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Biomedical subjects

N C Brown

Publications and source records attributed to N C Brown.

At least 55 records · Page 3Linked to original sources
PubMed1985

The utility of combinations of drugs directed at specific sites of the same target enzyme--ribonucleotide reductase as the model.

Ribonucleotide reductase is a key enzyme in DNA replication and, as such, has been a target for antitumor agents. This enzyme is composed of two nonidentical protein subunits which can be specifically and independently inhibited. Combinations of drugs directed at the effector-binding and non-heme iron subunits of ribonucleotide reductase resulted in the synergistic inhibition of L1210 cell growth and synergistic L1210 cell kill. These combinations included dAdo/EHNA/IMPY/Desferal; dAdo/EHNA/hydroxyurea/Desferal (the EHNA was required to protect dAdo from deamination while Desferal modulated the effects of IMPY or hydroxyurea); 2-F-araA/IMPY/Desferal and 2-F-2'-dAdo/IMPY/Desferal (EHNA was not required to protect 2-F-araA or 2-F-2'-dAdo from deamination); and dGuo/8-AGuo/IMPY/Desferal (8-AGuo was required to protect dGuo from phosphorolysis). Although thymidine alone inhibited L1210 cell growth, it was not possible to potentiate the effects of thymidine with the pyrimidine nucleoside phosphorylase inhibitors, acyclothymidine, 5-chlorouracil and 2,6-dihydroxypyridine. Combinations of drugs directed at the ribonucleotide reductase and DNA polymerase sites were studied for their effects on L1210 cell growth. With these combinations, no synergistic inhibition of L1210 cell growth was observed. The combinations of aphidicolin and IMPY/Desferal and aphidicolin and dAdo/EHNA inhibited L1210 cell growth in an additive manner; the combinations of IMPY/Desferal and BuAU or IMPY/Desferal and BuPdG resulted in antagonistic inhibition of L1210 cell growth. From these results it is clear that combination chemotherapy directed at independent sites of the same key target enzyme can result in strong synergistic inhibition of cell growth and cytotoxicity offering a clear therapeutic advantage. In contrast, the combinations directed at sequential key enzymes (e.g. ribonucleotide reductase and DNA polymerase) did not result in synergistic inhibition of cell growth. The utility of combinations of drugs directed at specific but independent sites of the target enzyme (e.g. ribonucleotide reductase) has been demonstrated in tumor cell systems in culture and now must be demonstrated in vivo.

Animals↗
PubMed1984

Butylphenyl dGTP: a selective and potent inhibitor of mammalian DNA polymerase alpha.

BuPdGTP , the 2'-deoxyribonucleoside 5'-triphosphate of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n- butylphenyl )guanine, was examined with respect to its mechanism and its capacity to inhibit the mammalian DNA polymerases, pol alpha, pol beta, and pol gamma. BuP dGTP was specifically inhibitory for pol alpha, with no discernible activity on pol beta and pol gamma. The potency of BuP dGTP is unprecedented, with an apparent Ki less than 10 nanomolar. The unusual potency of the BuP dGTP is derived primarily from the 5' alpha and beta phosphoryl moieties, whose binding to enzyme complements that of the base-linked butylphenyl substituent. BuP dGTP is competitive with dGTP and apparently not subject to polymerization. Experiments employing BuP dGTP in the presence of a non-complementary template suggest that the core polymerase or an associated coprotein contains dNTP binding sites which recognize specific nucleic acid bases. The partial sensitivity of selected, non-mammalian DNA polymerases suggests that modification of the N2 substituent of dGTP will be a useful route to the design of novel, polymerase-specific affinity-probes.

Animals↗
PubMed1982

Design and characterization of N2-arylaminopurines which selectively inhibit replicative DNA synthesis and replication-specific DNA polymerases: guanine derivatives active on mammalian DNA polymerase alpha and bacterial DNA polymerase III.

The 2-amino substituted derivatives of guanine, N2-(p-n-butylphenyl)guanine (BuPG) and N2-(3',4'-trimethylenephenyl) guanine (TMPG), were synthesized and found to selectively inhibit, respectively, HeLa cell DNA polymerase alpha (po1 alpha) and B. subtilis DNA polymerase III (po1 III). Both purines, like their corresponding uracil analogs, BuAu and TMAU (2,9), were specifically competitive with dGTP in their inhibitory action on their target polymerases. BuPG, the pol alpha-specific purine, was also toxic for HeLa cells in vivo, selectively inhibiting DNA synthesis. These N2-substituted purines, in contrast to the 6-substituted uracils, provide a structural basis for the synthesis of nucleosides and nucleotides with considerable potential as probes for the analysis of the structure of specific replicative DNA polymerases and their function in cellular DNA metabolism.

Bacillus subtilis↗
PubMed1980

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.

Avian Myeloblastosis Virus↗
PubMed1980

Inhibitors of Bacillus subtilis DNA polymerase III. 6-Anilinouracils and 6-(alkylamino)uracils.

Substituted 6-anilinouracils were found to be potent inhibitors of the replication-specific enzyme, DNA polymerase III, from Bacillus subtilis. Inhibition potency was maximized by inclusion of small alkyl groups or halogens in the meta and para positions of the phenyl ring; polar substituents decreased activity considerably. Qualitative structure--activity relationships indicated that the meta position can tolerate larger groups, suggesting that this position may be suitable for the introduction of a group capable of irreversibly binding to the enzyme. Several 6-(alkylamino)uracils were weak inhibitors of DNA polymerases III; the optimum alkyl groups for enzyme binding were n-pentyl and n-hexyl, which apparently can occupy the planar enzyme binding site. The varied activities of 6-anilinouracils on a mutant DNA polymerase, resistant to 6-(phenylhydrazino)- and 6-(benzylamino)uracils bearing a p-OH or NH2 group, have altered previous postulates for the structural basis of inhibitor resistance and have permitted construction of a refined model for inhibitor conformation in the latter series.

Bacillus subtilis↗
PubMed1979

Antibody to B. subtilis DNA polymerase III: use in enzyme purification and examination of homology among replication-specific DNA polymerases.

Bacillus subtilis DNA polymerase III (pol III), an arylhydrazinopyrimidine-sensitive, replication-specific enzyme, was used to generate a non-precipitating rabbit antibody which specifically inhibited pol III activity in vitro. The antibody was used to examine structural relationships among several DNA polymerases, and it was linked covalently to agarose; the antibody:agarose was employed to develop a rapid, selective method of purification of catalytically active B. subtilis pol III.

Animals↗
PubMed1978

Bacillus subtilis dnaF: a mutation of the gene specifying the structure of DNA polymerase III.

The characteristics of Bacillus subtilis dnaF, a mutation specifying a temperature sensitive phenotype, were examined to determine its relationship to polC, the gene specifying the structure of DNA polymerase III (pol III). Exposure of growing cells bearing dnaF to non-permissive temperature inhibited replicative DNA synthesis and specifically depressed the expression of pol III activity in crude extracts. Highly purified pol III derived from cells bearing dnaF was temperature.sensitive in its polymerase activity, indicating that dnaF is a specific, polC mutation which specifies a structurally altered enzyme.

Bacillus subtilis↗
PubMed1978

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.

Bacillus subtilis↗
PubMed1977

Inhibitors of Bacillus subtilis DNA polymerase III. Structure-activity relationships of 6-(phenylhydrazino)uracils.

6-(Phenylhydrazino)uracils inhibit the replication-specific enzyme DNA polymerase III of Bacillus subtilis by forming a strong, reversible complex with template-primer DNA and enzyme. The phenyl ring interacts with a hydrophobic enzyme site which, on the basis of structure-activity relationships of substituted analogues, appears to possess the following characteristics: (1) planarity or near-planarity; (2) a finite capacity to accommodate bulky substituents; and (3) location near the domain of the enzyme active site. A mutant DNA polymerase III, derived from a mutant strain of B. subtilis selected for resistance to 6-(p-hydroxyphenylazo)pyrimidines, is resistant only to inhibitors bearing p-hydroxy or amino groups and is hypersensitive to inhibitors containing nonpolar substituents; these results suggest the existence of mutable, secondary regions of the binding site which interact with para substituents and, thus, influence the strength of the primary phenyl-enzyme interaction.

Bacillus subtilis↗
PubMed1977

Inhibitors of Bacillus subtilis DNA polymerase III. 6-(arylalkylamino)uracils and 6-anilinouracils.

6-(Benzylamino)uracils and substituted 6-anilinouracils have been found to be potent inhibitors of Bacillus subtilis DNA polymerase III by a mechanism identical with that of 6-(phenylhydrazino)uracils. Higher phenylalkylamino homologues are progressively weaker inhibitors of the enzyme. Examination of the effects of substituents on the activity of 6-(benzylamino)uracils against wild-type and mutant enzymes and preliminary results for 6-anilinouracils have permitted further dissection of the mechanism of inhibition. The experimental results indicate that (1) the polymerase inhibitor binding site is compact, accommodating only small alterations in the distance between the uracil and phenyl rings, (2) the phenyl ring, which provides the major contribution to inhibitor-enzyme binding, adopts a specific active conformation, and (3) an enzyme site which interacts with substituents in the phenyl ring forms a part of the active site of DNA polymerase III.

Aniline Compounds↗
PubMed1977

Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105.

The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.

Bacillus subtilis↗
PubMed1976

Inhibition of Bacillus subtilis DNA polymerase III by arylhydrazinopyrimidines. Novel properties of 2-thiouracil derivatives.

6-(p-Tolylhydrazino)-uracil, 6-(p-tolylhydrazino)-isocytosine and 6-(p-tolylhydrazino)-2-thiouracil were synthesized and compared with respect to their chemical properties, their activity as inhibitors of DNA polymerase III of Bacillus subtilis, and their capacity to induce the formation of a complex between polymerase III and template DNA. As expected from earlier studies of analogous hydroxyphenylhydrazino compounds, the effects of the uracil derivative were reversed specifically by dGTP and those of the isocytosine derivative were reversed specifically by dATP. In contrast, reversal of the effects of the thiouracil derivative required both dGTP and dATP. The unique capacity of the 2-thiouracil analog to mimic either purine deoxyribonucleotide appears to reside in its ability to undergo tautomerism between the 2-thione and 2-thiol forms, which can pair with, respectively, template cytosine and thymine.

Bacillus subtilis↗
PubMed1976

Mapping of the gene specifying DNA polymerase III of Bacillus subtilis.

polC, the gene specifying the structure of the replication-specific DNA polymerase III of B. subtilis, was mapped by exploiting azp-12, a mutation conferring resistance to azopyrimidine which determines a mutant, azopyrimidine-resistant enzyme. azp-12 was located in the area of the pyrA locus and is between spcB1 and recA1. azp-12 was linked by transformation to four other mutations which influence the in vitro behaviour of DNA polymerase III--polC25, polC26, mut-1(ts), and DNAF133; the close linkage of these five mutations strongly suggests that they are alleles of the same gene.

Azo Compounds↗
PubMed1975

Inhibition of Bacillus subtilis deoxyribonucleic acid polymerase III by phenylhydrazinopyrimidines. Demonstration of a drug-induced deoxyribonucleic acid-enzyme complex.

The interaction of 6-(phenylhydrazino)-pyrimidines and Bacillus subtilis DNA polymerase III was examined in experiments exploiting agarose gel filtration of mixtures of drug, DNA, and purified enzyme. 6-(p-Hdroxyphenylhydrazino)-uracil and 6-(p-hydroxyphenylhydrazino)-isocytosine were used as model inhibitors; both drugs induced the formation of a distinct polymerase-DNA complex. Comples formation required the inhibitory, hydrazino forms of the drugs and a form of DNA suitable as a primer-template for DNA polymerase III. dGTP and dATP, which respectively, competitively antagonize the inhibitory effects of the uracil and isocytosine derivatives, antagonized in an equally specific manner the respective capacities of these compounds to induce complex formation. Experiments exploiting both wild type and drug-resistant, mutant polymerases indicated that drug concentrations required for the half-maximal induction of complex formation were nearly identical with the apparent inhibitor constants (Ki) determined independently by kinetic analysis of enzyme inhibition. These results and those of experiments exploiting defined homopolymer-oligomer combinations as template-primers support a model of inhibitor action in which arylhydrazinopyrimidine forms a reversible, ternary complex with the enzyme and an appropriate timplate pyrimidine residue in an area adjacent to the 3-hydroxyl primer terminus.

Bacillus subtilis↗
PubMed1973

Hydroxyphenylazopyrimidines: characterization of the active forms and their inhibitory action on a DNA polymerase from Bacillus subtilis.

The active forms of 6-(p-hydroxyphenylazo)-uracil and 6-(p-hydroxyphenylazo)-isocytosine were isolated and identified as their respective hydrazino derivatives. These arylhydrazino pyrimidines selectively inhibited a chromatographically distinct DNA polymerase from Bacillus subtilis. The actions of the reduced drugs on this polymerase were identical to those observed on ATP-dependent DNA synthesis in toluene-treated cells; dGTP competitively antagonized the inhibitory activity of the uracil derivative, and dATP competitively antagonized that of the isocytosine derivative. Analysis of the interactions of the arylhydrazinopyrimidines and nucleic acid bases by nuclear magnetic resonance suggested that hydroxyphenylhydrazino-uracil and hydroxyphenylhydrazino-isocytosine pair in a novel manner with, respectively, cytosine and thymine. A mechanism of inhibitor action, involving binding of the reduced drugs to enzyme and the pyrimidines of DNA template, is proposed.

Adenosine Triphosphate↗