Three closely related fungal metabolites, The mammalian isoprenoid pathway not only produces sterols but also produces dolichol, ubiquinone, the farnesyl group of heme A, the farnesyl and geranylgeranyl groups of prenylated proteins, and the isopentenyl side chain of isopentenyl adenine. The pathways for the synthesis of these other isoprenoids diverge from the synthesis ofcholesterol either at or before the farnesyl diphosphate (FPP) branch point. Thus, squalene synthase, which catalyzes the reductive dimerization of 2 mol of FPP to 1 mol of squalene (2, 3), is the first committed step in sterol synthesis. A specific inhibitor of squalene synthase should serve to inhibit cholesterol synthesis and not adversely affect the synthesis of other isoprenoids. FPP, the substrate for squalene synthase, is water soluble and may be readily metabolized (4). Thus, squalene synthase offers a potential target for the safe and specific inhibition of cholesterol synthesis.In this report we describe the isolation, structure, physical characterization, and biological properties of three structurally similar fungal metabolites that are potent inhibitors of squalene synthase. These metabolites, zaragozic acid A (5-7), zaragozic acid B (8, 9), and zaragozic acid C (10-12), had been reported previously only in the patent literature; however, during the review process of this manuscript, three manuscripts (13-15) appeared on the squalestatins: squalestatin I is identical with zaragozic acid A, squalestatin II is des-4'-acetylzaragozic acid A, and squalestatin III is des-6-acylzaragozic acid A. This class of squalene synthase inhibitors has potential utility as cholesterol-lowering agents. MATERIALS AND METHODSZaragozic Acid A, Cultures, and Media. An unidentified sterile fungal culture, ATCC 20986, isolated from a water sample taken from the Jalon river in Zaragoza, Spain (hence the name zaragozic acids), was used to produce zaragozic acid A. The culture was maintained at 25°C on medium B agar slants composed of 4 g of yeast extract, 10 g of malt extract, 4 g of dextrose, and 20 g of agar per liter at pH 7.0.Zaragozic acid A was produced in a two-tiered fermentation process consisting of mycelial growth and development in medium A of ref. 1 and product formation in medium C. Medium C contained 5 g of malt extract, 1 g of peptone, 15 g of dextrose, 1 g of KH2PO4, and 0.5 g of MgSO4 7H20 per liter. Fermentations consisted of mycelial growth in medium A for 72 hr at 250C with agitation, followed by inoculation (5-10%) of medium C. Maximum product was obtained from 14-day agitated fermentations at 250C.Isolation of Zaragozic Acid A. To isolate zaragozic acid A, 23 liters of harvested broth was filtered through Celite, and the mycelial cake was extracted twice with 7 liters of 50%o aqueous methanol. The filtrate was combined with the extracts, diluted with water to a final composition of 25% methanol, and adsorbed on a 1.5-liter column of Mitsubishi HP-20 resin. After a column wash with 6 liters of4:6 (vol/vol) methanol/water, crud...

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...

Since the completion of the two preceding papers of this series1' 2 we have had the opportunity of examining, by direct base analysis, the composition of two -preparations of separated strands isolated, as described before,1 from the DNA of two strains of B. subtilis. The results will be found in Tables 1 and 2. TABLE 1. Composition of native DNA and of separated DNA strands of B. subtilis. Bases, (mole %) I -Strain W23

No abstract