Mode of action of 2-amino-6-chloro-1-deazapurine

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5'-phosphate which accumulated to concentrations as high as 1000 nmoles/10(9) cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibit the synthesis of 5'-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel mechanism of mycophenolic acid resistance in the protozoan parasite Tritrichomonas foetus

Tritrichomonas foetus relies primarily on the salvage of hypoxanthine to supply purine nucleotides. Mycophenolic acid disrupts T. foetus growth by specifically inhibiting inosine-5'-monophosphate (IMP) dehydrogenase, thereby blocking the biosynthesis of guanine nucleotides from hypoxanthine. We have cloned a T. foetus strain (mpar) that was 50-fold more resistant to mycophenolic acid than wild type (IC50 = 1 mM for mpar vs 20 microM for wild type). None of the usual mechanisms of drug resistance could be identified. IMP dehydrogenase isolated from T. foetus mpar was indistinguishable from the wild type enzyme. No difference in mycophenolic acid uptake or metabolism was detected between the wild type and mpar strains. Mycophenolic acid (100 microM) completely blocked the conversion of adenine and hypoxanthine to guanine nucleotides in T. foetus mpar, although no inhibition of T. foetus mpar growth was observed at this concentration. These observations indicate that the major purine salvage pathways must be altered in T. foetus mpar so that guanine nucleotide biosynthesis no longer requires IMP dehydrogenase. T. foetus mpar incorporated xanthine more efficiently into the nucleotide pool relative to hypoxanthine and guanine than wild type. Xanthine incorporation via XMP provided an IMP dehydrogenase independent route to guanine nucleotides that would enable the parasite to become mycophenolic acid resistant. No difference could be detected between wild type and mpar hypoxanthine-guanine-xanthine phosphoribosyltransferases, the key enzyme in purine base incorporation into nucleotides. Two alterations were identified in the purine salvage network of mpar: it was deficient in hypoxanthine transport and had diminished adenine deaminase activity. The apparent net result of these two changes was to lower the intracellular concentration of hypoxanthine in mpar. Hypoxanthine and adenine inhibited the incorporation of xanthine into the nucleotide pool in wild type T. foetus, but not in mpar. The mpar strain, therefore, can salvage xanthine more efficiently from a mixture of purines and thus bypass the drug block at IMP dehydrogenase.     

Biotransformation of 9-beta-D-arabinofurano-syladenine by rat and human erythrocytes

Studies were performed to test whether 9-β-d-arabinofuranosyladenine (ara-A) would accumulate in erythrocytes as a result of phosphorylation to the nucleotide level. When [³H]ara-A was incubated with whole blood from rat, monkey or man for 2 hr at 37°, the drug rapidly equilibrated between erythrocytes and plasma but did not concentrate in the cells. Incubation of [³H]ara-A with rat and human erythrocyte lysates for 2 hr followed by Chromatographie analysis showed that 2–5 per cent of ara-A was converted to nucleotides. In contrast, 10–35 per cent of [¹⁴C]adenosine was converted to adenine nucleotides under the same conditions. Incubation of [³H]ara-A with human erythrocyte lysates for 18 hr resulted in a conversion of approx. 40 per cent of the labeled drug to nucleotides. Additional chromatography revealed, however, that the nucleotide fraction contained almost no arabinosyl nucleotides. Rather, 90 per cent of the label in the nucleotide fraction was identified as IMP. These results indicate that only a minor amount, if any, of ara-A was phosphorylated by erythrocyte enzymes to yield arabinosyl nucleotides. An alternative pathway converted much of the labeled drug to ribosyl nucleotides via the deamination of ara-A to ara-hypoxanthine, cleavage to hypoxanthine and conversion of the free hypoxanthine to IMP.     

Nucleosides of 2-azapurines. 7 H-Imidazo[4,5-d]-1,2,3-triazines. 2.

A number of nucleosides of 2-azaadenine (4-amino-7H-imidazo[4,5-d]-1,2,3-triazine) were prepared by a previously described route, and some of these were deaminated by means of adenosine deaminase. Alternatively, nucleosides of 2-azahypoxanthine (7H-imidazo[4,5-d]-1,2,3-triazin-4(3h)-one) were prepared from hypoxanthine nucleosides by a 2-azahypoxanthine (7H imidazo[4,5]-1,2,3-triazin-4(3H)-one) were prepared from hypoxanthine nucleosides by a ring-opening and reclosure sequence. The cytotoxicity of these compounds to human epidermoid carcinoma No. 2 cells in culture and to certain resistant sublines thereof was determined. 2-Azaadenine nucleosides chan be metabolized to nucleotides, the cytotoxic agents, by two pathways, but the activity of the 2-azahypoxanthine nucleosides appears to result only from cleavage back to 2-azahypoxanthine.     

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP)

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.     

Differential effects of inhibitors of purine metabolism on two trichomonad species

Tritrichomonas foetus and Trichomonas vaginalis are both incapable of de novo purine nucleotide synthesis. Previous studies indicated that T. foetus relies mainly on the salvage of hypoxanthine and subsequent conversion of IMP to AMP and GMP, whereas T. vaginalis depends on direct conversions of exogenous adenosine to AMP and guanosine to GMP without much interconversion between the two nucleotides. These two different types of purine salvage suggest the possibility of differential sensitivities between the two species of trichomonad flagellates toward different purine antimetabolites. Mycophenolic acid, hadacidin, 8-azaguanine, and formycin B inhibited the growth of T. foetus but had no effect on T. vaginalis. Mycophenolic acid acted by blocking conversion of IMP to GMP, hadacidin inhibited conversion of IMP to AMP, and 8-azaguanine was incorporated into the T. foetus nucleotide pool, likely via hypoxanthine phosphoribosyl transferase. Formycin B was converted to 5'-monophosphate in T. foetus and inhibited the conversion of IMP to AMP. Its precise mechanism of action on T. foetus remains, however, to be elucidated. Alanosine, whose ribonucleotide derivative is a potent inhibitor of adenylosuccinate synthetase, had no effect on the growth or hypoxanthine incorporation in T. foetus, which may be due to the lack of conversion of alanosine to the ribonucleotide because of the absence of de novo purine nucleotide synthesis in parasites. Four adenosine analogs, adenine arabinoside, tubercidin, sangivamycin, and toyocamycin, were found inhibitory to the growth of T. vaginalis but showed little effect on T. foetus growth. Further investigations suggested that these four compounds acted on T. vaginalis by blocking incorporation of adenosine into the adenine nucleotide pool.     

Biochemical properties of the nucleoside of 3-amino-1,5-dihydro-5-methyl-1,4,5,6,8-pentaazaacenaphthylene (NSC-154020)

A study of 3-amino-1, 5-dihydro-5-methyl-l-β-d-ribofuranosyl-1, 4, 5,6,8- pentaazaacenaphthylene (NSC-154020), a “tricyclic” nucleoside with activity against certain experimental tumors, was undertaken to determine if it differed in biochemical properties from structurally related7- deazapurine nucleosides with established biological activity, such as sangivamycin. In cultured L1210 cells, [¹⁴C-methyl]-NSC-154020 was converted to a single metabolite with the properties of a 5'-monophosphate as shown by (a) similarity to AMP in migration on paper chromatograms and in retention time when subjected to high pressure liquid chromatography (h.p.l.c.) on an ion exchange column and (b) conversion to a compound with the properties of NSC-154020 upon treatment with alkaline phosphatase or 5'-nucleotidase. In cultured H.Ep.-2 cells, the principal metabolite of NSC-154020 was also the monophosphate. H.Ep.-2 cells contained in addition a variable amount of a second metabolite which also had the retention time (on h.p.l.c. analysis) of a monophosphate and which was converted by the action of alkaline phosphatase or 5'-nucleotidase to a compound that migrated like NSC-154020 upon chromatography in three solvent systems. This second metabolite is as yet unidentified. In crude extracts of L1210 cells, addition of adenosine or 6-(methylthio)purine ribonucleoside decreased the phosphorylation of NSC-154020. NSC-154020 was a substrate for adenosine kinase 110-fold purified from H.Ep.-2 cells; the K_m was 215 μM and the V_max was 1.8 times greater than that of adenosine. No ¹⁴C from labeled NSC-154020 was found in the polynucleotides of either H.Ep.-2 cells or L1210 cells grown for 24 hr in the presence of the labeled nucleoside. Several different studies failed to reveal any selective sites of action for NSC-154020. In cultured L1210 cells it inhibited synthesis of DNA, RNA and protein and reduced ribonucleotide pools, but with little selectivity. The incorporation of [¹⁴C]formate into polynucleotides was inhibited more severely than that of hypoxanthine; this is evidence for a blockade of purine synthesis de novo, an effect also produced by many other analogs of purines and nucleosides. Sangivamycin produced generally similar inhibitions of incorporation of formate and hypoxanthine. However, the cytotoxicity of NSC-154020 and sangivamycin to L1210 cells was not prevented or reversed by 5-amino-4-imidazolecarboxamide (AIC), adenine, guanine, hypoxanthine, uridine, or AIC + uridine; therefore, inhibition of de novo synthesis of purine and pyrimidine nucleotides is not a primary site of action of these compounds. Although the loci of action of NSC-154020 are not yet defined, the fact that it is not metabolized to polyphosphates indicates that its mechanism of action probably differs significantly from those of the related compounds, tubercidin and sangivamycin, which are converted to polyphosphates and incorporated into RNA and DNA.     

Primary screening and inhibition of macromolecular biosynthesis in Ehrlich ascites cells by benzo(C)fluorene derivatives

The main objective of the present investigation was to screen a series of new benzo(c)fluorene compounds for in vitro activity. It can be stated that each of the 9 newly synthesized benzo(c)fluorene derivatives was about 10 times as active as tilorone. To elucidate the biochemical mode of action, the effects of 2 new compounds (13468 and 14200) on biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (DNA, RNA), [14C]-thymidine (DNA), [14C]uridine (RNA) and [14C]valine (protein) were studied in concentration and time dependence. Both compounds inhibited the incorporation of the 4 precursors into the TCA-insoluble fraction of Ehrlich ascites carcinoma cells.     

Effects of diazoacetyl-glycine amide on purine nucleotide metabolism in Ehrlich ascites tumour cells

On the basis of the known inhibitory activity of diazoacetyl-glycine amide (DGA) on DNA and purine nucleotide synthesis, a series of experiments was performed to study, in greater detail, the effects of the drug on purine nucleotide metabolism. DGA produces a marked inhibition of de novo utilization of glycine and formate and a less pronounced inhibition in the “salvage” utilization of hypoxanthine for IMP synthesis. The synthesis of adenine and guanine nucleotides from hypoxanthine is also depressed by DGA, and the drug also causes unbalance in the ratio of the concentration of adenine and guanine nucleotides and a drop in the energy status of nucleotides. These findings and those already reported are related to a rather unspecific interaction of the drug with cellular components.     

9-Hydroxybenfluron: cytostatic effects and inhibition of macromolecular biosynthesis in Ehrlich ascites and P388 murine leukemia cells.

Primary screening in vitro and study on the mode of action of 9-hydroxybenfluron (HBF) in both murine P388 leukemia and Ehrlich ascites carcinoma cells have been performed. Metabolite HBF is approximately twice as effective as a reference drug (benfluron). To elucidate the biochemical mode of action, the effect of HBF on the biosynthesis of macromolecules indicated by the incorporation rate of [14C]adenine (in DNA and RNA), [14C]thymidine (in DNA), [14C]uridine (in RNA) and [14C]valine (in protein) was studied in concentration and time dependence. HBF inhibited incorporation of all four precursors into the trichloroacetic acid-insoluble fraction of Ehrlich ascites cells. The fact that incorporation of these four precursors is inhibited suggests that the effect of HBF lies at an underlying level of energy generation or transfer rather than at specific reactions in the biosynthesis of DNA and proteins.     

Purine and pyrimidine salvage pathways in Leishmania donovani

Leishmania donovani, grown in culture, salvaged radiolabeled purine bases which were distributed into adenine and guanine ribonucleotides and into the RNA of these cells. De novo synthesis of purines in L. donovani does not occur [J. J. Marr, R. L. Berens and D. J. Nelson, Biochim. biophys. Acta544, 360 (1978)]. [8-¹⁴C]Adenine was rapidly deaminated to hypoxanthine via the action of an adenine aminohydrolase (EC 3.5.4.2). [8-¹⁴C]Guanine was also rapidly deaminated by guanase (EC 3.5.4.3) to form xanthine in these cells. Therefore, the formation of nucleotides of hypoxanthine and xanthine are the first committed steps of purine salvage in L. donovani. While purines are efficiently conserved by this parasite, the salvage of pyrimidines is not so dramatic. [2-¹⁴C]Orotic acid was converted to OMP and then incorporated into the pyrimidine nucleotides and into RNA, indicating the existence of the later steps of de novo pyrimidine synthesis. [6-¹⁴C]Thymidine was salvaged by L. donovani, being incorporated into the thymine deoxyribonucleotides and into DNA. The major pathway of thymidine metabolism in this parasite, however, was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (EC 2.4.2.4).     

Relationships between the chemical structure and cytotoxicity of 4-alkylmorpholine N-oxides.

The main objective of the present investigation was to screen a series of 4-alkylmorpholine N-oxides for in vitro cytotoxicity and to find out whether there is a quantitative structure-activity correlation (QSAR) between cytotoxic effect represented here by inhibition of incorporation of [14C]adenine into nucleic acid or [14C]valine into proteins in Ehrlich ascites carcinoma (EAC) cells and structure (as a structural parameter the number of carbon atoms m in the alkyl chain was used). On the basis of primary screening, one of the most active compounds, namely 4-dodecylmorpholine N-oxide, was chosen for further biochemical study. The drug inhibited the incorporation rate of [14C] precursors (adenine, thymidine, uridine, valine) into appropriate macromolecules of Ehrlich cells, the extent of inhibition being dependent on both time and concentration of the compound in the incubation medium. The lengthening of the alkyl chain in 4-alkyl-morpholine N-oxides positively affected their cytotoxic activity in Ehrlich cells. For these compounds the optimal m-value is 15-16.     

Regulation of pyrimidine biosynthesis in cultured L1210 cells by 3-deazauridine

The effect of 3-deazauridine on the synthesis of uracil nucleotides by de novo and salvage pathways was investigated in intact cultured L1210 cells. De novo pyrimidine biosynthesis, as measured by sodium [14C]bicarbonate incorporation into uracil nucleotides, was inhibited 40-85% at intracellular 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. The inhibition was not due to an increase in the size of the uracil nucleotide pool since this pool was only 97-66% of control level at 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. Furthermore, intracellular 3-deazauracil nucleotide concentrations of 0.5 to 5.2 nmoles/10(6) cells inhibited the salvage of [14C]uridine by 25-75%. The data indicate that 3-deazauridine may potentiate its own inhibition of CTP synthetase by reducing the concentration of competing uracil nucleotides by inhibiting de novo pyrimidine biosynthesis and pyrimidine salvage. It is postulated that the biochemical mechanism by which 3-deazauridine inhibits uracil nucleotide synthesis is by acting as a fraudulent allosteric regulator of carbamyl phosphate synthetase II and uridine/cytidine kinase.     

Human B lymphocytes and thymocytes but not peripheral blood mononuclear cells accumulate high dATP levels in conditions simulating ADA deficiency

Inherited adenosine deaminase (ADA) deficiency is associated with a lymphospecific cytotoxicity affecting both dividing and non-dividing cells. The metabolic basis for this was investigated using different cell types and the potentially toxic metabolite 2'-deoxyadenosine (dAR) in short-term experiments under physiological conditions simulating ADA deficiency (1 mM Pi 8.7 microM dAR). In the uncultured cells, [8-14C] dAR alone was metabolized almost completely only by thymocytes and tonsil-derived B-lymphocytes. The greater percentage of counts (greater than 75%) were in the medium (deoxyinosine, hypoxanthine). Cellular counts were predominantly in adenine nucleotides, and to a lesser extent guanine nucleotides. Interestingly, both thymocytes and tonsil-derived B-lymphocytes, and a partially ADA deficient B lymphoblast line, accumulated detectable amounts of dATP even in the absence of ADA inhibition. Peripheral blood lymphocytes (PBMs) did not, and showed little dAR metabolism. In experiments simulating ADA deficiency varying amounts of 2'-deoxycoformycin (2'dCF) were needed to completely inhibit ADA (20-60 microM), with thymocytes requiring the highest amount. ADA inhibited thymocytes and tonsillar B-lymphocytes accumulated very high dATP levels, which were sustained to an equal extent by both over a 60-min period; PBMs accumulated the lowest values. Results in cultured cells reflected findings in previous studies. Some counts were also found in ATP by a route excluding ADA or PNP. These results again question the hypothesis that B-cells are more resistant than T-cells to the toxic effects of dAR because of an inability to accumulate and sustain elevated dATP levels and underline the lack of comparability between enzyme activity in intact as distinct from lysed cells. They cast doubt on the validity of cultured cells as a model for ADA deficiency and suggest the observed toxicity in some instances might result from altered ATP or GTP pools through inadequate ADA inhibition. They indicate that combined immunodeficiency in ADA deficiency could relate to an equal sensitivity of B-cells and T-cell precursors to the toxic effects of dATP accumulation.     

Inhibition of utilization of hypoxanthine and guanine in cells treated with the carbocyclic analog of adenosine. Phosphates of carbocyclic nucleoside analogs as inhibitors of hypoxanthine (guanine) phosphoribosyltransferase

In cell cultures treated with the carbocyclic analog of adenosine (C-Ado, (+/-)-aristeromycin), the utilization of hypoxanthine and guanine has been observed to be blocked. In an attempt to define the mechanism of this inhibition, we have reexamined the metabolism of C-Ado and its effects on the metabolism of guanine and hypoxanthine. In cultures of L1210 cells, C-Ado at a concentration of 25 microM inhibited the utilization of hypoxanthine and guanine for nucleotide synthesis by more than 90% but produced little or no inhibition of the utilization of these bases in cultures of L1210/MeMPR cells which lack adenosine kinase and cannot phosphorylate C-Ado. In cultures of mammalian cells (L1210, HEp-2, and colon-26 cells), C-Ado was converted to the triphosphate (as previously observed) and also to the triphosphate of the carbocyclic analog of guanosine. The presence of coformycin in the medium at a concentration sufficient to inhibit AMP deaminase almost completely prevented the formation of carbocyclic GTP; thus, the deamination of C-Ado monophosphate is essential for the formation of phosphates of carbocyclic guanosine. Since hypoxanthine (guanine) phosphoribosyltransferase is known to be subject to end product inhibition, it was considered likely that phosphates of carbocyclic guanosine or carbocyclic inosine, present in C-Ado-treated cells, were responsible for inhibition of utilization of hypoxanthine and guanine. The 5'-phosphates of the carbocyclic analogs of inosine and guanosine were synthesized and found to be effective inhibitors of the phosphoribosyltransferase. Carbocyclic GMP was a better inhibitor than carbocyclic IMP and was also superior to GMP and IMP; the concentration of C-GMP that produced a 50% inhibition of GMP formation was approximately 1 microM. It is probable that the presence of phosphates of carbocyclic guanosine accounts for the inhibition of utilization of hypoxanthine and guanine in C-Ado-treated cells.     

Terpentecin, an inhibitor of DNA synthesis

Terpentecin at a concentration of 0.78 microgram/ml decreased the number of viable cells of Escherichia coli NIHJ to less than one thousandth the starting number in an hour when added to an exponentially growing culture in a nutrient broth. During this time, the turbidity of the cell suspension kept increasing as fast as the control. Microscopic inspection of the cells exposed to terpentecin under these conditions revealed that the cells were elongated. Terpentecin at a concentration of 6.25 micrograms/ml inhibited incorporation of [14C]thymidine into the acid-insoluble material of cells of E. coli NIHJ by 70% in 30 minutes in contrast to little or no inhibition of the incorporation of [14C]uridine or [14C]leucine. Under similar conditions, terpentecin did not inhibit either membrane transport (uptake) of [14C]thymidine into the cells or the metabolic conversion of the precursor into various cellular acid-soluble components. Terpentecin at a higher concentration (70 micrograms/ml) inhibited by 40% in 30 minutes the incorporation of [methyl-3H]thymidine triphosphate into the DNA fraction of toluene-treated cells of E. coli JE6296 (pol A-). Terpentecin showed stronger antibacterial activities against Bacillus subtilis M45T (rec-) and E. coli BE1121 (rec A-) than against their corresponding wild type strains. However, terpentecin showed no mutagenicity by the Ames test with Salmonella typhimurium strains TA100, TA98, TA92, TA1538, TA1537 and TA1535, and with E. coli WP2 (uvr A). Terpentecin at a lower concentration (0.07 micrograms/ml) inhibited growth in vitro of mouse leukemia L1210 cells by 50%. With the mammalian cells again the incorporation of [14C]thymidine into the acid-insoluble cell material was inhibited more strongly than incorporation of [14C]uridine and [14C]leucine. There was no sign of mutagenicity by the micronucleus test using mice.     

Effects of 8-azaadenosine and formycin on cell lethality and the synthesis and methylation of nucleic acids in human colon carcinoma cells in culture

The cytocidal and biochemical effects of formycin and 8-azaadenosine in the presence and absence of the adenosine deaminase inhibitor, 2′-deoxycoformycin, were studied in human colon carcinoma (HT-29) cells in culture. Logarithmically growing cells were unaffected by 24-hr exposure to either 10^?6M formycin or 8-azaadenosine, but 1 to 1.4 log reductions in colony formation were produced by 10^?5M of each analog. In the presence of 10^?6M 2′-deoxycoformycin, a 3- and 30-fold potentiation of the cytocidal activity of 8-azaadenosine and formycin, respectively, was produced. Inhibition of DNA synthesis but not RNA synthesis by 8-azaadenosine paralleled its cytocidal activity; however, neither variable correlated closely with the cytotoxic effects of formycin. In addition, the methylation of nuclear RNA was unaffected by both drugs while the methylation of 5-methyl-deoxycytidine in DNA was inhibited to a lesser extent than DNA synthesis. Measurements of the incorporation of [³H]formycin and [³H]8-azaadenosine into nuclear RNA and DNA in the presence and absence of 2′-deoxycorformycin indicated that formycin substitution in RNA and DNA was enhanced 10- and 20-fold, respectively, while [³H]8-azaadenosine incorporation into both nucleic acids was increased 6- to 7-fold. These results suggest that the incorporation of formycin into nucleic acids, particularly DNA, correlates closely with its lethal effect on cell viability. On the other hand, the cytocidal activity of 8-azaadenosine more clearly parallels its inhibitory effect on DNA synthesis rather than its substitution into nucleic acids.     

Initial studies on the mechanism of action of a new oncolytic thiazole nucleoside, 2-beta-D-ribofuranosylthiazole-4-carboxamide (NSC 286193)

Studies on the mechanism of action of a new oncolytic nucleoside, 2-β-d-ribofuranosylthiazole-4-carboxamide, have been undertaken using P388 murine leukemia cells growing in culture. The title compound was cytotoxic at micromolar levels, but a number of simple substitutions of both the ring and sugar moieties nullified cytotoxicity. Cytofluorimetric analysis revealed that the drug arrests cells in the “S phase” of the cell cycle. At antiproliferative concentrations, the agent inhibited the synthesis of both RNA and DNA. The macromolecular incorporation of preformed pyrimidines, including thymidine, was inhibited by the drug but, among the purines, this effect extended only to members of the adenine family and, in fact, the utilization of guanine and its congeners was reproducibly stimulated. When an examination was made of the ability of a comprehensive series of preformed purines and pyrimidines to overcome the inhibition of thymidine incorporation provoked by exposure to the thiazole nucleoside, the guanines were notably effective, but xanthosine also was shown to be an active antidote. Confirmation that the drug was producing a state of guanine deprivation was provided by high performance liquid chromatography (HPLC) analysis of acid-soluble extracts: a time-dependent fall in the concentrations of GMP and GTP ensued upon exposure to the drug; on the other hand, IMP concentrations increased by ～15-fold. Pursuant to these findings, an examination was made of the enzymologic steps unique to the biosynthesis of guanine nucleotides in cells exposed to cytotoxic concentrations of the drug. No prominent inhibition of GMP synthetase could be demonstrated in vitro or in culture, but the specific activity of IMP dehydrogenase underwent substantial reductions in both of these cases. HPLC analyses of extracts of cultures exposed to supralethal concentrations of the title compound provided evidence of modest anabolism to the 5′-monophosphate among other products; in vitro a chemically synthesized sample of 2-β-d-ribofuranosylthiazole-4-carboxamide-5′-monophosphate was twenty times more potent than the parent nucleoside in inhibiting IMP dehydrogenase. On kinetic analysis, this inhibition was non-competitive with IMP as the variable substrate.     

N6-methyl-AMP aminohydrolase activates N6-substituted purine acyclic nucleoside phosphonates

In this study we present the identification and characterization of the enzyme involved in the N6-cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (N6-cyclopropyl-PMEDAP) conversion to biologically active 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) as well as abacavir 5'-phosphate to carbovir 5'-phosphate. This enzyme was purified from rat liver to homogeneity; it appears to be composed from six 42 kDa subunits and its native form has the molecular weight 260 kDa. This so far unknown enzyme catalyzes conversion of both N6-methyl-AMP and N6-methyl-dAMP to IMP and/or dIMP, respectively. The enzyme acts as 6-(N-substituted amino)purine 5'-nucleotide aminohydrolase with the reaction mechanism very similar to AMP deaminase. The enzyme does not deaminate AMP and dAMP, or the corresponding nucleosides. It is inhibited by deoxycoformycin 5'-phosphate but not by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).     

Effect of reserpine on adenosine uptake and metabolism,and subcellular transport of platelet adenosine triphosphate in washed rabbit platelets

There is a slow exchange of adenine nucleotides between the metabolically active (cytoplasmic) pool and the releasable amine storage organelle pool of blood platelets. Reserpine is known to inhibit serotonin uptake into platelet storage organelles. Therefore, we have determined whether reserpine also inhibits the uptake of adenine nucleotides from the cytoplasm into the storage organelles of rabbit platelets. Transport of adenine nucleotides from the metabolically active pool into the releasable amine storage granule pool was followed by labeling the metabolically active pool of adenine nucleotides by incubating the platelets with [¹⁴C]adenosine or [¹⁴C]adenine. Practically complete release of amine storage granule constituents was induced at various times in aliquots of the labeled platelet suspensions by treatment with a high concentration of thrombin (0.45 units/ml. The fraction of the total labeled [¹⁴C]ATP released was taken as a measure of ATP transport from the metabolically active pool into the releasable pool. Reserpine (0.2 and 2 μM) decreased the rate of ATP transport into the storage granules by about 50 per cent. Platelets obtained from rabbits that had received 5 mg/kg of reserpine intraperitoneally 18 hr prior to the collection of blood released less ATP and ADP than control platelets from animals that had not received any drugs. This was not due to inhibition of the release reaction by reserpine. Since reserpine reduces the amount of adenine nucleotides in the storage granules, we conclude that if it affects the rate of efflux of adenine nucleotides from the granules at all, this effect must be slight compared with the inhibition of the uptake into the granules. Reserpine was also found to decrease the incorporation of [8-¹⁴C]adenosine into platelet adenine nucleotides by inhibiting adenosine uptake into the platelets noncompetitively (K_i = 2 μM). Inosine uptake was also inhibited by reserpine. The effect of reserpine on adenosine uptake was reversible. In contrast, the effect of reserpine on ATP transfer from the metabolically active pool into the releasable pool was irreversible. This is in keeping with earlier observations that some reserpine binds to platelets reversibly and some binds irreversibly.