Stereochemical considerations in the enzymatic phosphorylation and antiviral activity of acyclonucleosides. I. Phosphorylation of 2'-nor-2'-deoxyguanosine.

The antiviral compound 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (2'-nor-2'-deoxyguanosine, 2'-NDG) is phosphorylated by the HSV-1-induced thymidine kinase to the monophosphate (2'-NDG-MP) and this is further phosphorylated by cellular kinases to the triphosphate (2'-NDG-TP) which is a potent inhibitor of DNA polymerases. Since phosphorylation of 2'-NDG creates a chiral center in the molecule, it was of interest to examine whether both monophosphate enantiomers were produced by the viral thymidine kinase, whether they both could be further phosphorylated by cellular kinases and, if so, whether the respective triphosphates were equally inhibitory to the DNA polymerases. The time course of the phosphorylation by GMP kinase of a chemically synthesized, racemic 2'-NDG-MP was compared to that of a 2'-NDG-MP preparation obtained by enzymatic phosphorylation of 2'-NDG with HSV-1 thymidine kinase. The results indicated that the two enantiomeric monophosphates were phosphorylated by GMP kinase with different rates and that phosphorylation of 2'-NDG by HSV-1 thymidine kinase gave only one of the isomers, whose structure was determined to be S. Both enantiomeric diphosphates were further phosphorylated to the respective triphosphates and it was shown that, in contrast to the triphosphate obtained from the 2'-NDG-MP prepared by viral thymidine kinase which was a potent inhibitor of HSV-1 DNA polymerase, the triphosphate obtained from the slow-reacting R isomer had little or no inhibitory activity against this enzyme.

Hepatic responses to inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase: a comparison of atorvastatin and simvastatin.

We have compared the cellular responses to simvastatin (Simva) and atorvastatin (Atorva), two potent HMG-CoA reductase inhibitors. The two drugs exhibited similar IC50's for inhibition of either rat or human reductase, and single oral dosing in rats showed the compounds to be nearly equipotent at inhibiting hepatic cholesterol synthesis. Treatment of rats with Simva or Atorva in the feed for four days yielded comparable inductions of hepatic reductase activity and reductase protein. For example, 0.05% Simva induced reductase activity 27.3 +/- 9.1 fold and 0.05% Atorva induced activity 26.9 +/- 4.7 fold. This adaptive response was also studied in HepG2 cells, a human hepatoblastoma line, cultured for 24 h in delipidated serum and then for an additional 24 h with Simva or Atorva. Over a broad range (10 nM-10 microM), both drugs caused similar inductions of reductase activity, reductase protein, and reductase mRNA. Under all conditions, the drugs induced similar changes in the ratio of mRNA/protein suggesting that Simva and Atorva have similar effects on both transcriptional and post-transcriptional regulatory machinery. Moreover, reductase in cells treated with Simva or Atorva for 22 h responded similarly to subsequent challenge with 25-hydroxycholesterol. Finally, we measured the ability of the two reductase inhibitors to reduce ApoB secretion by HepG2 cells. Simva and Atorva at 0.5 microM inhibited ApoB secretion nearly identically, 38% and 42% respectively. We conclude that these two drugs induce similar adaptive responses in cells and that their actions are qualitatively and mechanistically identical. Human studies have shown that plasma is cleared of Atorva much more slowly than it is of Simva. The large pharmacokinetic difference in man, rather than some difference in mechanism, is the most likely explanation for the finding that the equipotent dose ratio for cholesterol lowering in humans of Simva to Atorva is about 2/1.

Enzymatic phosphorylation of the antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine.

The antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (iNDG) is phosphorylated by HSV1 thymidine kinase, and its phosphorylated products inhibit DNA polymerase activity. iNDG exists in two enantiomeric forms, each with a primary and a secondary hydroxyl; thus, a number of possibilities for preferential phosphorylation exist, which were explored in this study. HSV1 thymidine kinase phosphorylates the primary hydroxyl of both the R and the S isomers of iNDG. This was established by comparison with analogues in which either the primary or the secondary hydroxyl was replaced by fluorine or hydrogen and also by a study of the NMR spectrum of the monophosphate. GMP kinase phosphorylates the R and the S monophosphates to the respective diphosphates. Further phosphorylation, however, is much more efficient with the S than with the R isomer. Furthermore, (S)-iNDG triphosphate is a more potent inhibitor of HSV1 DNA polymerase than (R)-iNDG triphosphate. These differences in the biochemical specificities of the two isomers account for the observed higher antiviral potency of (S)-iNDG as compared to that of (R)-iNDG.

3-Hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors. 7. Modification of the hexahydronaphthalene moiety of simvastatin: 5-oxygenated and 5-oxa derivatives.

Modification of the hexahydronaphthalene ring 5-position in simvastatin 2a via oxygenation and oxa replacement afforded two series of derivatives which were evaluated in vitro for inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acutely in vivo for oral effectiveness as inhibitors of cholesterogenesis in the rat. Of the compounds selected for further biological evaluation, the 6 beta-methyl-5-oxa 10 and 5 alpha-hydroxy 16 derivatives of 3,4,4a,5-tetrahydro 2a, as well as, the 6 beta-epimer 14 of 16 proved orally active as hypocholesterolemic agents in cholestyramine-primed dogs. Subsequent acute oral metabolism studies in dogs demonstrated that compounds 14 and 16 evoke lower peak plasma drug activity and area-under-the-curve values than does compound 10 and led to the selection of 14 and 16 for toxicological evaluation.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 9. The synthesis and biological evaluation of novel simvastatin analogs.

Substitution of hydroxy and hydroxyalkyl functionality at C-7 of the hexahydronaphthalene nucleus of simvastatin has provided novel analogs. The synthetic strategy employed epoxidation or Lewis acid-catalyzed aldol reaction of the 8-keto silyl enol ether as a key reactive intermediate. These analogs were evaluated as potential hypocholesterolemic agents via initial determination of their ability to inhibit HMG-CoA reductase in vitro. Oral activity of these compounds was determined in an acute rat model and a three-week study in cholestyramine-primed dogs. Compounds were identified that possessed in vitro and in vivo activity comparable to that of simvastatin.

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

Inverse relationship between galactokinase activity and 2-deoxygalactose resistance in Chinese hamster ovary cells.

Galactokinase activity is reduced in 12 independent clones of Chinese hamster ovary cells resistant to 2-deoxygalactose. The frequency of resistant colonies is increased with chemical mutagens. The resistant phenotype is stable in the absence of selection. There is an inverse correlation between the levels of galactokinase activity and the cloning efficiency in deoxygalactose. Cells with high resistance have 1% or less of the enzyme activity observed in the parental cells; while cells with low resistance have 10-30% galactokinase activity. Studies with tetraploid hybrid cells reveal that resistance to deoxygalactose is a recessive trait and that cells with high resistance do not complement those with low resistance. In cell lines with low resistance, the Km for galactose, Ki for deoxygalactose, Km for ATP, and thermolability were not significantly altered compared to sensitive parental cells. Although the possibility of mutation at the structural gene locus has not been ruled out, the reduced enzyme activity may also be due to mutation at a regulatory site which affects the number of galactokinase molecules per cell.

Massive production of farnesol-derived dicarboxylic acids in mice treated with the squalene synthase inhibitor zaragozic acid A.

The zaragozic acids are potent inhibitors of squalene synthase. In vivo studies in mice confirmed our earlier observations that inhibition of squalene synthase by zaragozic acid A was accompanied by an increase in the incorporation of label from [3H]mevalonate into farnesyl-diphosphate (FPP)-derived isoprenoic acids (J. D. Bergstrom et al., 1993, Proc. Natl. Acad. Sci. USA 90, 80-84). Farnesyl-diphosphate-derived metabolites appear transiently in the liver. We were unable to detect any farnesol formation in the zaragozic acid-treated animals which indicates that FPP is readily converted to farnesoic acid and dicarboxylic acids in the liver. These metabolites were found to be produced only in the liver and not in the kidney. trans-3,7-Dimethyl-2-octaen-1,8-dioic acid and 3, 7-dimethyloctan-1,8-dioic acid were identified as the major end products of farnesyl-diphosphate metabolism in the urine of mice treated with zaragozic acid A. Quantitative analysis of these FPP-derived dicarboxylic acids by gas-liquid chromatography revealed that approximately 11 mg of total dicarboxylic acids is excreted per day into the urine of a mouse after 3 days of treatment with zaragozic acid A.

Tissue selectivity of the cholesterol-lowering agents lovastatin, simvastatin and pravastatin in rats in vivo.

Tissue selectivity of lovastatin, simvastatin and pravastatin was determined in male rats. Peak levels of active drug were found in all tissues examined between 0.5 and 2 hours after oral administration. The area under the curve describing 24 hour exposure of the tissues to drug indicated that the drugs were preferentially concentrated in the liver. However, the concentration of pravastatin was approximately 50% that of either lovastatin or simvastatin in the liver and 3-6 times higher in peripheral tissues. These studies demonstrate that the hydrophobic prodrugs, lovastatin and simvastatin show greater selectivity than the hydrophilic agent pravastatin towards the liver which is the target organ for inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Comparison of the modes of antiviral action of 2'-nor-deoxyguanosine and its cyclic phosphate, 2'-nor-cyclic GMP.

The metabolisms of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) and its cyclic phosphate, 9-[(2-hydroxy-1,3,2-dioxophosphorinan-5-yl) oxymethyl]guanine P-oxide (2'-nor-cGMP), were compared in cultures of primary rabbit kidney cells infected with herpes simplex virus type 1 (HSV-1). 2'-Nor-cGMP was taken up by the cells essentially intact, after which it was opened to the acyclic monophosphate and phosphorylated further, ultimately to the triphosphate. Formation of the triphosphate was independent of HSV thymidine kinase expression, unlike what is observed with 2'NDG. In addition, there was a direct correlation between the antiviral activity of 2'NDG and the level of triphosphate formed in HSV-1-infected cells, whereas such a correlation was absent with 2'-nor-cGMP. In vivo experiments indicated that only a small percentage of free 2'NDG was formed in the bloodstream of mice after oral administration of 2'-nor-cGMP. Incubation of 2'-nor-cGMP with crude extracts of HSV-1-infected or uninfected HeLa cells resulted in the direct production of 2'NDG triphosphate. The possibility that the triphosphate of 2'NDG produced from 2'-nor-cGMP was the enantiomer of the triphosphate made from 2'NDG by viral and cellular kinases was investigated and disproved. Taken together, these data indicate that (i) 2'-nor-cGMP does not act simply as a prodrug of 2'NDG, (ii) 2'-nor-cGMP does not require viral thymidine kinase for its activity, and (iii) 2'-nor-cGMP may have an additional, triphosphate-independent mode of action.

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells.

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.

A comparison of the antiviral agents 2'-nor-2'-deoxyguanosine and acyclovir: uptake and phosphorylation in tissue culture and kinetics of in vitro inhibition of viral and cellular DNA polymerases by their respective triphosphates.

A comparative study was conducted between the antiherpetic agents 2'-nor-2'-deoxyguanosine (2'NDG) and acyclovir (ACV) with respect to 1) the relative rates of uptake and phosphorylation to the "active" triphosphate species in tissue culture and 2) the in vitro inhibition of viral and cellular DNA polymerases by their respective triphosphates. The results indicated that a) six hours after HSV1 infection of primary rabbit kidney cells there was seven times more 2'NDG-triphosphate in the cells than ACV triphosphate; b) the relative rate of triphosphate formation in HSV1-infected versus uninfected cells was 4.5 times higher for 2'NDG than for ACV and c) the triphosphate of 2'NDG (2'NDG-TP) was a more selective inhibitor of the viral compared to the cellular DNA alpha-polymerase than the triphosphate of ACV (ACV-TP). The Km/Ki ratios for 2'NDG-TP and ACV-TP (in the competitive inhibition of dGTP) were 3.10 and 1.37, respectively, for the highly purified HSV1 polymerase; and 0.05 and 1.11, respectively, for the partially-purified HeLa alpha-polymerase. Neither triphosphate inhibited the HeLa DNA beta-polymerase to any significant extent. These results are in line with the findings [Ashton et al. (1982), Biochem. Biophys. Res. Commun. 108, 1716-1721] that 2'NDG has superior in vivo antiherpetic activity compared to ACV without apparent toxicity.

Farnesol-derived dicarboxylic acids in the urine of animals treated with zaragozic acid A or with farnesol.

Farnesyl diphosphate, the substrate for squalene synthase, accumulates in the presence of zaragozic acid A, a squalene synthase inhibitor. A possible metabolic fate for farnesyl diphosphate is its conversion to farnesol, then to farnesoic acid, and finally to farnesol-derived dicarboxylic acids (FDDCAs) which would then be excreted in the urine. Seven dicarboxylic acids were isolated by high performance liquid chromatography (HPLC) from urine of either rats or dogs treated with zaragozic acid A or rats fed farnesol. Their structures were determined by nuclear magnetic resonance analysis. Two 12-carbon, four 10-carbon, and one 7-carbon FDDCA were identified. The profile of urinary dicarboxylic acids from rats fed farnesol was virtually identical to that produced by treating with zaragozic acid A, establishing that these dicarboxylic acids are farnesol-derived. By feeding [1-14C]farnesol and comparing the mass of the dicarboxylic acids produced with the ultraviolet absorption of the HPLC peaks, a method to quantitate the ultraviolet-absorbing FDDCAs was devised. When rats were treated with zaragozic acid A, large amounts of FDDCAs were excreted in the urine. The high level of FDDCAs that were found suggests that their synthesis is the major metabolic fate for carbon diverted from cholesterol synthesis by a squalene synthase inhibitor. A metabolic pathway is proposed to explain the production of each of these FDDCAs.

Zaragozic acids: a family of fungal metabolites that are picomolar competitive inhibitors of squalene synthase.

Three closely related fungal metabolites, zaragozic acids A, B, and C, that are potent inhibitors of squalene synthase have been isolated and characterized. Zaragozic acids A, B, and C were produced from an unidentified sterile fungal culture, Sporormiella intermedia, and Leptodontium elatius, respectively. The structures of the zaragozic acids and their trimethyl esters were determined by a combination of physical and chemical techniques. The zaragozic acids are characterized by a novel 2,8-dioxobicyclo[3.2.1]octane-4,6,7- trihydroxyl-3,4,5-tricarboxylic acid core and differ from each other in the structures of the 6-acyl and 1-alkyl side chains. They were found to be potent competitive inhibitors of rat liver squalene synthase with apparent Ki values of 78 pM, 29 pM, and 45 pM, respectively. They inhibited cholesterol synthesis in Hep G2 cells, and zaragozic acid A was an inhibitor of acute hepatic cholesterol synthesis in the mouse (50% inhibitory dose of 200 micrograms/kg of body weight). Inhibition of squalene synthase in cells and in vivo was accompanied by an accumulation of label from [3H]mevalonate into farnesyl diphosphate, farnesol, and organic acids. These data indicate that the zaragozic acids are a previously unreported class of therapeutic agents with potential for the treatment of hypercholesterolemia.