4862F, a new inhibitor of HIV-1 protease, from the culture of Streptomyces I03A-04862.

We have isolated an extraordinary pentapeptide, called 4862F, from the culture broth of Streptomyces albosporus I03A-04862 by Diaion HP-20 macroporous adsorbent resin column, ODS-A and Sephadex LH-20 chromatography, followed by preparative HPLC. This peptide shows inhibitory activity against HIV-1 protease. The structure was elucidated by spectroscopic approaches, including ESI-MS and various NMR methods. Absolute configuration of the amino acid residues in 4862F was defined using Marfey's method, and the structure was identified as N,N,N-(trimethylated)-Tyr-L-Leu-L-Val-L-Leu-(dehydrated)-His. The peptide 4862F displays inhibitory activity against HIV-1 protease, with IC50 values of 15.26 nM, using a fluorescence-based assay.

Geldanamycin, a ligand of heat shock protein 90, inhibits herpes simplex virus type 2 replication both in vitro and in vivo.

Previously, we discovered geldanamycin, a ligand of heat shock protein 90, effectively inhibited herpes simplex virus type 1 replication in vitro and in vivo (mouse encephalitis model). In this study, we demonstrate that geldanamycin has very strong activities against herpes simplex virus type 2 in vitro and in vivo (mouse vagina model). In mouse vagina model, administration of geldanamycin suspension to vagina after virus infection protected the infected mice from death and increased the average survival days in a dose-dependent manner. Geldanamycin also significantly reduced virus shedding from mouse vagina. All geldanamycin-treated groups were statistically significant when compared with the infected control group. The high-dose group of geldanamycin (5.72 mg kg(-1)) was better than acyclovir group (2.86 mg kg(-1)). All geldanamycin vaginal administration mock-infected groups did not show significant body weight loss. Although geldanamycin has strong antiviral activities against various DNA and RNA viruses, geldanamycin is not suitable for systemic administration because of its high toxicity. We consider that geldanamycin is a candidate of topical usage for the treatment of herpes simplex virus type infections.

Geldanamycin is effective in the treatment of herpes simplex virus epithelial keratitis in a rabbit model.

BACKGROUND: To evaluate the efficacy of geldanamycin eye drops against herpes simplex virus epithelial keratitis in a rabbit model. METHODS: New Zealand white rabbits were randomized into four groups and infected with herpes simplex virus type 1; geldanamycin topical eye drops was initiated 24 h after the infection and maintained for 12 consecutive days. Four groups of rabbits received 5 µg/mL geldanamycin, 10 µg/mL geldanamycin, 0.1% acyclovir and escipient (a kind of artificial tears), respectively. The severity of herpes simplex virus type 1 epithelial keratitis was measured by slit-lamp and scored for statistics analysis. The virus shedding in eye swabs was isolated, and tissue culture infective dose (TCID50) was determined. RESULTS: Geldanamycin (10 µg/mL) treatment reduced significantly the severity of herpes simplex virus type 1 epithelial keratitis than the other three groups. Geldanamycin (5 µg/mL) was as effective as acyclovir (0.1%) treatment. The effect of geldanamycin against herpes simplex virus type 1 epithelial keratitis correlated with accelerated clearance of virus of the rabbits. CONCLUSION: Geldanamycin is a promising treatment option against herpes simplex virus type 1 epithelial keratitis. Geldanamycin (10 µg/mL) is better than acyclovir and geldanamycin (5 µg/mL) in the rabbit model. The optimal concentration of this drug in human is still to be determined.

Novel 4,5-dihydro-thiazinogeldanamycin in a gdmP mutant strain of Streptomyces hygroscopicus 17997.

Novel geldanamycin derivative, 4,5-dihydro-thiazinogeldanamycin (3), was characterized from the gdmP mutant in Streptomyces hygroscopicus 17997, besides expected 4,5-dihydro-geldanamycin (2). The presence of this compound would suggest an unknown post-PKS modification in geldanamycin biosynthesis. Compound 3 exhibited moderate anti-HSV-1-virus activity and higher water solubility than geldanamycin (1). Cysteine served as a precursor to synthesize 3, whose formation required obligatory enzymatic assistance.

[Synthesis and antiviral activities of geldanamycin analog TC-GM in vitro].

In order to find antiviral compounds with novel structures, geldanamycin and lamivudine with different antiviral mechanisms were conjunctively synthesized to acquire a new compound TC-GM, and the antiviral activity of TC-GM was measured. The antiviral activity against HIV-1 was examined by p24 antigen ELISA kit. The activity against HBV was examined by dotblot. The activity against HSV and CoxB virus was examined by CPE. TC-GM exhibited broad-spectrum antiviral activities similarly like geldanamycin. TC-GM inhibited the replication of different viruses, including HIV-1, HBV, HSV 1 and 2, CoxB6. TC-GM showed more potent inhibitory activity against HIV-1 and HBV than other detected virus.

[Anti-HIV-1 activity and structure-activity relationship of pyranocoumarin analogs].

The purpose of this study is to find out anti-HIV-1 reverse transcriptase (RT)/protease (PR) activity and inhibition of virus replication in cell cultures of novel coumarin analogs and determine their structure-activity relationship. Coumarin derivatives have been demonstrated to inhibit the activity of HIV-1 RT/PR in cell free system. It also shows inhibition effects to HIV-1 replication in cell culture. Based on the Chinese traditional pharmacological characteristics and protein three dimension computer aided design, analogs of tetracyclic dipyranocoumarin were synthesized from natural leading compounds. We studied the relationship of antiviral effects and chemical structures via HIV-1 PR/RT enzyme models and cell culture model system. Seven compounds were designed and tested. Several compounds showed anti-HIV-1 activity in varying degrees, especially V0201 showed much higher anti-HIV-1 activity with 3.56 and 0.78 micromol x L(-1) of IC50 against HIV-1 PR/RT and 0.036 micromol x L(-1) against HIV-1 replication in PBMC cultures. V0201 with a novel structure may be a new leading compound. These new compounds are valuable for development of new anti-HIV drugs in the future.

Synthesis and biological evaluation of heat-shock protein 90 inhibitors: geldanamycin derivatives with broad antiviral activities.

BACKGROUND: Previous studies have suggested that geldanamycin (GA) inhibits the replication of several viruses in vitro. Here, we aimed to synthesize and evaluate the antiviral activity of 17-amino-17-demethoxygeldanamycin derivatives. METHODS: A series of 17-substituted and 17-,19-disubstituted GA derivatives were screened for antiviral activities against eight different viral strains, including herpesvirus, hepatitis virus, retrovirus and picornavirus. RESULTS: Most of the tested compounds showed inhibitory activity against the viruses and showed reduced cytotoxicity in vitro as compared with the parent compound GA. In vivo efficacy evaluation results showed that compound 6 noticeably inhibited duckling hepatitis B virus DNA replication in duckling serum after oral administration. Viral rebound did not occur after termination of the treatment. The modified GA derivatives also showed median lethal dose values that were higher than that of the parent GA in mice intraperitoneally treated with the study compounds. CONCLUSIONS: Targeting heat-shock protein 90 could be a new antiviral approach that is not prone to the development of drug resistance. The 17-amino-17-demethoxygeldanamycin derivatives could be novel agents with potential antiviral activity.

[Antiviral activities of cycloheximide and its derivatives].

Cycloheximide (CHX) inhibits protein synthesis in most eukaryotic cells and it is a well-known tool commonly used in biochemical research. In this paper, the antiviral spectrum of CHX against several DNA and RNA viruses have been evaluated. CHX showed strong inhibitory activities against several RNA viruses such as HIV-1, influenza viruses, coxsackie B virus, enterovirus (EV71) and several DNA viruses such as HSV and HCMV. Especially the strong inhibitory activities of CHX against coxsackie B virus and enterovirus caught our attention, since effective drugs available in clinic are limited. The SAR of CHX derivatives also has been discussed in the paper. The hydroxyl group at C-2' and carbonyl group at C-2" of CHX are essential for its antiviral activity. And modification to these groups results its derivatives' antiviral activities reduced or lost.

Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro.

Chlorogenic acid and its related compounds are abundant plant polyphenols that have a diverse antiviral activity. In this study, HepG2.2.15 cells and duck hepatitis B virus infection model were used as in vitro and in vivo models to evaluate their anti-HBV activity. In the cell model, all the three compounds inhibited HBV-DNA replication as well as HBsAg production. Chlorogenic acid and caffeic acid also reduced serum DHBV level in DHBV-infected duckling model. Moreover, the anti-HBV activity of crude extracts of coffee beans, which have a high content of chlorogenic acid, was studied. Both the extracts of regular coffee and that of decaffeinated coffee showed inhibitory effect on HBV replication.

N-(4-Hydroxy-pheneth-yl)acetamide.

In the title compound, C(10)H(13)NO(2), the occurrence of inter-molecular N-H⋯O and O-H⋯O hydrogen bonds between the hydr-oxy and acetamido groups results in the formation of tetra-mers with an R(4) (4)(25) graph-set motif. These tetra-mers are further assembled, building up a corrugated sheet parallel to (001).

Improved synthesis and pharmacological evaluation of racemic 11 -demethylcalanolide A.

An improved and practical synthesis of racemic 11-demethylcalanolide A [(+/-)-1] was developed. This improved process involved Pechmann reaction on phloroglucinol with ethyl butyrylacetate to give 5,7,-dihydroxy4-n-propylcoumarin (3). Poly phosphoric acid (PPA) catalyzed acylation of compound (3) with crotonic acid, then intramolecular cyclization was achieved simultaneously in one step to afford the key intermediate chromanone (4). A microwave assisted synthetic method preparing chromene (6) using chromenynation of chromanone (4) with 1, 1-diethoxy-methyl-2-butene was conducted. Luche reduction of chromene (6) using NaBH4 with CeCl3 x 7H2O preferably gave (+/-)-1. The overall yield of this four step synthesis of (+/-)-1 was around 32% increasing one fold more than that of the previous method. An in vitro investigation showed that (+/-)-1 exhibited inhibitory activities against both wild-type and drug-resistant HIV-1 in HIV-1 RT and cell culture assay, and significant synergistic effects in combination with AZT, T-20, and indinavir. Its LD50 of acute toxicity in mice by intragastric administration and by intraperitoneal injection were 735.65 mg kg(-1) and 525.10 mg x kg(-1), respectively. The Cmax and AUC(0-infinity) were 0.54 microg x mL(-1) and 1.08 (microg x mL(-1) x h, respectively. The dynamics study of the inhibition of mice sera on HIV-1 RT showed that mice treated with 100 mg x kg(-1 (+/-)-1 once intraperitoneally were similar to that of 5 mg x kg(-1) of known clinical effective anti-HIV-1 drug neverapine. The results suggested that further investigation of the anti-HIV candidate (+/-)-1 was warranted.

Geldanamycin, a ligand of heat shock protein 90, inhibits the replication of herpes simplex virus type 1 in vitro.

Geldanamycin (GA) is an antibiotic targeting the ADP/ATP binding site of heat shock protein 90 (Hsp90). In screening for anti-herpes simplex virus type 1 (HSV-1) candidates, we found GA active against HSV-1. HSV-1 replication in vitro was significantly inhibited by GA with an 50% inhibitory concentration of 0.093 microM and a concentration that inhibited cellular growth 50% in comparison with the results seen with untreated controls of 350 microM. The therapeutic index of GA was over 3700 (comparable to the results seen with acyclovir). GA did not inhibit HSV-1 thymidine kinase. Cells infected with HSV-1 demonstrated cell cycle arrest at the G(1)/S transition; however, treatment with GA resulted in a cell cycle distribution pattern identical to that of untreated cells, indicating a restoration of cell growth in HSV-1-infected cells by GA treatment. Accordingly, HSV-1 DNA synthesis was suppressed in HSV-1(+) cells treated with GA. The antiviral mechanism of GA appears to be associated with Hsp90 inactivation and cell cycle restoration, which indicates that GA exhibits broad-spectrum antiviral activity. Indeed, GA exhibited activities in vitro against other viruses, including severe acute respiratory syndrome coronavirus. Since GA inhibits HSV-1 through a cellular mechanism unique among HSV-1 agents, we consider it a new candidate agent for HSV-1.

[Inhibition of duck hepatitis B virus DNA replication by antisense phosphorothioate oligodeoxynucleotides in vitro and in vivo].

BACKGROUND: To determine the feasibility of inhibition of duck hepatitis B virus (DHBV) DNA replication by antisense phosphorothioate oligodeoxynucleotides corresponding to DHBV transcription region. METHODS: The authors designed three antisense phosphorothioate oligodeoxynucleotides which correspond to DHBV PreS1,PreS2 and S antigen gene promotors respectively. The DNA replication level was detected with Southern blot method and cpm calculation. RESULTS: Primary duck hepatocyte culture was treated with 1.5 micromol/L antisense oligodeoxynucleotides in vitro, all the antisense fragments caused a firm inhibition of viral DNA replication and the inhibition rates were 61.5%, 69.3% and 62.4%, respectively. In vivo, the animals were treated with 10 microgram/g PreS1 antigen gene promotor antisense oligodeoxynucleotides per day for 6 days and a very strong inhibition rate of 87.9% was obtained. CONCLUSIONS: The results demonstrated the potential clinical application of antisense phosphorothioate oligodeoxynucleotides in clinics.

[The establishment and application of antiviral screening model targeted on SAH hydrolase].

BACKGROUND: To establish a SAH hydrolase antiviral screening in vitro model for screening of broad spectrum antiviral agents. METHODS: SAH hydrolase was purified from rat livers by (NH4) 2SO4 fractionation, DEAE52,hydroxyapatite and Sephadex G-100 chromatography successively. The activity of SAH hydrolase was estimated by radio labeled substrate in synthesis direction by TLC. RESULTS: Purified SAH hydrolase showed a single band in SDS-PAGE electrophoresis with silver nitrate staining, the apparent molecular weight is 45 000. The Km for adenosine is (6.32 +- 0.17) micromol/L. The IC50 of S-DNPA, a known inhibitor of SAH hydrolase, was 7.6 micromol/L estimated in our system. The structure and activity relationships shown by racemic and regiosomer analogs of S-DHPA indicated that the structural specificity of SAH hydrolase was high. 42 compounds had been screened in the system and no compound showed more inhibitory activity against SAH hydrolase than S-DNPA. CONCLUSIONS: An in vitro antiviral screening model has been established using SAH hydrolase. It can also be used to study kinetics of enzyme inhibition.