity is inhibited irrespective of the purity of the enzyme used or of the amount or type of template/primer or substrate present. The inhibitory effect of the drug is only observed in the presence of dithiothreitol. The primary site of action of beta-lapachone appears to be the enzyme protein, as is also borne out by the specificity of its action. Eukaryotic DNA-dependent DNA polymerase-beta, prokaryotic DNA-dependent DNA polymerase I, several other nucleic acid polymerases and some completely unrelated enzymes are not affected. Reverse transcriptase and DNA-dependent DNA polymerase-alpha may be in someway related in possessing similarly exposed '--SH structures' in their active sites. beta-lapachone thus affords a novel means of studying such interrelationships and of further characterizing enzymes.

On the kinetics of the rifampicin-RNA-polymerase complex. Differences between crude and purified enzyme fractions.

The antibiotic rifampicin forms a very tight complex with DNA-dependent RNA polymerase of Escherichia coli. The rate constants of association and dissociation of this complex have been measured and found to be dependent on the purity of the enzyme. Thus a crude RNA polymerase (fraction-3 enzyme) has rate constants different from those of an enzyme further purified by DEAE-cellulose chromatography (fraction-4 enzyme). The complex produced by the antibiotic and the fraction-3 enzyme is about ten times more stable and is formed about ten times more slowly than the complex with fraction-4 enzyme. It has been shown that the RNA present in the crude enzyme and removed by chromatography on DEAE-cellulose is the cause of the change in the kinetics of the complex. tRNA of rat liver and crude rat liver RNA added to purified RNA polymerase have a similar effect. Mg2+, which has no intrinsic influence, augments the effect of the nucleic acids, whereas monovalent cations do not. Since nucleic acids increase the stability of the complex, but at the same time decrease the rat of its formation, the equilibrium constant, Keq, remains almost the same. The possible effects of nucleic acids on the rifampicin binding site are discussed.

Root induction on several Solanaceae species by Agrobacterium rhizogenes and the determination of root tropane alkaloid content.

Using Agrobacterium rhizogenes, roots were induced on explants of 24 different Solanaceae species and established as in vitro cultures. Some of the root clones produced tropane alkaloids at levels similar to roots of the corresponding intact plants and maintained these levels over several passages.

Highly efficient uptake of a rifamycin derivative via the FhuA-TonB-dependent uptake route in Escherichia coli.

Rifamycin CGP 4832 is a semisynthetic rifamycin derivative. It is at least 200 times more active than rifampicin against Escherichia coli and related bacteria. This increased activity is shown here to be due to the efficient uptake of CGP 4832 across the E. coli outer membrane via the ferrichrome transport system comprising the outer membrane FhuA (TonA) protein, the ferrichrome receptor, and the inner membrane TonB protein. CGP 4832 competed with ferrichrome and other iron siderophore complexes, and with bacteriophage T5 and colicin M for binding sites on the FhuA protein. Mutations in fhuA or tonB genes reduce CGP 4832 sensitivity to a level comparable to that to rifampicin. There is no evidence that CGP 4832 or rifampicin utilize the inner membrane ferrichrome transport system to gain entry into the cytoplasm.

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.

Pharmaco-EEG and Psychometric study of the effect of single doses of temazepam and nitrazepam.

A double-blind, daytime, placebo-controlled study was carried out in 12 healthy volunteers to investigate the effects of single doses of 5, 15 and 30 mg temazepam and of 5 and 10 mg nitrazepam on the EEG, psychomotor performance, subjective mental and emotional status, blood pressure and heart rate. Each subject received all 6 treatments in a random sequence at intervals of 1 week. The EEG tracings were evaluated quantitatively by spectral analysis. Psychomotor performance was assessed by means of the tapping test. Subjective mental and emotional status were assessed using the Bond and Lader analogue self-rating scale. Procedures were carried out before and at 1/2, 1, 2, 4, 6, 7, 8 and 9 h after drug administration, with the exception of the tapping test, which was carried out before and, again, after 2 and 7 h. EEG estimates of equipotency, based on magnitude of peak effect, were as follows: 15 mg temazepam approximately 5 mg nitrazepam; and 30 mg temazepam greater than or equal to 10 mg nitrazepam. At these approximately equipotent doses, temazepam had a somewhat earlier onset of action on the EEG, a clearly shorter duration of EEG action, and lesser impairment of psychomotor performance than nitrazepam. Qualitatively, both drugs had similar effects on the subjective mental and emotional states of the subjects. There were no clinically relevant changes in mean or individual sitting and standing blood pressure values. After temazepam, but not after nitrazepam, heart rate increased (maximal mean change 10 bpm) as part of a normal startle response to arousal. The results suggest that temazepam has less hangover potential than nitrazepam.

Determination of the central effects of the asthma prophylactic ketotifen, the bronchodilator theophylline, and both in combination: an application of quantitative electroencephalography to the study of drug interactions.

Ketotifen (Zaditen) is a new, orally active benzocycloheptathiophene derivative for use in the prophylaxis of asthma. Besides possessing anti-allergic properties, it also has H1-receptor mediated antihistaminic activity. Drugs which block H1-receptors are known to have some sedative properties. On the other hand the bronchodilator theophylline is a CNS stimulant. We investigated whether these side-effects could be reduced by concomitant administration. In a balanced-sequence crossover study, 12 healthy males received placebo, 1 mg ketotifen + 300 mg theophylline, 1 mg ketotifen + 600 mg theophylline, and each drug separately at 1-week intervals. Quantified electroencephalograms, cardiovascular and behavioral measurements, symptom reports, and blood drug assays were used to assess the drug interaction. Results showed EEG and behavioral effects with both ketotifen and theophylline alone which were less evident with the drugs in combination. Blood drug levels were not altered by combined drug administration. These findings suggest a mutual attenuation of the CNS effects of ketotifen and theophylline at therapeutic doses and encourage their combined use in asthma therapy. The combined effect may be optimized by modifying formulations or timing. The applicability of quantitative EEG to such problems is well demonstrated.