AIMSynthesis of 1-(3-phthalimido-2-oxobutyl)-4-substituted- phenylpiperazines (5-15).

METHODSThe starting material nitrogen mustard hydrochloride (16), reacted with the corresponding substituted anilines to afford piperazine hydrochlorides (17-27), which were then coupled with 1-bromo-3-phthalimidobutan-2-one (4) to give the target compounds.

RESULTSEleven target compounds (5-15) were synthesized, which were characterized by 1HNMR, IR and elemental analysis.

CONCLUSIONAnti-HIV-1 RT using HIV reverse transcriptase P-66 protein test showed that compounds 11, 14, 10 and 13 possessed inhibitory effects against HIV-1 reverse transcriptase (RT), with IC50 29.80, 35.20, 43.77 and 63.76 mumol.L-1, respectively.

HIV Reverse Transcriptase ; antagonists & inhibitors ; metabolism ; Inhibitory Concentration 50 ; Molecular Structure ; Piperazines ; chemical synthesis ; chemistry ; pharmacology ; Reverse Transcriptase Inhibitors ; chemistry ; pharmacology

This study is to investigate the mechanism of action of lindenane disesquiterpenoid shizukaol F on HIV-1 replication. Real time quantity PCR, ELISA assay and fluorescence methods were used to test HIV-1 reverse transcription process, RNA-dependent DNA polymerase activity, and RNase H activity, respectively. It showed that shizukaol F inhibited LTR/Gag production of HIV-1 reverse transcription with an IC50 of 9.11 micromol x L(-1). This result is consistent with its inhibitory effect on HIV-1 replication (IC50 of 6.12 micromol x L(-1)). Mechanism studies showed that compound shizukaol F inhibited HIV-1 RT-RNase H with IC50 of 26.4 micromol x L(-1), but had no effect on HIV-1 RT RNA-dependent DNA polymerase activity. In conclusion, shizukaol F is a new structural type HIV-1 RNase H inhibitor. This discovery will provide a clue for new type of reverse transcriptase inhibitors development.
Cell Line, Tumor ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; HEK293 Cells ; HIV Reverse Transcriptase ; metabolism ; HIV-1 ; physiology ; Humans ; Inhibitory Concentration 50 ; Magnoliopsida ; chemistry ; Molecular Structure ; Plants, Medicinal ; chemistry ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; pathology ; Reverse Transcriptase Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Ribonuclease H ; antagonists & inhibitors ; metabolism ; Sesquiterpenes ; chemistry ; isolation & purification ; pharmacology ; Virus Replication ; drug effects

It was recently shown that several new synthetic 2-alkylsulfanyl-6-benzyl-3, 4-dihydropyrimidin-4(3H)-one (S-DABO) derivatives demonstrated anti-HIV-1 activity. Three of the derivatives namely RZK-4, RZK-5 and RZK-6 were used in this study to explore their inhibitory effects on a variety of HIV strains. These compounds at a concentration of 200 microg mL(-1) almost completely inhibited the activity of recombinant HIV-1 reverse transcriptase. All of the three compounds reduced replication of HIV-1 laboratory-derived strains, low-passage clinical isolated strain, and the drug resistant strain. In particular RZK-6 showed potent activity against the HIV-1 drug resistant strain. In general, the antiviral activities are similar in magnitude to nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor approved by FDA. The therapeutic indexes of these compounds were remarkable, ranging from 3704 to 38462 indicating extremely low cytotoxicity. These results suggest that the three S-DABO derivatives in this study have good potential for further development in anti-HIV-1 therapy. It may be particularly useful to target at the non-nucleoside reverse transcriptase inhibitors resistant HIV-1 strain.
Anti-HIV Agents ; chemical synthesis ; chemistry ; pharmacology ; Benzyl Compounds ; chemical synthesis ; chemistry ; pharmacology ; Cell Line ; Drug Resistance, Viral ; HIV Reverse Transcriptase ; antagonists & inhibitors ; metabolism ; HIV-1 ; drug effects ; Humans ; Pyrimidinones ; chemical synthesis ; chemistry ; pharmacology ; Reverse Transcriptase Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Virus Replication ; drug effects

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.
Anti-HIV Agents ; chemical synthesis ; chemistry ; pharmacology ; Cells, Cultured ; HIV Core Protein p24 ; metabolism ; HIV Protease ; metabolism ; HIV Reverse Transcriptase ; metabolism ; HIV-1 ; physiology ; Humans ; Inhibitory Concentration 50 ; Leukocytes, Mononuclear ; cytology ; metabolism ; virology ; Pyranocoumarins ; chemical synthesis ; chemistry ; pharmacology ; Reverse Transcriptase Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Structure-Activity Relationship ; Virus Replication ; drug effects

BACKGROUND/AIMS: Cyclooxygenase-2 (COX-2) inhibitors reportedly inhibit the growth of hepatocellular carcinoma (HCC) via caspase-dependent or caspase-independent apoptosis, which is due to COX-2 being associated with hepatocarcinogenesis. Survivin is highly expressed in most human cancers, but the mechanism regulating survivin expression remains unclear. We investigated the regulatory expression of survivin in selective-COX-2-inhibitor-induced growth inhibition of hepatoma cells. METHODS: After treatment with NS-398 (a selective COX-2 inhibitor) at various concentrations (10, 50, 100, 150, and 200 micrometer), the growth inhibition of Hep3B hepatoma cells was assessed by an MTT cell-viability assay, DNA fragmentation gel analysis, and flow cytometry. The expression of survivin transcript was analyzed by reverse-transcription polymerase chain reactions. RESULTS: NS-398 inhibited the growth of hepatoma cells by an amount dependent on the concentration and the time since treatment. Apoptotic DNA ladder and flow-cytometry shifting to the sub-G1 phase were revealed in NS-398-induced growth inhibition of hepatoma cells. NS-398 suppressed the expression of the survivin gene in a concentration- and time-dependent manner. CONCLUSIONS: Survivin was down-regulated in the growth inhibition of hepatoma cells induced by a selective COX-2 inhibitor, NS-398, in a concentration- and time-dependent manner. These results suggest the therapeutic inhibition of COX-2 via suppression of survivin in HCC.
Carcinoma, Hepatocellular/enzymology/*metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cyclooxygenase 2 Inhibitors/chemistry/*pharmacology ; G1 Phase ; Humans ; Liver Neoplasms/enzymology/*metabolism/pathology ; Microtubule-Associated Proteins/*antagonists & inhibitors/metabolism ; Neoplasm Proteins/*antagonists & inhibitors/metabolism ; Nitrobenzenes/chemistry/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfonamides/chemistry/*pharmacology ; Time Factors

To compare the anti-HIV-1 activities of (+/-)-11-demethyl-calanolide A and its mother compound (+/-)-calanolide A in vitro and in vivo, the inhibitory activities of the two compounds on HIV-1 reverse transcriptase (RT) were detected in vitro with isotope 3H assay. The cytotoxicity and inhibition of cytopathic effect (CPE) were studied in HIV-1 IIIB infected MT-4 cell cultures by MTT staining method; Mice were given with the two compounds 100 mg x kg(-1) once intraperitoneally, then the mouse sera taken on 30 min and 60 min after administration were detected for the inhibition of HIV-1 RT in vitro. The data showed that (+/-)-11-demethyl-calanolide A and (+/-)-calanolide A inhibited HIV-1 RT in vitro with 50% inhibitory concentration (IC50) of (3.028 +/- 2.514) micromol x L(-1) and (3.965 +/- 5.235) micromol x L(-1), and also inhibited CPE in HIV-1 IIIB infected MT-4 cell cultures with IC50 of (1.081 +/- 0.337) micromol x L(-1) and (1.297 +/- 0.076) micromol x L(-1), respectively. After intraperitoneal injection of 100 mg x kg(-1) of the two compounds in mice, all the mice sera taken 30 and 60 min afterward inhibited HIV-1 RT in vitro. In comparison with control mice sera, the inhibitory rates of the sera for (+/-)-11 -demethyl-calanolide A were (42.7 +/- 1.5)% at 30 min (P < 0.01) and (32.2 +/- 6.1)% at 60 min (P < 0.05), separately, while the inhibitory rates of the sera for (+/-)-calanolide A were (40.7 +/- 6.3)% at 30 min (P < 0.01) and (29.2 +/- 6.7)% at 60 min. The results suggested that (+/-)-11-demethyl-calanolide A is a new non-nucleoside HIV-1 RT inhibitor, its anti-HIV-1 activities in vitro, in cell cultures and in mice were slightly higher than that of its mother compound (+/-)-calanolide A and warrants further studies.
Animals ; Anti-HIV Agents ; pharmacology ; Cell Line, Tumor ; Female ; HIV Reverse Transcriptase ; antagonists & inhibitors ; metabolism ; HIV-1 ; drug effects ; Humans ; Immune Sera ; pharmacology ; Inhibitory Concentration 50 ; Male ; Mice ; Molecular Structure ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; pathology ; virology ; Pyranocoumarins ; chemistry ; pharmacology ; Reverse Transcriptase Inhibitors ; pharmacology ; Stereoisomerism

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.
Animals ; Caseins/metabolism ; Cathepsin B/antagonists&inhibitors/*genetics/isolation & purification/*metabolism ; Cysteine Proteinase Inhibitors/pharmacology ; DNA, Complementary/genetics ; Goat Diseases/*parasitology ; Goats ; Haemonchiasis/parasitology/*veterinary ; Haemonchus/*enzymology/genetics/isolation & purification ; Hemagglutination Tests/veterinary ; Hemoglobins/metabolism ; Hydrogen-Ion Concentration ; Immunoglobulin G/metabolism ; Leucine/analogs & derivatives/pharmacology ; RNA, Helminth/chemistry/genetics ; Recombinant Proteins/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/veterinary

AIM: To evaluate the anti-HIV activity and mechanism of action of wikstroelide M, a daphnane diterpene from Daphne acutiloba Rehder (Thymelaeaceae). METHODS: The anti-HIV activities of wikstroelide M against different HIV strains were evaluated by cytopathic effect assay and p24 quantification assay with ELISA. The inhibitory effect of wikstroelide M on HIV reverse transcription was analyzed by real-time PCR and ELISA. The effect of wikstroelide M on HIV-1 integrase nuclear translocation was observed with a cell-based imaging assay. The effect of wikstroelide M on LEDGF/p75-IN interaction was assayed by molecular docking. RESULTS: Wikstroelide M potently inhibited different HIV-1 strains, including HIV-1IIIB, HIV-1A17, and HIV-19495, induced a cytopathic effect, with EC50 values ranging from 3.81 to 15.65 ng·mL⁻¹. Wikstroelide M also had high inhibitory activities against HIV-2ROD and HIV-2CBL-20-induced cytopathic effects with EC50 values of 18.88 and 31.90 ng·mL⁻¹. The inhibitory activities of wikstroelide M on the three HIV-1 strains were further confirmed by p24 quantification assay, with EC50 values ranging from 15.16 to 35.57 ng·mL⁻¹. Wikstroelide M also potently inhibited HIV-1IIIB induced cytolysis in MT-4 cells, with an EC50 value of 9.60 ng·mL⁻¹. The mechanistic assay showed that wikstroelide M targeted HIV-1 reverse transcriptase and nuclear translocation of integrase through disrupting the interaction between integrase and LEDGF/p75. CONCLUSION Wikstroelide M may be a potent HIV-1 and HIV-2 inhibitor, the mechanisms of action may include inhibition of reverse trascriptase activity and inhibition of integrase nuclear translocation through disrupting the interaction between integrase and LEDGF/p75.
Anti-HIV Agents ; pharmacology ; therapeutic use ; Cell Line ; Daphne ; chemistry ; Diterpenes ; pharmacology ; HIV Infections ; drug therapy ; virology ; HIV Integrase ; metabolism ; HIV Integrase Inhibitors ; pharmacology ; therapeutic use ; HIV Reverse Transcriptase ; antagonists & inhibitors ; HIV-1 ; drug effects ; enzymology ; HIV-2 ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Virus Integration ; drug effects ; Virus Replication ; drug effects

BACKGROUND/AIMS: To develop a novel treatment method for hepatitis B virus (HBV) infection, we aimed to make a human monoclonal antibody inhibiting reverse transcriptase (RT) activity of P protein which was important in HBV replication by using phage display technique. Therefore, we analysed the usability of human monoclonal antibody as a protein based gene therapy. METHODS: Reverse transcriptase/polymerase (RT/POL) functional motif of P protein of HBV was cloned in pMAL-c vector and expressed as maltose binding fusion protein form. The RT/POL recombinant protein (pMRT/POL) was purified by amylose resin column. Using human single chain Fv phage antibody library with 1.1x10(10) size, human antibody against pMRT/POL was selected with BIAcore panning. Selected antibody fragments were analyzed for the activity of RT inhibition. Finally, they were analyzed for the affinity with BIAcore and the complementarity determining regions with nucleotide sequencing. RESULTS: pMRT/POL recombinant protein expressed in E. coli showed RT activity, 1microgram of recombinant protein had an activity equivalent to 5 unit of MMLV RT. By BIAcore panning, we could select 3 clones; POL-A5, POL-B8 and POL-B12. Each clone's RT inhibiting activity were 52-82%, affinity against antigen were 8.15x10(-8) M to 1.75x10(-6) M. CONCLUSIONS: Human monoclonal antibodies produced in this study showed low affinity, but efficiently inhibited the activity of RT in vitro. If POL-A5, POL-B8, and POL-B12 can be converted to intracellular antibody form, it can be used for protein-based gene therapy by inhibiting the replication through the neutralization of polymerase protein of HBV.
Antibodies, Monoclonal/biosynthesis/genetics/*pharmacology ; Complementarity Determining Regions/chemistry ; Gene Products, pol/*antagonists & inhibitors/genetics/immunology ; Genetic Vectors ; Hepatitis B virus/enzymology/genetics ; Humans ; Peptide Library ; RNA-Directed DNA Polymerase/genetics/*immunology ; Recombinant Fusion Proteins/biosynthesis/genetics ; Reverse Transcriptase Inhibitors/chemistry/metabolism/*pharmacology

VPAC2 is a co-receptor of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) and mediates multiple bio-functions. In order to construct the CHO line expressing VPAC2 stably, pcDNA-VPAC2 was used to transfect CHO cells. The positive clones were selected by G418 and the clone VPAC2-CHO with high sensitivity to PACAP38 was picked out by its ability to promoting the concentration of cAMP. RT-PCR, Western blot and Immunofluorescenece assay were used to identify the express of VPACS. Binding competition with VPAC2 agonist and the bioactivity of mediating the ligand to promote the concentration of cAMP showed that VPAC2 was expressed effectively in VPAC2-CHO. The results of Scatchard analysis revealed that VAPC2-CHO expressed a receptor density of (1.1 +/- 0.2) pmol/mg protein, respectively, with Kd values of (0.55 +/- 0.10) nmol/L for PACAP38 used as a tracer. The construction of CHO cells expressing VPAC2 specially and functionally lays a foundation not only for the further research on the characters and functions of VPAC2 but also for the screening and characterization of novel agonists of antagonists for VPAC2.
Animals ; Binding, Competitive ; CHO Cells ; Cell Membrane ; drug effects ; metabolism ; Cricetinae ; Cricetulus ; Cyclic AMP ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Iodine Radioisotopes ; chemistry ; Pituitary Adenylate Cyclase-Activating Polypeptide ; chemistry ; metabolism ; pharmacology ; Receptors, Vasoactive Intestinal Peptide, Type II ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection