Synthesis and antiviral evaluation of analogs of adenosine-N1-oxide and 1-(Benzyloxy)adenosine.

The activity of a series of compounds related to adenosine-N1-oxide (1) and 1-(benzyloxy)adenosine (42) against vaccinia virus has been determined both in vitro and in a vaccinia mouse tailpox model. Significant activities have been found both in vitro and in vivo for a number of the synthetic compounds.

Safety factors involved in the extraction of biologically active proteins from human immunodeficiency virus.

The disruption of the viral coat of human immunodeficiency virus by Triton X-100, a nonionic detergent, is a time-dependent process which requires incubation times of 30 min or longer. Conditions for the production of a noninfectious sample from a viral pellet that can be used to measure reverse transcriptase activity were determined.

Polyamine pools in HIV-infected cells.

The polyamines putrescine, spermine, and spermidine, present in all living cells, have been implicated in the replication of some herpesviruses and retroviruses, and elevated levels of these polyamines have been found in the lymphocytes of patients infected with HIV-1. We have examined the effect of HIV-1 infection on polyamine pools in cell culture. HIV-1 did not significantly affect the polyamine pools in CEM cells. Consistent with this observation, inhibitors of the two key enzymes of this pathway, ornithine decarboxylase and S-adenosylmethionine decarboxylase, did not prevent viral-induced cytopathic effects (CPE) in this cell line. Our results indicate that inhibitors of this pathway will not be therapeutically useful in the treatment of AIDS.

Optimization of the reverse transcriptase assay for the detection of viral burden in mice infected with Rauscher murine leukemia virus.

Rauscher murine leukemia virus induces an erythroleukemia in susceptible strains of mice that is associated with splenomegaly and viremia. This animal model has been used for evaluating the in vivo efficacy of potential anti-HIV agents. The in vivo antiviral activity of therapeutic agents has usually been determined by measuring a reduction in the spleen weights of compound-treated mice or by quantitating viremia with the UV-XC plaque assay. The UV-XC assay, however, is time-consuming and labor-intensive. Virions of Rauscher murine leukemia virus, like other retroviruses, contain the enzyme reverse transcriptase. Quantitating the level of this enzyme in infected mouse sera provides a more rapid measure of viremia in the animal. We have examined the effects of several reagents, including detergent, KCl, EGTA, dGMP, spermine, as well as protease and RNase inhibitors, on the reverse transcriptase assay. The optimized assay method was effective in evaluating the antiviral activity of AZT in the Rauscher murine leukemia virus in vivo model. The assay is also amenable to automation if large numbers of assays are required.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin.

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

Characterization of S-adenosylmethionine decarboxylase induced by human cytomegalovirus infection.

Infection of human diploid embryonic lung (MRC5) cells by human cytomegalovirus (HCMV), strain AD169, increased the activity of a key enzyme in the synthesis of polyamines: S-adenosylmethionine decarboxylase (E.C. 4.1.1.50). The initial peak of S-adenosylmethionine decarboxylase activity occurred about 15 h postinfection. S-Adenosylmethionine decarboxylase was purified using a highly specific affinity chromatography step from HCMV-infected and control uninfected MRC5 cells. No difference was found between the two enzymes in their stability to heat or effect of pH on activity. Both enzymes were activated only by putrescine. The appKm for S-adenosylmethionine for the virus-induced enzyme was 1.7 times higher than the appKm for the control enzyme. The most dramatic difference observed was in the effect of high salt concentration on enzyme activity. S-Adenosylmethionine decarboxylase from HCMV-infected cells was unaffected by 0.8 M NaCl, whereas the enzyme from uninfected cells was inhibited by 50% at 0.45 M NaCl and was significantly inhibited at a concentration of 0.8 M NaCl. Thus, different forms of S-adenosylmethionine decarboxylase probably exist in infected and uninfected MRC5 cells.

Lack of synergy in the inhibition of HIV-1 reverse transcriptase by combinations of the 5'-triphosphates of various anti-HIV nucleoside analogs.

3'-Deoxy-3'-azidothymidine (AZT) has been shown to synergistically inhibit the replication of human immunodeficiency virus type 1 (HIV-1) in cell culture when combined with several other 2',3'-dideoxynucleoside analogs. In an effort to understand the biochemical mechanism of this synergy, we have examined the effect of combinations of the 5'-triphosphate of AZT (AZT-TP) with either ddCTP, ddATP, or the 5'-triphosphate of the carbocyclic analog of 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir) on both the RNA-directed and DNA-directed DNA polymerase activity of HIV-1 reverse transcriptase. Kinetic studies, which evaluated the ability of these combinations to competitively inhibit the enzyme, showed that AZT-TP could not bind to the enzyme with either the RNA or DNA template at the same time as either of the other three inhibitors. None of these analogs could affect the incorporation of another analog into the DNA chain by the HIV-1 reverse transcriptase. These results indicated that synergistic inhibition of the HIV-1 reverse transcriptase is not responsible for the synergistic antiviral activity seen in cell culture with combinations of these nucleoside analogs.

Phosphorylation of the enantiomers of the carbocyclic analog of 2'-deoxyguanosine in cells infected with herpes simplex virus type 1 and in uninfected cells. Lack of enantiomeric selectivity with the viral thymidine kinase.

CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.

Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents.

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.

Thiazolobenzimidazole: biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor.

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

Metabolism of carbovir, a potent inhibitor of human immunodeficiency virus type 1, and its effects on cellular metabolism.

Carbovir (CBV) [the (--)-enantiomer of the carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine] is a potent inhibitor of human immunodeficiency virus type 1 (HIV) replication in vitro. We have characterized the metabolism of CBV and its effect on cellular metabolism in an effort to better understand its mechanism of action. CBV was primarily metabolized to the 5'-triphosphate of CBV (CBV-TP) to concentrations sufficient to inhibit HIV reverse transcriptase. Infection of CEM cells with HIV did not affect the metabolism of CBV. In CEM cells, there was no evidence of the degradation of CBV by purine nucleoside phosphorylase. The half-life of CBV-TP in CEM cells was 2.5 h, similar to that of the 5'-triphosphate of zidovudine (AZT). However, unlike the levels of the 5'-triphosphate of AZT, CBV-TP levels declined without evidence of a plateau. CBV did not affect the metabolism of AZT, and AZT did not affect the metabolism of CBV. A small amount of CBV was incorporated into DNA in intact CEM cells, and this incorporation was increased by incubation with mycophenolic acid, an inhibitor of IMP dehydrogenase. CBV specifically inhibited the incorporation of nucleic acid precursors into DNA but had no effect on the incorporation of radiolabeled precursors into RNA or protein. CBV did not decrease the level of TTP, dGTP, dCTP, or dATP. These results suggested that the cytotoxicity of CBV was due to the inhibition of DNA synthesis. Further studies are necessary to identify the target(s) responsible for growth inhibition.

Activity of triciribine and triciribine-5'-monophosphate against human immunodeficiency virus types 1 and 2.

Triciribine (TCN) and its 5'-monophosphate (TCN-P) are novel tricyclic compounds with known antitumor activity; TCN-P is currently in phase II human clinical trials. We now report that these compounds have potent and selective activity against HIV-1 and HIV-2. Using a syncytial plaque assay, TCN and TCN-P were active against HIV-1 at 0.01-0.02 microM and had differential selectivities of 2250 and 1900, respectively, compared to 1850 for AZT. In contrast, TCN and TCN-P had minimal selectivity against human cytomegalovirus (50 and 27, respectively). TCN and TCN-P markedly inhibited HIV-1-induced p24 core antigen production, reverse transcriptase, and infectious virus production in a dose-dependent manner using HIV-1 acutely infected CEM-SS, H9, and persistently infected H9IIIB and U1 cells. In acutely infected PBL cells, TCN and TCN-P inhibited reverse transcriptase and infectious virus production but not p24 core antigen production. Using a microtiter XTT assay, TCN and TCN-P were active against a panel of HIV-1 and HIV-2 strains at IC50 values ranging from 0.02 to 0.46 microM. Evaluation of matched pairs of predrug and postdrug therapy HIV-1 isolates established that AZT-resistant and TIBO-resistant variants of HIV-1 were sensitive to TCN or TCN-P. Furthermore, unlike AZT and other fraudulent nucleosides, neither TCN, TCN-P, nor TCN-TP inhibited the viral reverse transcriptase. Thus, even though triciribine is a nucleoside chemically, it does not act biologically by classic nucleoside modalities but rather by a unique mechanism yet to be elucidated.

Antiviral (RNA) activity of selected Amaryllidaceae isoquinoline constituents and synthesis of related substances.

A series of 23 Amaryllidaceae isoquinoline alkaloids and related synthetic analogues were isolated or synthesized and subsequently evaluated in cell culture against the RNA-containing flaviviruses (Japanese encephalitis, yellow fever, and dengue viruses), bunyaviruses (Punta Toro, sandfly fever, and Rift Valley fever viruses), alphavirus (Venezuelan equine encephalomyelitis virus), lentivirus (human immunodeficiency virus-type 1) and the DNA-containing vaccinia virus. Narciclasine [1], lycoricidine [2], pancratistatin [4], 7-deoxypancratistatin [5], and acetates 6-8, isonarciclasine [13a], cis-dihydronarciclasine [14a], trans-dihydronarciclasine [15a], their 7-deoxy analogues 13b-15b, lycorines 16 and 17, and pretazettine [18] exhibited consistent in vitro activity against all three flaviviruses and against the bunyaviruses, Punta Toro and Rift Valley fever virus. Activity against sandfly fever virus was only observed with 7-deoxy analogues. In most cases, however, selectivity of the active compounds was low, with toxicity in uninfected cells (TC50) occurring at concentrations within 10-fold that of the viral inhibitory concentrations (IC50). No activity was observed against human immunodeficiency virus-type 1, Venezuelan equine encephalomyelitis virus, or vaccinia viruses. Pancratistatin [4] and its 7-deoxy analogue 5 were evaluated in two murine Japanese encephalitis mouse models (differing in viral dose challenge, among other factors). In two experiments (low LD50 viral challenge, variant I), prophylactic administration of 4 at 4 and 6 mg/kg/day (2% EtOH/saline, sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 100% and 90%, respectively. In the same model, prophylactic administration of 5 at 40 mg/kg/day in hydroxypropylcellulose (sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 80%. In a second variant (high LD50 viral challenge), administration of 4 at 6 mg/kg/day (ip, twice daily for 9 days, day -1 to +7) resulted in a 50% survival rate. In all cases, there was no survival in the diluent-treated control mice. Thus, 4 and 5 demonstrated activity in mice infected with Japanese encephalitis virus but only at near toxic concentrations. To our knowledge, however, this represents a rare demonstration of chemotherapeutic efficacy (by a substance other than an interferon inducer) in a Japanese-encephalitis-virus-infected mouse model.

Interference with HIV-1 reverse transcriptase-catalyzed DNA chain elongation by the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine.

In an effort to better understand features in nucleotide analogs that result in the inhibition of HIV-1 reverse transcriptase, we have evaluated this enzyme with the 5'-triphosphate of the carbocyclic analog of 2'-deoxyguanosine (CdG-TP). CdG-TP was a reasonably potent competitive inhibitor of the incorporation of dGTP into DNA by HIV-1 reverse transcriptase using either a RNA or DNA template (Ki, 1 microM). CdG-TP was a good substrate for HIV-1 reverse transcriptase on both templates, but the DNA chain was poorly extended beyond the incorporation of CdG. These results indicate that substitution of ribose with a cyclopentane ring in nucleotides is not well tolerated by HIV-1 reverse transcriptase.

5,5-disubstituted hydantoins: syntheses and anti-HIV activity.

A series of 5,5-disubstituted hydantoin derivatives was synthesized by alkylating 5,5-bis(mercaptomethyl)-2,4-imidazolidinedione (3) with various halomethylaromatic or halomethylheteroaromatic precursors, or by using the Buchener-Berg procedure on the required ketone. When evaluated for their ability to inhibit HIV-induced cell killing and virus production in CEM or MT-2 cells only compounds 2, 4n, 4o, and 4i demonstrated modest activity, the latter with an IC50 = 53 microM.

3'-Azido-3'-deoxythymidine-induced reduction in the ability of uninfected CD4-expressing cells to participate in syncytium formation.

A cocultivation assay system consisting of uninfected human T cells and cells chronically infected with human immunodeficiency virus type 1 has been used to investigate syncytium formation in short-term assays. Continuous treatment or short-term pretreatment of uninfected CD4-expressing human T-cell lines with 3'-azido-3'-deoxythymidine (AZT) reduces the ability of these cells to participate in syncytium formation when mixed with chronically infected cells. The effect of AZT on syncytium formation is observed both as a reduction in the number of syncytia and as a reduction in the size of the syncytia that are detected. This syncytium-reducing effect of AZT is dose and time dependent and does not result from a modulation of CD4 antigen expression on the cell surface of uninfected, treated cells. Maximum syncytium reduction is observed with the continuous presence of AZT; however, pretreatment for times as short as 15 min results in a significant reduction in syncytium formation. Since reverse transcription is not required for efficient syncytium formation, the syncytium-reducing effect of AZT on uninfected human cells may represent an antiviral property of AZT with important therapeutic potential.

Incorporation of the carbocyclic analog of 2'-deoxyguanosine into the DNA of herpes simplex virus and of HEp-2 cells infected with herpes simplex virus.

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.

Novel method for evaluating antiviral drugs against human cytomegalovirus in mice.

A virus-host cell system in which human cytomegalovirus-infected human cells are entrapped in agarose plugs has been developed. This model provides an inexpensive method for the in vivo evaluation (with outbred, immunocompetent mice) of antiviral drugs against human viruses such as cytomegalovirus that replicate primarily or only in human cells.

Significant anti-HIV activity of new modified polyanionic polymers in vitro.

The anti-HIV activities of two new polyanionic polymers (AM 242 and AM 612) were investigated in cell culture-based and biochemical antiviral assays. These compounds inhibited the reverse transcriptases from HIV-1 and HIV-2, using enzyme purified from virions and either a ribosomal RNA or gapped duplex DNA as the template. With the ribosomal RNA template, AM 242 and AM 612 had ID50 values of 1.1 and 0.10 micrograms/ml against the HIV-1 reverse transcriptase. In vitro cell based assays determined that both compounds significantly inhibited both the cytopathic effects associated with HIV-1 infection and the replication of virus in infected cells. AM 242 had an IC50 of approximately 1.0 micrograms/ml, while that of AM 612 was 0.19 micrograms/ml. These two active polyanionic polymers were effective in inhibiting the growth of a panel of HIV-1 isolates and were also active against HIV-2. Although the compounds were toxic at high concentration, they had antiviral activity over a wide range of nontoxic concentrations, yielding a high selectivity index. AM 612 was 100% protective for CEM cells from 320 ng/ml to 1 microgram/ml. Both compounds caused a significant increase in cellular proliferation as determined by the concentration-dependent increase in incorporation of radioactive precursors into cellular macromolecules.