Drug-enzyme Interactions Research Articles

Differential susceptibility of dihydroorotate dehydrogenase/oxidase to brequinar sodium (NSC 368 390) in vitro

To verify the assumption of a specific and potent drug action on de novo pyrimidine biosynthesis, isolated dihydroorotate dehydrogenase (DHO-DH) (EC 1.3.3.1) was exposed to Brequinar Sodium TM (6-fluoro-2-(2′-fluoro-1, 1′-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt, NSC 368 390) (Brequinar). The membrane-bound DHO-DH was purified to apparent homogeneity (25,000-fold) from rat liver mitochondria in six steps via detergent extraction and subsequent chromatography using the dye ligand Matrex TM Gel Orange A. Using molecular mechanistic studies (MM2) this ligand was found to mimic closely the stereochemical conformation of Brequinar. SDS-PAGE revealed two protein bands for the purified enzyme with apparent molecular masses of 58 (major) and 68 kDa (minor). In vitro, two modes of action of the DHO-DH are possible: (i) acting as a dehydrogenase in the presence of ubiquinone as proximal electron acceptor and (ii) direct reaction with oxygen as oxidase. A novel assay for the measurement of the oxidase activity was adapted using leuco-dichlorofluorescein-diacetate. Inhibition experiments revealed a striking difference in the susceptibility of DHO-dehydrogenase/oxidase to Brequinar : apparent K i = 6.09 ± 0.05 (SD) nM (DHO; ubiquinone n = 10), but K i = 3.10 ± 0.09 (SD) mM (DHO; O 2). Analyses of initial velocity experiments showed non-competitive inhibition of Brequinar with respect to the substrate dihydroorotic acid in both assays (dehydrogenase and oxidase). The inhibitory effect of the latter was compared to that of the competitive inhibitor 5-aza-dihydroorotate (apparent K i = 15 ± 0.25 (SD) μM). The present kinetic data on the action of the purified rodent DHO-DH with Brequinar and computer-aided analyses provide a better insight into the drug-enzyme interaction.

The effect of delmopinol on glucosyltransferase adsorbed on to saliva-coated hydroxyapatite

The aim was to explore the effects of delmopinol, a substituted amino-alcohol compound recently reported as a potential antiplaque agent, on GTF activity in solution and when adsorbed on to sHA. Delmopinol was without a significant effect on GTF activity in solution. In contrast, a reduction in the bound glucans synthesized by the adsorbed GTF was found in the presence of delmopinol. Delmopinol did not displace the adsorbed GTF from the sHA, nor was there significant desorption of glucans from sHA. The total glucan synthesis (bound and unbound) was reduced in the presence of delmopinol. Inhibition of GTF was not reversed by sucrose. Inhibition of GTF activity by delmopinol apparently results from drug-enzyme interaction on the surface of sHA beads. These observations provide further support for the important differences in the properties of adsorbed GTF and GTF in solution, illustrating that GTF-drug interaction differs between enzyme adsorbed to surfaces and enzyme in solution.

Mechanism of resistance of non-cycling mammalian cells to 4′-[9-acridinylamino]methanesulphon- m-anisidide: Role of DNA topoisomerase II in log- and plateau-phase CHO cells

CHO-AA8 cells were used as a model system to study the role of DNA topoisomerase II in the resistance of non-cycling cells to amsacrine. Plateau-phase AA8 cells have previously been shown to be resistant to amsacrine and to contain fewer DNA breaks than log-phase cells after drug treatment (Robbie, M.A., Baguley, B.C., Denny, W.A., Gavin, J.R. and Wilson, W.R. (1988) Cancer Res., in press). The phage P4-unknotting activity of nuclear extracts decreased 2-fold when AA8 cells entered into the non-cycling state, but there was no difference in sensitivity to amsacrine between log- and plateau-phase nuclear extracts. Drug stimulation of protein-DNA complex formation was similar in whole cells, isolated nuclei and nuclear extracts from either log- or plateau-phase cells. However, stimulation of complex formation in cells, nuclei or nuclear extracts was approx. 4-fold lower in plateau-phase than in log-phase. The data presented suggested that drug-enzyme interaction was altered in plateau-phase cells.

DNA topoisomerase II: A primer on the enzyme and its unique role as a multidrug target in cancer chemotherapy

Based on the weight of evidence accrued in the past eight years, there is little question that the nuclear enzyme, topoisomerase II, serves as a common intracellular target for the cytotoxic effect of drugs of widely varying structure. The enzyme appears to be unique as a chemotherapy target in that it is recruited into a lethal process under the influence of drug. Its role contrasts sharply with other more classical chemotherapy targets, such as dihydrofolate reductase, whose activity must be successfully inhibited for the expression of cytotoxicity. Resistance to inhibitors of this enzyme frequently results from marked elevations in intracellular enzyme content. In contrast, the presence of topoisomerase is required for drug effect, and, in general, the greater the cellular content of the enzyme, the more sensitive the cell will be to these agents. However, important issues remain unresolved. The biochemical events that are initiated by cleavable complex formation and result in cell death must be more fully defined. It is likely a better understanding of the drug-enzyme interaction will be required for rational drug development. Finally, those aspects of the drug-topoisomerase interaction that confer therapeutic selectivity and/or clinical resistance are of paramount importance if the phenomenon is ever to be fully exploited.

Interaction of N-trifluoroacetyladriamycin-14- O-hemiadipate with chicken leukemia RNA polymerase : Formation of drug-enzyme complex

The biochemical mechanism of the N-trifluoroacetyladriamycin-14- O-hemiadipate-induced inhibition of RNA synthesis in vitro by chicken (myeloblastosis) leukemia RNA polymerase II was studied. The inhibition was found to be dependent upon preincubation of the drug with the enzyme prior to enzyme assays, suggesting that drug-enzyme interactions occur. A drug-enzyme association complex was subsequently isolated through glycerol gradient sedimentation and further characterized by fluorescent microscopic studies. The drug was dissociated from the complex upon sodium dodecyl sulfate (SDS)-gel electrophoresis, revealing the non-covalent nature of the binding between the drug and the RNA polymerase.

Role of the cardiolipin-adriamycin complex in mitochondrial toxicity

It is demonstrated here that adriamycin (ADM), a widely used antimitotic agent, has a similar affinity for its target (DNA) and for a mitochondrial membrane component (cardiolipin). This cardiolipin—adriamycin complex is responsible for the inhibition of cytochrome c oxidase. The activity change of cytochrome oxidase evaluated for several adriamycin derivatives in beef heart mitochondria indicates that the inhibition is not a consequence of a direct drug—enzyme interaction but of a modification of the enzyme lipid environment. The adriamycin—cardiolipin complex segregates in a separate lipid phase and the new enzyme environment does not allow its activation. Cardiolipin (CL) is essential for cytochrome c oxidase activity. This inactivation could explain the adriamycin cardiotoxicity. Indeed, it is significant that the most cardiotoxic adriamycin derivatives form the strongest complexes with cardiolipin. A precise knowledge of this inhibition mechanism is a prerequisite for the synthesis of new non-cardiotoxic compounds.

Inhibition of DNA synthesis in murine tumor cells by geldanamycin, an antibiotic of the benzoquinoid ansamycin group.

The mechanism of action of geldanamycin, a benzoquinoid ansamycin, was investigated with murine lymphoblastoma L5178Y cells. The agent inhibited the cell growth at concentrations over 0.01 micrograms/ml. The antibiotic blocked DNA synthesis more markedly than RNA and protein syntheses. Mitosis was not significantly affected by the drug in the cells synchronized with demecolcine (Colcemid). The antibiotic did not interfere with in vitro assembly of tubulin. In the synchronized cells, strong inhibition of DNA synthesis was observed when geldanamycin was introduced into the culture prior to S phase of the cell cycle. The degree of inhibition was stronger with prolongation of incubation period and with increase of DNA synthesis rate. The results suggested that initiation of DNA synthesis or S phase is affected by the drug. DNA degradation was not significantly induced in vivo by the antibiotic. Geldanamycin blocked DNA polymerase alpha more markedly than beta and gamma. The degree of inhibition depended up on concentrations of enzyme but not upon those of template, suggesting a drug-enzyme interaction. IC50 for DNA polymerase alpha was 10 micrograms/ml and for DNA polymerase beta 100 micrograms/ml at low concentrations of enzyme. The inhibition of DNA polymerase alpha by the antibiotic was non-competitive and Ki was 20 microM.

Inhibition of DNA polymerases alpha and gamma by rifamycin derivative AF/013.

The nature of the inhibitory effects of rifamycin derivative AF/013 (O-n-octyloxime of rifamycin SV) on DNA polymerases alpha and gamma were studied. Lineweaver-Burk analysis of the inhibition of DNA polymerases with respect to a substrate and template-primer showed a different mode of inhibition by AF/013 for each: the inhibition of DNA polymerase gamma was competitive with both dTTP and poly(rA)-oligo(dT), while that of DNA polymerase alpha was competitive with activated calf thymus DNA and non-competitive with dTTP. Further analysis of the competitive mode of the inhibition of DNA polymerase alpha, using poly(dT)-oligo(rA) as a template-primer, demonstrated that the primer molecule competed with AF/013. A change of effective divalent metal ion (Mn2+ in place of Mg2+) in the reaction mixture did not alter this competitive mode of inhibition with respect to the template-primer. The results of experiments to obtain further insight into the mechanism of drug-enzyme interaction suggest that AF/013 binds tightly to DNA polymerase alpha, and inhibits the process of chain elongation with DNA polymerases alpha and gamma.

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors ( K i : 2–3 × 10 −12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors ( K i = 3–300 × 10 −8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.

Drug‐Receptor and Drug‐Enzyme Interactions: A Basic Approach

International Journal of Quantum ChemistryVolume 10, Issue 3 p. 555-557 AnnouncementFree Access Drug-Receptor and Drug-Enzyme Interactions: A Basic Approach First published: May 1976 https://doi.org/10.1002/qua.560100319AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Volume10, Issue3May 1976Pages 555-557 RelatedInformation

Congress on drug receptor and drug enzyme interaction: A basic approach: Namen, Belgium, May 19–21, 1976

Congress on drug receptor and drug enzyme interaction: A basic approach: Namen, Belgium, May 19–21, 1976

An International Meeting on Drug Receptor and Drug Enzyme Interactions. A basic approach

An International Meeting on Drug Receptor and Drug Enzyme Interactions. A basic approach

Mechanisms of drug-enzyme interactions.

Mechanisms of drug-enzyme interactions.