In vitro anti-human immunodeficiency virus (HIV) activities of transition state mimetic HIV protease inhibitors containing allophenylnorstatine.

Transition state mimetic tripeptide human immunodeficiency virus (HIV) protease inhibitors containing allophenylnorstatine [(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] were synthesized and tested for activity against HIV in vitro. Two compounds, KNI-227 and KNI-272, which were highly potent against HIV protease with little inhibition of other aspartic proteases, showed the most potent activity against the infectivity and cytopathic effect of a wide spectrum of HIV strains. As tested in target CD4+ ATH8 cells, the 50% inhibitory concentrations of KNI-227 against HIV type 1 LAI (HIV-1LAI), HIV-1RF, HIV-1MN, and HIV-2ROD were 0.1, 0.02, 0.03, and 0.1 microM, respectively, while those of KNI-272 were 0.1, 0.02, 0.04, and 0.1 microM, respectively. Both agents completely blocked the replication of 3'-azido-2',3'-dideoxythymidine-sensitive and -insensitive clinical HIV-1 isolates at 0.08 microM as tested in target phytohemagglutinin-activated peripheral blood mononuclear cells. The ratios of 50% cytotoxic concentrations to 50% inhibitory concentrations for KNI-227 and KNI-272 were approximately 2,500 and > 4,000, respectively, as assessed in peripheral blood mononuclear cells. Both compounds blocked the posttranslational cleavage of the p55 precursor protein to generate the mature p24 Gag protein in stably HIV-1-infected cells. The n-octanol-water partition coefficients of KNI-227 and KNI-272 were high, with log Po/w values of 3.79 and 3.56, respectively. Degradation of KNI-227 and KNI-272 in the presence of pepsin (1 mg/ml, pH 2.2) at 37 degrees C for 24 h was negligible. Current data warrant further careful investigations toward possible clinical application of these two novel compounds.     

Inhibition of infectivity of human immunodeficiency virus by a novel nucleoside, oxetanocin, and related compounds.

Oxetanocin is a novel nucleoside containing a 4-membered sugar, oxetanosyl-N-glycoside, and adenine. The effects of oxetanocin and related compounds on the infectivity of human immunodeficiency virus (HIV) were examined. They inhibited HIV infectivity in vitro. Allopurinol and mycophenolic acid produced additive anti-HIV effects when added with these compounds.     

In vitro inhibition of human immunodeficiency virus (HIV) type 1 replication by C2 symmetry-based HIV protease inhibitors as single agents or in combinations.

C2 symmetry-based human immunodeficiency virus (HIV) protease inhibitors were examined in vitro as single agents or in combination with 3'-azido-2',3'-dideoxythymidine (AZT) or 2',3'-dideoxyinosine for activity against HIV type 1 (HIV-1). Ten C2 symmetry-based or pseudo-C2 symmetry-based HIV protease inhibitors were active against a laboratory strain (HIV-1IIIB) in the HIV-1 cytopathic effect inhibition assay. Three inhibitors, A75925, A76928, and A77003, selected to represent a range of aqueous solubility and antiviral activity, were active against four different HIV-1 strains tested. These three inhibitors exhibited a significant inhibition of the cytopathic effect of HIV-1 against the CD4+ ATH8 cell line, with 90% inhibitory concentrations ranging from 0.1 to 4 microM. Cellular toxicity was negligible at up to 20 microM. Furthermore, they completely inhibited the replication of monocytotropic strain HIV-1Ba-L in purified monocytes and macrophages at 0.75 to 2 microM. Potent inhibitory activity against a primary HIV-1 isolate and an AZT-resistant HIV-1 variant was also observed for all three inhibitors in phytohemagglutinin-activated peripheral blood mononuclear cells. When these three HIV protease inhibitors and AZT or 2',3'-dideoxyinosine were used in combinations against a primary HIV isolate in phytohemagglutinin-activated peripheral blood mononuclear cells and the results were analyzed with the COMBO program package, their antiviral activities were identified to be synergistic in some cases and additive in others. The present data warrant further investigations of these compounds as potential antiviral agents for the therapy of HIV infections.     

Evaluation of SQ 34,514: pharmacokinetics and efficacy in experimental herpesvirus infections in mice.

The new antiviral nucleoside SQ 34,514 [(1R-1 alpha, 2 beta, 3 alpha)-2-amino-9- [2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one], the active R isomer of racemic SQ 33,054 (cyclobut-G), was evaluated for efficacy in the treatment of herpesvirus infections in mice. SQ 34,514 was orally efficacious in a herpes simplex virus type 1 (HSV-1) systemic infection, an intracerebral HSV-2 infection, a vaginally induced HSV-2 infection in ovariectomized mice, and in a systemic murine cytomegalovirus infection. SQ 34,514 compared favorably with acyclovir and ganciclovir in the treatment of these experimental infections. In mice, SQ 34,514 had an oral bioavailability of 80% based on urinary excretion. SQ 34,514 may have potential value in the therapy of HSV and cytomegalovirus infections in humans.     

Effects of acyclovir, oxetanocin-G, and carbocyclic oxetanocin-G in combinations on the replications of herpes simplex virus type 1 and type 2 in Vero cells.

9-(2-hydroxyethoxymethyl)guanine (acyclovir, ACV) and novel nucleosides, 9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanocyl)guanine (oxetanocin-G, OXT-G) and (+)-9-[(1R, 2R, 3S)-2, 3-bis(hydroxymethyl)cyclobutyl]guanine (carbocyclic oxetanocin-G, carbocyclic OXT-G) possessed substantial antiviral activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). ACV inhibited only viral thymidine kinase positive (TK+) herpes viruses, although the latter two compounds inhibited the replications of the TK deficient (TK-) mutants of HSV-1 and HSV-2 as well as the TK+ parent strains in vitro. The TK- mutants of HSV-1 and HSV-2 (HSV-1 TK- and HSV-2 TK-) were as susceptible to OXT-G as the TK parent strains. However, the TK- mutants were less susceptible to carbocyclic OXT-G than the TK+ parent strains. We demonstrated synergistic inhibition of the replications of HSV-1 and HSV-2 by ACV and OXT-G in combination, additive inhibition of HSV-1 and HSV-2 by ACV and carbocyclic OXT-G in combination, synergistic inhibition of HSV-1 by OXT-G and carbocyclic OXT-G in combination, and additive inhibition of HSV-2 by these two compounds. We investigated the metabolism of ACV and OXT-G in HSV-1 TK(+)-, HSV-1 TK(-)- and mock-infected Vero cells by thin layer chromatography. ACV-triphosphate increased more in HSV-1 TK(+)-infected Vero cells than in HSV-1 TK(-)- and mock-infected Vero cells. The metabolism of OXT-G had almost the same pattern in HSV-1 TK(+)-, HSV-1 TK(-)- and mock-infected Vero cells. These results suggest that ACV is phosphorylated by virus-induced TK, and OXT-G is phosphorylated by cellular nucleoside and nucleotide kinases.     

Antiretroviral activities of protease inhibitors against murine leukemia virus and simian immunodeficiency virus in tissue culture.

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polyproteins in cultures of human immunodeficiency virus type 1 (HIV-1)-infected T lymphocytes and, as a result, inhibit the infectivity of HIV-1 for such cultures. The ability of HIV-1 protease inhibitors to suppress replication of the C-type retrovirus Rauscher murine leukemia virus (R-MuLV) and the HIV-related lentivirus simian immunodeficiency virus (SIV) was examined in plaque reduction assays and syncytium reduction assays, respectively. Three of seven compounds examined blocked production of infectious R-MuLV, with 50% inhibitory concentrations of < or = 1 microM. Little or no cellular cytotoxicity was detectable at concentrations up to 100 microM. The same compounds which inhibited the infectivity of HIV-1 also produced activity against SIV and R-MuLV. Electron microscopic examination revealed the presence of many virions with atypical morphologies in cultures treated with the active compounds. Morphometric analysis demonstrated that the active compounds reduced the number of membrane-associated virus particles. These results demonstrate that synthetic peptide analog inhibitors of retroviral proteases significantly inhibit proteolytic processing of the gag polyproteins of R-MuLV and SIV and inhibit the replication of these retroviruses. These results are similar to those for inhibition of HIV-1 infectivity by these compounds, and thus, R-MuLV and SIV might be suitable models for the in vivo evaluation of the antiretroviral activities of these protease inhibitors.     

Inhibition of human herpesvirus 6 replication by 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine (2HM-HBG) and other antiviral compounds.

The in vitro susceptibilities of human herpesvirus 6 to foscarnet; the guanosine analogs acyclovir, ganciclovir, and two isomers of 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine; and the thymidine analogs 3'-azido-3'-deoxythymidine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil were investigated. All compounds except 3'-azido-3'-deoxythymidine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil inhibited human herpesvirus 6 replication. The highest in vitro selectivity was obtained for 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine.     

Blockade of rat alpha3beta4 nicotinic receptor function by methadone, its metabolites, and structural analogs

The opioid agonist properties of (+/-)-methadone are ascribed almost entirely to the (-)-methadone enantiomer. To extend our knowledge of the pharmacological actions of methadone at ligand-gated ion channels, we investigated the effects of the two enantiomers of methadone and its metabolites R-(+)-2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolinium perchlorate (EDDP) and R-(+)-2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline hydrochloride (EMDP), as well as structural analogs of methadone, including (-)-alpha-acetylmethadol hydrochloride (LAAM) and (+)-alpha-propoxyphene, on rat alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs) stably expressed in a human embryonic kidney 293 cell line, designated KXalpha3beta4R2. (+/-)-methadone inhibited nicotine-stimulated 86Rb+ efflux from the cells in a concentration-dependent manner with an IC50 value of 1.9 +/- 0.2 microM, indicating that it is a potent nAChR antagonist. The (-)- and (+)-enantiomers of methadone have similar inhibitory potencies on nicotine-stimulated 86Rb+ efflux, with IC50 values of approximately 2 microM. EDDP, the major metabolite of methadone, is even more potent, with an IC50 value of approximately 0.5 microM, making it one of the most potent nicotinic receptor blockers reported. In the presence of (+/-)-methadone, EDDP, or LAAM, the maximum nicotine-stimulated 86Rb+ efflux was markedly decreased, but the EC50 value for nicotine stimulation was altered only slightly, if at all, indicating that these compounds block alpha3beta4 nicotinic receptor function by a noncompetitive mechanism. Consistent with a noncompetitive mechanism, (+/-)-methadone, its metabolites, and structural analogs have very low affinity for nicotinic receptor agonist binding sites in membrane homogenates from KXalpha3beta4R2 cells. We conclude that both enantiomers of methadone and its metabolites as well as LAAM and (+)-alpha-propoxyphene are potent noncompetitive antagonists of alpha3beta4 nAChRs.     

A novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI), GRL-98065, is potent against multiple-PI-resistant human immunodeficiency virus in vitro

We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC(50)], 0.0002 to 0.0005 microM) with minimal cytotoxicity (50% cytotoxicity, 35.7 microM in CD4(+) MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to a 5 microM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 microM concentration of lopinavir or atazanavir (EC(50), 0.0015 to 0.0075 microM), although it was less active against HIV-1(NL4-3) selected by amprenavir (EC(50), 0.032 microM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active-site amino acids (Asp29 and Asp30) is important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.     

Phosphonylmethoxyethyl purine derivatives, a new class of anti-human immunodeficiency virus agents

A study of the structure-activity relationship of a series of newly synthesized phosphonylmethoxyalkyl purine and pyrimidine derivatives revealed that several adenine derivatives substituted at the N9 position by a 2-phosphonylmethoxyethyl (PME) group inhibited human immunodeficiency virus (HIV)-induced cytopathogenicity and HIV antigen expression in vitro at concentrations significantly below the toxicity threshold for the host cells. In terms of anti-HIV potency in MT-4 cells, the PME 2,6-diaminopurine derivative (50% effective dose [ED50], 1 microM) ranked first, followed by the PME adenine derivative (ED50, 2 microM [MT-4]) and the PME 2-monoaminopurine derivative (ED50, 45 microM). Antiretroviral activity was also demonstrated in ATH8 and H9 cells, which were de novo infected with HIV, and extended to C3H mouse fibroblasts infected with Moloney murine sarcoma virus. Unlike 2',3'-dideoxyadenosine, these compounds were not found to be degraded by deaminases derived from bovine intestine.     

NP-06: a novel anti-human immunodeficiency virus polypeptide produced by a Streptomyces species.

From an extract of a Streptomyces culture, we identified and purified a novel compound, NP-06, which is active against human immunodeficiency virus (HIV) in vitro. Analyses indicate that NP-06 is a hydrophobic 21-mer oligopeptide, N terminally cyclized through the side chain of Asp-9, containing two intramolecular cystine linkages with a molecular weight of 2,163.4. The 50% inhibitory concentrations were 2.8 and 1.3 microM when NP-06 was tested for in vitro anti-HIV-1 activity in ATH8 cells and phytohemagglutinin-activated peripheral blood mononuclear cells, respectively, NP-06 appears to block the early stage of HIV-1 infection, most likely at the stage of virus-cell fusion.     

Mode of action, toxicity, pharmacokinetics, and efficacy of some new antiherpesvirus guanosine analogs related to buciclovir.

9-[4-Hydroxy-3-(hydroxymethyl)butyl]guanine (3HM-HBG), (RS)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine ([+/-]2HM-HBG), and cis-9-(4-hydroxy-2-butenyl)guanine (2EN-HBG), new acyclic guanosine analogs structurally related to buciclovir (BCV [(R)-9-(3,4-dihydroxybutyl)guanine]), were evaluated in parallel with buciclovir as anti-herpes simplex virus (HSV) agents. In cell cultures, replication of different strains of HSV type 1 (HSV-1) and HSV-2 was inhibited at nontoxic drug concentrations. The concentrations giving 50% inhibition of plaque formation were, however, dependent on virus strain and cell type. In most cell types, the order of activity against HSV-1 strains was 3HM-HBG greater than (+/-)2HM-HBG greater than BCV greater than 2EN-HBG, whereas the drugs showed an approximately equivalent activity against HSV-2 strains in different cells. The cytotoxic effects of the drugs were also cell type dependent, the order of activity being BCV greater than 3HM-HBG = (+/-)2HM-HBG greater than 2EN-HBG. At growth-inhibitory concentrations, the guanosine analogs BCV, 3HM-HBG, and (+/-)2HM-HBG showed clastogenic effects in human lymphocytes, mainly because of the induction of chromatid breaks. When evaluated for their anti-HSV effects in systemic HSV-1 infections in mice, the order of activity was BCV = 3HM-HBG greater than (+/-)2HM-HBG greater than 2EN-HBG, and in mice infected systemically with HSV-2, only BCV and 3HM-HBG showed efficacy. The differences between efficacy in vitro and in vivo could be explained in part by differences in kinetics of the drugs in mouse plasma, as the more efficacious drugs, BCV and 3HM-HBG, showed lower clearances and longer half-lives than the less efficacious ones, (+/-)2HM-HBG and 2EN-HBG. When used topically against a cutaneous HSV-1 infection in guinea pigs, 3HM-HBG showed an effect equivalent to that of BCV, whereas (+/-)2HM-HBG and 2EN-HBG were inactive. Mechanistically, the guanosine analogs were characterized by a high affinity for the viral thymidine kinase and a low affinity fo a cellular thymidine kinase and by their inhibition of viral DNA synthesis in infected cells.     

In vitro antiviral activity of penciclovir, a novel purine nucleoside, against duck hepatitis B virus.

The in vitro antihepadnavirus activities of the purine nucleoside analogs ganciclovir (9-[2-hydroxy-1-(hydroxymethyl)ethoxymethyl]guanine) and penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were compared in primary duck hepatocyte cultures congenitally infected with the duck hepatitis B virus (DHBV). Both compounds inhibited DHBV DNA replication to a comparable extent during continuous short-term treatment of the cultures. However penciclovir was more active both during longer-term continuous treatment (50% inhibitory concentrations: penciclovir, 0.7 +/- 0.1 microM; ganciclovir, 4.0 +/- 0.2 microM) and in washout experiments (50% inhibitory concentrations: penciclovir, 3.0 +/- 0.4 microM; ganciclovir, 46 +/- 1.5 microM) designed to compare the persistence of inhibitory activity after removal of the extracellular compound. The effects on viral protein synthesis were similar to the effects on viral DNA replication. These data suggest that penciclovir or its oral form, famciclovir, may have clinical utility in the treatment of chronic hepatitis B virus infection.     

Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes.

The inhibitory activities of acyclovir (ACV), 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), ganciclovir (GCV), 9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine (OXT-G), and (+)-9-[(1R,2R,3S)-2,3-bis(hydroxymethyl)Cyclobutyl]guanine (cOXT-G) on the replication of wild-type and thymidine kinase (TK)-negative strains of herpes simplex virus types 1 and 2 and varicella-zoster virus (VZV) and the wild-type strain of human cytomegalovirus were tested to clarity whether the phosphorylation of these compounds is catalyzed by viral TK or other enzymes. ACV and BV-araU had little effect on the replication of TK-negative virus strains. On the other hand, GCV, OXT-G, and cOXT-G inhibited the replication of TK-negative VZV at concentrations 10 times higher than those at which they inhibited wild-type VZV, indicating that a kinase other than TK phosphorylates GCV and OXT-G in VZV-infected cells. GCV phosphorylation activity was not detected in VZV-infected cell lysates; therefore, this activity was evaluated in COS 1 cells expressing viral TK and viral protein kinase (PK). The COS 1 cells expressing VZV TK were shown to be susceptible to all compounds tested. In contrast, VZV Pk-expressing COS 1 cells were susceptible to only GCV, OXT-G, and cOXT-G. These results suggest that VZV PK phosphorylates some nucleoside analogs, for example, GCV, OXT-G, and cOXT-G. This phosphorylation pathway may be important in the anti-VZV activities of some nucleoside analogs.     

Sodium Lauryl Sulfate Abrogates Human Immunodeficiency Virus Infectivity by Affecting Viral Attachment

The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1(NL4-3) with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 microM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 microM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.