No abstract

A guanosine analog, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG), was found to inhibit herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2, cytomegalovirus, and Epstein-Barr virus replication by >50% at concentrations that do not inhibit cell growth in culture. The potency of the drug against all of these viruses is greater than that of 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir). DHPG was active against HSV-1 growth during the early phase of virus replication and had no activity when added at a later time after infection. Its antiviral activity was irreversible. Thymidine partially neutralized its action. The anti-HSV-1 activity of DHPG was dependent on the induction and the properties -of virus-induced thymidine kinase. Virus variants that induced altered virus thymidine kinase and became resistant to acyclovir were still as sensitive to DHPG as the parental virus. DHPG is active against five different HSV variants with induced altered DNA polymerase and resistance to acyclovir. Cells infected by herpesviruses, which are important causes of human diseases, undergo many biochemical changes, among which are changes in several virus-specified enzymes that are involved in either deoxynucleotide or DNA metabolism (1). Nucleoside analogs with unique behavior toward such enzymes have been discovered to have selective anti-herpesvirus activity (2-9). Among the nucleoside analogs discovered, 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir), one of the new nucleoside analogs with such activity, was recently licensed in the United States for the treatment of primary genital herpesvirus infection and disseminated herpetic infection. Some of the other compounds are at different stages of clinical development. Recently, an analog of guanosine, 9-[(1,3-dihydroxy-2-propoxy)-methyl]guanine (DHPG), with the structure shown in Fig. 1, was synthesized independently by Syntex (Palo Alto, CA) (10) and Biologics (Toronto, ON, Canada) (11) and was found to have anti-herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) activity. HSV that lacked the ability to induce virally coded thymidine kinase (TK) was found to be resistant to DHPG. Nevertheless, phosphonoacetate-resistant HSV, which is crossresistant to acyclovir, was as sensitive to DHPG as the parental virus, whereas a marginal activity of DHPG against Epstein-Barr virus (EBV) and no activity against human cytomegalovirus (CMV) were reported (11).In view of the potency and the unique spectrum of antiviral activity of DHPG, we examined: (i) the activity of this compound against several unique HSV mutants that had either altered TK or DNA polymerase; (ii) its behavior toward HSV-induced TK and its mode of action against HSV-1; and (iii) sensitivity of several strains of CMV and EBV to the drug. The results of the studies are reported in this communication.

The development of specific inhibitors that can block nuclear factor-B (NF-B) activation is an approach for the treatment of cancer, autoimmune, and inflammatory diseases. Several diterpenoids, oridonin, ponicidin, xindongnin A, and xindongnin B were isolated from the herb Isodon rubescens. These compounds were found to be potent inhibitors of NF-B transcription activity and the expression of its downstream targets, cyclooxygenase-2 and inducible nitric-oxide synthase. The mechanisms of action of the diterpenoids against NF-B are similar, but significant differences were also identified. All of the diterpenoids directly interfere with the DNA-binding activity of NF-B to its response DNA sequence. Oridonin and ponicidin have an additional impact on the translocation of NF-B from the cytoplasm to nuclei without affecting IB-␣ phosphorylation and degradation. The effect of these compounds on the interaction of NF-B with consensus DNA sequences is unique. Different inhibitory effects were observed when NF-B bound to various DNA sequences. Both p65/p65 and p50/p50 homodimers, as well as p65/p50 heterodimer association with their responsive DNA, were inhibited. Kinetic studies on NF-B-DNA interaction indicate that the diterpenoids decrease the B max app but have no effect on K d app . This suggests that this class of compounds interacts with both p65 and p50 subunits at a site other than the DNA binding site and subsequently modulates the binding affinity of the transcription factor toward DNA with different NF-B binding sequences. The diterpenoid structure could therefore serve as a scaffold for the development of more potent and selective NF-B inhibitors that target regulated gene transcription.

We examined the effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) on viral DNA, RNA, protein, and enzyme synthesis in HeLa cells infected with herpes simplex virus type 1 and type 2. DHPG inhibited virus DNA synthesis in a dose-dependent fashion. This inhibition was not due to the lack of deoxynucleoside triphosphates which are required for DNA synthesis. This compound has no apparent effect on early and late viral RNA synthesis, viral protein synthesis, or viral thymidine kinase, DNA polymerase, and DNase induction in virus-infected cells.In recent years, several nucleoside analogs have been found to have selective antiherpesvirus activities. Among these, a new guanosine analog, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), was shown to have a broad and unique spectrum of antiherpesvirus activity at concentrations which were not toxic to several mammalian cells in culture (1,4,15,20,23,24). Its antiviral activity in vivo was also demonstrated (8,16,23). In herpes simplex virus (HSV)-infected cells, DHPG was phosphorylated to DHPG monophosphate (DHPGMP) by virus-induced thymidine kinase (1,4,23) and further phosphorylated to DHPG diphosphate (DHPGDP) and DHPG triphosphate (DHPGTP) by cellular kinases (2,8). DHPGTP could substitute for dGTP and be incorporated into DNA by virus-induced DNA polymerase in an efficient manner. The incorporation of DHPG could slow down DNA chain elongation. Furthermore, DHPG-terminated DNA could act as a potent inhibitor of virus-induced DNA polymerase by competing with activated DNA template (9). Early studies by other investigators indicated that DHPG inhibited viral DNA synthesis (23) Santa Barbara, Calif. All chemicals used were reagent grade or better. Cells and virus. Cells were grown at 37°C in RPMI 1640 medium containing 100 p.g of kanamycin per ml and supplemented with 5% horse serum or 5% fetal calf serum for HeLa S3 or Vero cells, respectively. All cultures were found to be mycoplasma free when tested by the 4,6-diamidino-2-phenylindole fluorescence technique (21). HSV-1 (strain KOS) and HSV-2 (strain 333) were maintained as previously described (3).Preparation of samples for isopycnic gradient centrifugation. HeLa S3 cells were plated in a six-well dish at 106 cells per 10-cm2 well. Once the cells were attached to the dish, the growth medium was removed and replaced with Joklik modified (low-phosphate) medium containing 1% fetal calf serum. HSV-1 (KOS) was added at 3 PFU per cell and incubated at 37°C for a 1-h adsorption period. The medium containing virus was then removed, and the cell layer was washed with phosphate-buffered saline (PBS) (0.14 M NaCl, 4.0 mM KCI, 0.5 mM Na2HPO4, 0.15 M KH2PO4) and replaced with 1 ml of medium containing various concentrations of DHPG. After a 2-h incubation period, 25 ,uCi of 32P04 was added to each well. At 8 h after virus infection the samples were harvested by scraping the cells into the medium and centrifuging at 2,000 rpm for 10 min. The cell pellets were washed two times in cold PBS and lysed overnight in buffer (10 mM...