Shan Cen

Small Molecular Compounds Inhibit HIV-1 Replication through Specifically Stabilizing APOBEC3G

APOBEC3G (hA3G) is a host inhibitor for human immunodeficiency virus, type 1 (HIV-1). However, HIV-1 Vif binds hA3G and induces its degradation. We have established a screening system to discover inhibitors that protect hA3G from Vif-mediated degradation. Through screening, compounds IMB-26 and IMB-35 were identified to be specific inhibitors for the degradation of hA3G by Vif. The inhibitors suppressed HIV-1 replication in hA3G-containing cells but not in those without hA3G. The anti-HIV effect correlated with the endogenous hA3G level. HIV-1 particles from hA3G(+) cells treated with IMB-26/35 contained a hA3G level higher than that from those without IMB-26/35 treatment and showed decreased infectivity. IMB-26/35 bound directly to the hA3G protein, suppressed Vif/hA3G interaction, and therefore protected hA3G from Vif-mediated degradation. The compounds were safe with an anti-HIV therapeutic index >200 in vitro. LD50 of IMB-26 in mice was >1000 mg/kg (intraperitoneally). Therefore, IMB-26 and IMB-35 are novel anti-HIV leads working through specific stabilization of hA3G.

Oxymatrine inhibits hepatitis B infection with an advantage of overcoming drug-resistance

Oxymatrine (OMTR) is an anti-hepatitis drug used in China. Its mechanism of action is unknown. Recently, we found that OMTR inhibits hepatitis B virus (HBV) via down-regulating the expression of heat-stress cognate 70 (Hsc70), a host protein required for HBV DNA replication. Goal of this study was to assess the effect of OMTR on clinical HBV drug-resistance. OMTR monotherapy (oral, 12 months) reduced blood HBV DNA by 96% and HBeAg by 70% in the chronic hepatitis B (CHB) patients resistant to lamivudine (n = 17), equal to its efficacy in the naïve CHB cohort (n = 20). Liver biopsy study showed that OMTR treatment caused a decrease of Hcs70 mRNA in liver cells, parallel with a reduction of intracellular HBV DNA. Combination of lamivudine with OMTR (n = 15) (oral, 12 months) showed an enhanced anti-HBV effect as compared to lamivudine monotherapy (n = 25). The incidence of drug resistance against lamivudine in the combination group was significantly lower than that in the lamivudine group (1/15 vs 7/25; p<0.01). The results were further confirmed in vitro. Treatment of HBV(+) HepH2215 cells with sub-optimal dose of OMTR for 8 months suppressed HBV replication without inducing drug resistance, whereas lamivudine monotherapy caused drug-resistant mutation in 3 months. Combination of OMTR with lamivudine prevented HBV from developing drug resistance.

Heat Stress Cognate 70 Host Protein as a Potential Drug Target against Drug Resistance in Hepatitis B Virus

ABSTRACT Heat stress cognate 70 (Hsc70) is a host protein associated with hepatitis B virus (HBV) replication. The goal of this study was to investigate whether Hsc70 could be an anti-HBV drug target. Our results showed that introducing Hsc70 increased HBV replication in HBV+ human hepatocytes (HepG2.2.15 cells). The coiled-coil region on Hsc70 (nucleotides 1533 to 1608; amino acids 511 to 536) was the key sequence for HBV replication. Knockdown of Hsc70 expression by RNA interference (RNAi) largely inhibited HBV replication with no cytotoxicity to the host. Using an Hsc70 mRNA screening assay, the natural compound oxymatrine (OMTR) was found to be a selective inhibitor for Hsc70 expression. Then, OMTR was used to investigate the potential of Hsc70 as an anti-HBV drug target. OMTR inhibited Hsc70 mRNA expression by 80% and HBV DNA replication by over 60% without causing cytotoxicity. The anti-HBV effect of OMTR appeared to be mediated by destabilizing Hsc70 mRNA. The half-life (T1/2) of Hsc70 mRNA decreased by 50% in OMTR-treated hepatocytes. The Hsc70 mRNA 3′-untranslated-region (UTR) sequence was the element responsible for OMTRs destabilization activity. OMTR suppressed HBV de novo synthesis at the reverse transcription stage from pregenomic RNA (pgRNA) to DNA and was active against either wild-type HBV or strains resistant to lamivudine, adefovir, and entecavir. Therefore, host Hsc70 could be a novel drug target against HBV, and OMTR appears to inhibit HBV replication by destabilizing Hsc70 mRNA. As the target is not a viral protein, OMTR is active for either wild-type HBV or strains resistant to reverse transcriptase (RT) inhibitors.

Small molecular compounds that inhibit hepatitis C virus replication through destabilizing heat shock cognate 70 messenger RNA

Host heat shock cognate 70 (Hsc70) protein is packaged into hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA release into the cytoplasm in the next infection cycle. The goal of this study is to investigate whether chemically down‐regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Compounds were screened with an Hsc70 messenger RNA (mRNA) assay. IMB‐DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB‐DM122 inhibited HCV replication through destabilization of Hsc70 mRNA, and the half‐life of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3′ untranslated region sequence is the element responsible for the effect of IMB‐DM122 on Hsc70 mRNA. The compound appears to be highly efficient in inhibiting Hsc70‐related HCV replication. Treatment of the HCV‐infected hepatocytes with IMB‐DM122 reduced the virion encapsidation of Hsc70, and therefore disrupted HCV replication and the infection cycle. IMB‐DM122 showed considerable good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. Conclusion: Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation. (HEPATOLOGY 2010;)

In vivo antibacterial activity of nemonoxacin, a novel non-fluorinated quinolone

OBJECTIVES To evaluate the in vivo antibacterial efficacy of nemonoxacin, a novel C8-methoxy non-fluorinated quinolone in murine systemic and local infection models. METHODS The efficacy of nemonoxacin in systemic infections was evaluated in mouse peritonitis models using isolates of methicillin-susceptible Staphylococcus aureus (MSSA, n=1), methicillin-resistant S. aureus (MRSA, n=1), methicillin- and levofloxacin-resistant Staphylococcus capitis (levofloxacin-resistant MRSC, n=1), penicillin-intermediate Streptococcus pneumoniae (PISP, n=1), penicillin-resistant S. pneumoniae (PRSP, n=2), Enterococcus faecalis (n=2, including 1 vancomycin-resistant Enterococcus, VRE) and Escherichia coli (n=3). The local infections included mouse pulmonary infections caused by PRSP (n=1), Klebsiella pneumoniae (n=1) and mouse ascending urinary tract infection caused by E. coli (n=1). RESULTS In the mouse systemic infection model, nemonoxacin demonstrated potent activity against MSSA (ED(50) =2.08 mg/kg), MRSA (ED(50) =2.59 mg/kg), levofloxacin-resistant MRSC (ED(50) =2.52 mg/kg), PISP (ED(50) =5.47 mg/kg), PRSP (ED(50) =3.68-5.28 mg/kg) and E. coli (ED(50) =3.13-5.28 mg/kg), and moderate activity towards E. faecalis infection (ED(50) =8.48-15.16 mg/kg). The therapeutic efficacy of nemonoxacin was significantly higher (P<0.01) than that of levofloxacin in infections caused by Gram-positive isolates (MSSA, MRSA, levofloxacin-resistant MRSC, PISP, PRSP and E. faecalis), but less potent than that of levofloxacin against E. coli infection (P<0.01). Nemonoxacin in vivo efficacy results with Gram-positive isolates (2- to 5-fold ED(50) advantage over levofloxacin) are consistent with the MIC data (4- to 16-fold MIC advantage of nemonoxacin over levofloxacin). In the mouse pulmonary infection model, nemonoxacin showed potent activity towards PRSP (higher than levofloxacin) and K. pneumoniae (lower than levofloxacin) infections. In the mouse ascending urinary tract infection model, nemonoxacin exhibited potent activity against E. coli infection (lower than levofloxacin). CONCLUSIONS The results validated the potent efficacy of nemonoxacin in vivo. The higher efficacy of nemonoxacin than of levofloxacin towards infections caused by Gram-positive cocci (especially MRSA, levofloxacin-resistant MRSC, PRSP and VRE) warrants investigation of its clinical use.

In Vivo Antibacterial Activity of Vertilmicin, a New Aminoglycoside Antibiotic

ABSTRACT Vertilmicin is a novel aminoglycoside antibiotic with potent activity against gram-negative and -positive bacteria in vitro. In this study, we further evaluated the efficacy of vertilmicin in vivo in systemic and local infection animal models. We demonstrated that vertilmicin had relatively high and broad-spectrum activities against mouse systemic infections caused by Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. The 50% effective doses of subcutaneously administered vertilmicin were 0.63 to 0.82 mg/kg, 0.18 to 0.29 mg/kg, 0.25 to 0.99 mg/kg, and 4.35 to 7.11 mg/kg against E. coli, K. pneumoniae, S. aureus, and E. faecalis infections, respectively. The therapeutic efficacy of vertilmicin was generally similar to that of netimicin, better than that of gentamicin in all the isolates tested, and better than that of verdamicin against E. coli 9612 and E. faecalis HH22 infections. The therapeutic efficacy of vertilmicin was further confirmed in local infection models of rabbit skin burn infection and mouse ascending urinary tract infection.

A cell-based high-throughput approach to identify inhibitors of influenza A virus.

Influenza is one of the most common infections threatening public health worldwide and is caused by the influenza virus. Rapid emergence of drug resistance has led to an urgent need to develop new anti-influenza inhibitors. In this study we established a 293T cell line that constitutively synthesizes a virus-based negative strand RNA, which expresses Gaussia luciferase upon influenza A virus infection. Using this cell line, an assay was developed and optimized to search for inhibitors of influenza virus replication. Biochemical studies and statistical analyses presented herein demonstrate the sensitivity and reproducibility of the assay in a high-throughput format (Z′ factor value>0.8). A pilot screening provides further evidence for validation of the assay. Taken together, this work provides a simple, convenient, and reliable HTS assay to identify compounds with anti-influenza activity.

Surfactin derivatives from Micromonospora sp. CPCC 202787 and their anti-HIV activities

Micromonospora sp. CPCC 202787 is a strain isolated from soil, its EtOAc-soluble fraction of the extract from mycelia displayed significant inhibitory effect on HIV-1 replication in SupT1 Cells. That motivated us, and finally we obtained two new linear surfactins (1 and 2) and eight known cyclic surfactins (3− 10) (Figure 1). Surfactin, a kind of peptide consisting of seven amino acids and one 3β-hydroxy fatty acids, is not only supposed to be the effective lipopeptide biosurfactant, but also to be bioactive constituent deserving investigation due to its multiple bioactivities such as antibacterial, antiviral, antitumor and hemolytic etc.1 The linear surfactins have been reported for their surfactant property,2–4 whereas their NMR data and bioactivity apart from anti-TB5–7 are underreported. Strain CPCC 202787 was isolated from a soil sample collected in Daluo, Menghai, Jinghong of Yunnan province, China, and it was identified as Micromonospora sp. due to its identical ribosomal 16 s rRNA gene sequence to Micromonospora carbonacea (98%). Strain CPCC 202787 was cultured on Difco ISP medium 2 (0.4% yeast extract, 1.0% malt extract, 0.4% glucose and 2.0% agar power, pH 7.2) slant at 28 °C for 7 days. Then, the grown strain was inoculated in 500-ml Erlenmeyer flasks containing 100 ml of seed medium (0.5% glucose, 1% malt extract, 1% cottonseed powder, 2% soluble starch, 0.5% yeast extract, 0.05% K2HPO4, 0.5% (NH4)2SO4, 0.3% CaCO3 and 0.1% NaCl in deionized water; pH was adjusted to 7.5 before sterilization) at 28 °C on a rotary shaker (190 r.p.m.) for 72 h to prepare seed culture. Subsequently, 1 l seed culture was transferred into 25 l fermenter filled with 20 l of culture medium (same as above seed medium), and the fermentation was carried out at 28 °C for 90– 100 h with stirring at 200 r.p.m. The mycelia separated from 20 l culture by centrifugation were supersonic extracted with (CH3)2CO–H2O (v/v, 50:50) for three times, and the extract was further partitioned between EtOAc and H2O. The EtOAc-soluble fractioned was subjected to silica gel column chromatography eluted with petroleum ether− ethyl acetate (v/v, 10:1→ 7:1) to provide 22 fractions (Fr.1~Fr.22). By repeated separation on semi-preparative HPLC, compounds 1 (8.5 mg) and 2 (12.8 mg) were obtained from Fr.6–7, and compounds 3 (24.4 mg), 4 (7.5 mg), 5 (22.0 mg), 6 (7.7 mg), 7 (18.9 mg), 8 (32.6 mg), 9 (22.2 mg) and 10 (44.1 mg) were obtained from Fr.4− 5 finally. Compound 1 was a white solid with optical rotation of 1⁄2a 25 D + 7.3 (c= 0.1, CH3OH). The HRESIMS showed a [M–H] ion at m/z 1038.6736 (calcd for C52H92N7O14, 1038.6736), deducing the molecular formula of 1 to be C52H93N7O14. In the 1H NMR spectrum, seven characteristic NH doublets proton signals at δ 8.23 (1H, d, J= 7.4 Hz), 8.11 (1H, d, J= 8.2 Hz), 8.04 (1H, d, J= 8.0 Hz), 7.98 (1H, d, J= 8.0 Hz), 7.96 (1H, d, J= 7.7 Hz), 7.85 (1H, d, J= 8.6 Hz) and 7.73 (1H, d, J= 8.4 Hz) were observed, suggesting 1 had seven amino acid residues. Moreover, the large signal at δ 1.22 observed in the 1H NMR spectrum indicated the existence of a fatty acid chain in the molecular. The 13C NMR spectrum presented 10 carbonyl signals at δ 173.9, 173.8, 172.2, 172.0, 171.7, 171.6, 171.3, 171.2, 170.9 and 170.0. The above characteristic NMR data suggested 1 was a surfactin derivative. On the basis of combined analysis of 1H–1H COSY, TOCSY, HSQC and HMBC spectra (Figure 2), seven amino acid units consisting of one glutamic acid (Glu), one asparagine (Asp), one valine (Val) and four leucines (Leu) were identified. The sequence of the amino acid units was assigned by the ROESY correlations of δ 7.98 (Glu1-NH)/3.79 (fatty acid-C-3), 7.96 (Leu2-NH)/4.27 (Glu1-α-H), 8.04 (Leu3-NH)/4.26 (Leu2-α-H), 7.85 (Val4-NH)/4.34 (Leu3-α-H), 8.23 (Asp5-NH)/4.16 (Val4-α-H), 7.73 (Leu6-NH)/4.56 (Asp5-α-H), 8.11 (Leu7-NH)/4.34 (Leu6-α-H) (Figure 2), which was also supported by the observed HMBC correlations of δ 7.98 (Glu1-NH)/171.3 (fatty acid-C-3), 7.96 (Leu2-NH)/171.2 (Glu1-C=O), 8.04 (Leu3-NH)/172.0 (Leu2-C=O), 7.85 (Val4NH)/172.2 (Leu3-C=O), 8.23 (Asp5-NH)/170.9 (Val4-C=O), 7.73 (Leu6-NH)/170.0 (Asp5-C=O), 8.11 (Leu7-NH)/171.7 (Leu6-C=O). The molecular formula of 1 proved that a hydroxyl fatty acid chain length of 14 carbons were in the molecular, and the characteristic carbon signals at δ 38.5 (fatty acid-C-11), 27.4 (fatty acid-C-12), 22.5 (fatty acid-C-13) and 22.5 (fatty acid-C-14) suggested the iso-pattern of its terminal methyl.8 Consequently, 1 was elucidated as (CH3)2CH (CH2)8CH(OH) CH2CO-Glu-Leu-Leu-Val-Asp-Leu-Leu, a new [Leu7] linear surfactin with a iso-fatty acid chain length of 14 carbons and the fully assignment of NMR data were presented in Table 1.

Identification and characterization of loop7 motif and its role in regulating biological function of human APOBEC3G through molecular modeling and biological assay

Human APOBEC3G (hA3G) is a cytidine deaminase which inhibits HIV-1 replication. The HIV-1 accessory protein viral infectivity factor (Vif) counteracts with hA3G by targeting it for proteasomal degradation. In this work, we constructed and optimized molecular models of the hA3G dimer and the hA3G–Vif complex. The molecular modeling study revealed that the loop7 motif of hA3G appears on the interfaces of both the hA3G–Vif complex and the hA3G dimer. Biochemical analysis provided evidence suggesting that binding of Vif to hA3G results in steric blocking of hA3G dimerization, implying that monomeric hA3G serves as a substrate for Vif-mediated degradation. Furthermore, we presented evidence for the important roles of the loop7 motif, especially the central residues within the region, in hA3G dimerization, hA3G--Vif interaction, Vif-mediated hA3G degradation as well as subcellular localization of hA3G. This work highlights a multiple-task interface formed by loop7 motif, which regulates biological function of hA3G, thus providing the feasibility of the strategy of blocking Vif-mediated A3G degradation by targeting the putative site around loop7.

In vitro Antibacterial Activity of Chinfloxacin, a New Fluoroquinolone Antibiotic

Background: Chinfloxacin is a novel synthetic fluoroquinolone with a structure similar to moxifloxacin. The in vitro activity of chinfloxacin was evaluated in the current study. Method: Chinfloxacin was tested against a total of 1,739 clinical isolates representing 23 species using the agar dilution method. Studies of bactericidal activity, including minimum bactericidal concentrations (MBC) and time-kill curve determinations, were conducted according to the recommendations of the Clinical and Laboratory Standards Institute. Results: Minimum inhibitory concentrations (MIC)50s and MIC90s of chinfloxacin were found to be the same or 2-fold lower than those of moxifloxacin against gram-positive isolates except for Streptococcus pyogenes (against which chinfloxacin showed similar MIC50 as moxifloxacin but 2-fold higher MIC90), and the same as or 2-fold higher than those of moxifloxacin against gram-negative isolates. Chinfloxacin showed potent bactericidal activity with MBC/MIC ratios in the range of 1–2 for almost all the isolates tested. Time-kill curves further demonstrated chinfloxacin as a concentration-dependent bactericidal agent usually effective at concentrations of 2 MIC or higher. Conclusion: Chinfloxacin showed similar in vitro activity as moxifloxacin.