9 -{[2 -Hydroxy-I -(hydroxymethyl)ethoxy]methyllguanine (2'-nor-2'-deoxyguanosine; 2'NDG) selectively inhibits the replication of herpes group viruses. In cell culture studies 2'NDG was at least 10-fold more potent than acyclovir (ACV) in inhibition of human cytomegalovirus replication and Epstein-Barr virus-induced lymphocyte transformation and was about as effective as ACV in inhibition of herpes simplex viruses 1 and 2 and varicella zoster virus. Orally administered 2'NDG was 6-to 50-fold more efficacious than ACV in treating systemic or local HSV-1 infection or HSV-2 intravaginal infection in mice. The mode of action of 2'NDG appears to involve phosphorylation by herpes simplex virus thymidine kinase and subsequent phosphorylations by cellular kinases to produce 2'NDG triphosphate, which is a potent inhibitor of herpes virus DNA polymerase. Compared to ACV, 2'NDG was a more efficient substrate for HSV-1 thymidine kinase (Vma./Km for 2'NDG 30-fold higher than that for ACV), whereas 2'NDG monophosphate is a more efficient substrate for GMP kinase (Vm./Km for 2'NDG monophosphate 492-fold higher than that for ACV monophosphate). The combined effect is more rapid production of the inhibitory triphosphate from 2'NDG than from ACV.As part of our studies on the structure-activity relationships of herpes virus encoded thymidine kinase (TK) and DNA polymerase, a nucleoside analog, 9-{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyl}guanine (2'-nor-2'-deoxyguanosine; 2'NDG) (1-3) was synthesized. 2'NDG is an efficient substrate for the herpes simplex virus 1 (HSV-1) TK and is readily converted to the triphosphate, a potent inhibitor of viral DNA polymerase (1).In the present studies, a chemical synthesis of 2'NDG, the characteristics of its selective phosphorylation by HSV-1 TK, and its rapid conversion to the triphosphate are more fully described. In addition, data are presented demonstrating that the rapid phosphorylation of 2'NDG is correlated with potent inhibition of herpes virus replication in cell cultures and both prophylactic and therapeutic efficacy against herpes virus infections in mice.MATERIALS AND METHODS Materials. Phosphocreatine, creatine kinase, ATP, deoxythymidine, and dGMP were purchased from Sigma; GMP kinase (hog brain) and NADH, from Boehringer Mannheim; lactate dehydrogenase, from Worthington; [methyl-3H] by determining the drug concentration (,ug/ml) required to confer a 50% plaque inhibition on duplicate cell monolayers [for HSV-1, VZV, HCMV, Mengo virus, and vaccinia virus]. For both assays, the antiviral compound was added to the maintenance medium at the time of infection. Viral cytopathic effect was evaluated after incubation for 5 days at 37°C, and plaque development was evaluated after incubation for 3 days (7 days for HCMV) at 370C. Inhibition of EBV replication was measured by prevention of transformation of normal cord lymphocytes to lymphoblastoid cells. In brief, lymphocyte-rich suspensions were prepared from fresh, heparinized human cord blood specimens by differential centrifu...

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 farnesolderived. By feeding [1-14 C]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.Squalene synthase is an attractive target for the development of a cholesterol synthesis inhibitor that could serve as a cholesterol lowering agent. Cholesterol synthesis inhibitors, such as lovastatin (1), a 3-hydroxy-3-methylglutaryl-coenzyme A inhibitor, are effective cholesterol-lowering agents in man and/or animals. Squalene synthase catalyzes the first committed step in cholesterol synthesis, and selective inhibition of this enzyme should result in inhibition of cholesterol synthesis without affecting the synthesis of other isoprenoids such as dolichol, ubiquinone, and the prenylated proteins. A novel class of fungal metabolites, known as zaragozic acids, has been recently discovered and characterized as potent inhibitors of squalene synthase (2-8). The zaragozic acids are subnanomolar inhibitors of squalene synthase in vitro, they inhibit cholesterol synthesis from acetate or mevalonate in cell culture and in animal models, and also have been shown to lower plasma cholesterol when administered orally in certain animal species (4, 5,8). Other classes of squalene synthase inhibitors have also been discovered (for a review, see Ref. 9).An important question to be answered for these compounds is the metabolic effect of inhibition of squalene synthase. The squalene synthase reaction consists of the reductive dimerization of two molecules of farnesyl diphosphate (FPP) 1 to form a molecule of squalene (10). The primary consequence of inhibition of this reaction would be an accumulation of FPP. Thus, the metabolic fate of FPP in the presence of a squalene synthase inhibitor is of interest. Previous wor...

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Immobilized insulin, prepared by coupling insulin directly to agarose or through hydrocarbon "connecting arms," was demonstrated to be capable of firmly binding intact adipocytes and their ghosts. Various lines of evidence indicate that the insulin receptor on the plasma membrane, in addition to the insulin coupled to the agarose, was responsible for the observed binding. This evidence includes: (a) the finding that increasing the "arm" length increased the binding capacities of insulinSepharose affinity chromatographic columns, (b) specific inhibition and reversal by insulin and antiserum to insulin of the binding, as compared to lesser effects by other peptide hormones, (c) the indication that only the plasmamembrane sacs, not the other cellular contaminants in the crude ghosts, are capable of binding, and (d) the impairmerit and restoration of trypsin-sensitive membrane binding sites that are also required for insulin biosensitivity. These findings support the idea that the insulin receptor is the trypsin-sensitive site. By use of the differential btloyant densities of the various cell-bead complexes that resulted from the interaction of adipocytes with insulinSepharose, a new procedure was developed to demonstrate and study the binding. These complexes could also be demonstrated by interference contrast microscopy. Binding readily occurred under conditions favorable for insulin stimulation of the cells. By coupling tracer amounts of [1251]insulin to Sepharose oj insulin-Sepharose, the effects of anti-insulin antisera, free insulin, and other peptide hormones and supplemental factors on the buoyant-density distribution of the complexes could be measured, as well as the effects of other ligands coupled to Sepharose.Despite the rapidly accumulating literature on the various applications of proteins coupled ("immobilized") to insoluble matrices, most of the published research on the insulin (I) receptor used free radioiodinated insulin, rather than immobilized native insulin, as the interacting hormonal agent. Except for our earlier preliminary report (1), there has been no published evidence demonstrating the ability of immobilized insulin to bind insulin receptor-containing intact cells or membranes. Other recent studies iih our laboratory (1, 2) and by Cuatrecasas (3) It had been indicated previously that insulin coupled to Sepharose (S) retains the biological properties characteristic of the native hormone (4-6). Because of the relatively large size of the Sepharose beads, it was concluded that the hormone acts on the surface membrane of the cell (4). However, since Oka and Topper (6) could observe no actual binding of insulin-Phe-Sepharose beads to mammary epithelial cells that were sensitive to the immobilized-but not the free-hormone, they questioned the involvement of binding, from a mechanistic point of view, in the biological response to the hormone.Specific binding of [ln2I]insulin(monoiodinated) to liverand fat-cell membranes and intact fat cells has been demonstrated (7-9) and shown to corr...