Towards new antibiotics targeting bacterial transglycosylase: Synthesis of a Lipid II analog as stable transition-state mimic inhibitor.

Described here is the asymmetric synthesis of iminosugar 2b, a Lipid II analog, designed to mimic the transition state of transglycosylation catalyzed by the bacterial transglycosylase. The high density of functional groups, together with a rich stereochemistry, represents an extraordinary challenge for chemical synthesis. The key 2,6-anti- stereochemistry of the iminosugar ring was established through an iridium-catalyzed asymmetric allylic amination. The developed synthetic route is suitable for the synthesis of focused libraries to enable the structure-activity relationship study and late-stage modification of iminosugar scaffold with variable lipid, peptide and sugar substituents. Compound 2b showed 70% inhibition of transglycosylase from Acinetobacter baumannii, providing a basis for further improvement.

Structure-based design of bacterial transglycosylase inhibitors incorporating biphenyl, amine linker and 2-alkoxy-3-phosphorylpropanoate moieties.

Transglycosylase (TGase) is essential to biosynthesis of peptidoglycan for formation of bacterial cell wall. Moenomycin is a potent TGase inhibitor, but not used in clinic treatment due to its poor pharmacokinetics. The E-F disaccharide, phosphoglycerate and lipid tail in moenomycin are crucial elements for TGase inhibition and antibacterial activity. Based on this scaffold, a series of truncated mimics comprising biphenyl, amine linker and 2-alkoxy-3-phosphorylpropanoate moieties were designed to test their TGase inhibitory activity. In this design, the phosphorylpropanoate group is a surrogate of phosphoglycerate with improved stability. A library of lipid tails can be constructed by a straightforward approach using Cu(I)-catalyzed (3 + 2) cycloaddition reactions, and the as-synthesized triazole ring can provide additional hydrogen bonds in the TGase active site. Our molecular docking experiments reveal that the biphenyl group provides π-π and π-cation interactions to act as a simplified alternative of the C-E disaccharide in moenomycin. To play the role of the oxonium transition state in transglycosylation, the amine linker exists as a positively charged species in physiological condition to attain electrostatic interactions with acidic residues. In this study, two biphenyl-linked 2-alkoxy-3-phosphorylpropanoate compounds (8 and 10) are found to exhibit modest inhibitory activity (IC50 ≈ 150 µM) against the TGase of Acinetobacter baumannii and good antibacterial activity against Staphylococcus aureus (MIC = 6.3 µM).

Affinity-Based Screen for Inhibitors of Bacterial Transglycosylase.

The rise of antibiotic resistance has created a mounting crisis across the globe and an unmet medical need for new antibiotics. As part of our efforts to develop new antibiotics to target the uncharted surface bacterial transglycosylase, we report an affinity-based ligand screen method using penicillin-binding proteins immobilized on beads to selectively isolate the binders from complex natural products. In combination with mass spectrometry and assays with moenomycin A and salicylanilide analogues (1-10) as reference inhibitors, we isolated four potent antibacterials confirmed to be benastatin derivatives (11-13) and albofungin (14). Compounds 11 and 14 were effective antibiotics against a broad-spectrum of Gram-positive and Gram-negative bacteria, including Acinetobacter baumannii, Clostridium difficile, Staphylococcus aureus, and drug-resistant strains with minimum inhibitory concentrations in the submicromolar to nanomolar range.

Effectiveness of 10-year vaccination (2001-2010) on Hepatitis A in Tianjin, China.

Vaccination is an effective strategy to prevent and control the transmission of hepatitis A. Hepatitis A immunization program has been taken into effect since 2001 in Tianjin, China. This study evaluated the effectiveness of strategies in the prevention and control of hepatitis A. Data of serological survey, annual hepatitis A incidence, immunization coverage and the positive rate of hepatitis A IgG before and after the immunization program in residents under 15 years old were used to do the analysis. The results indicated that hepatitis A vaccine induced a striking decrease of hepatitis A incidence and a significant increase in the positive rate of anti-HAV IgG among the children younger than 15 years old. Hepatitis A vaccination in children was proved to be effective in the prevention and control of hepatitis A in Tianjin, China.

New continuous fluorometric assay for bacterial transglycosylase using Förster resonance energy transfer.

The emergence of antibiotic resistance has prompted scientists to search for new antibiotics. Transglycosylase (TGase) is an attractive target for new antibiotic discovery due to its location on the outer membrane of bacteria and its essential role in peptidoglycan synthesis. Though there have been a few molecules identified as TGase inhibitors in the past thirty years, none of them have been developed into antibiotics for humans. The slow pace of development is perhaps due to the lack of continuous, quantitative, and high-throughput assay available for the enzyme. Herein, we report a new continuous fluorescent assay based on Förster resonance energy transfer, using lipid II analogues with a dimethylamino-azobenzenesulfonyl quencher in the lipid chain and a coumarin fluorophore in the peptide chain. During the process of transglycosylation, the quencher-appended polyprenol is released and the fluorescence of coumarin can be detected. Using this system, the substrate specificity and affinity of lipid II analogues bearing various numbers and configurations of isoprene units were investigated. Moreover, the inhibition constants of moenomycin and two previously identified small molecules were also determined. In addition, a high-throughput screening using the new assay was conducted to identify potent TGase inhibitors from a 120,000 compound library. This new continuous fluorescent assay not only provides an efficient and convenient way to study TGase activities, but also enables the high-throughput screening of potential TGase inhibitors for antibiotic discovery.

[Current status of hepatitis B immunization and strengthened immune memory among first-year middle school students in Tianjin].

OBJECTIVE: To investigate the immunization status of hepatitis B vaccine who were inoculated at birth, HBV infections and the vaccine booster effect in the first-year middle school students (12 - 14 years old). METHODS: A cluster, stratified simplified random sampling method was administrated. The sample size was at least 218, which was calculated by Epi Info 3.3.2 software at 53% the minimum acceptable anti-HBs positive rate and 95% confidence level. A total of 250 and 236 students participated in the infection status and booster immunization effects investigation. The HBsAg, anti-HBs and anti-HBc IgG were detected by Enzyme-linked immunosorbent assay (ELISA). HBV DNA was detected by fluorescence quantitative PCR, and the diagnostic test kit were produced respectively by ABBOTT, Diasorin and Beijing Wantai Biological Pharmacy Enterprise Co. RESULTS: For the immunization status before booster: the positive rate of anti-HBs was 62.80% (157/250), the GMT was 73.79 IU/L; the currently HBV infection rate (HBsAg and anti-HBc positive) was 2.80% (7/250). After injection, the anti-HBs positive rate was 94.92% (224/236). Compared with the before booster results, the significant difference was observed (χ(2) = 73.92, P = 0.00). The GMT was 521.15 IU/L, comparing with the before booster results, there was significant difference (t = 15.98, P = 0.00). The anti-HBs conversion rate (from negative to positive) was 91.86% (79/86) after immune-enhancement; of which, 11 students got the second dose of booster vaccine who are no-responders after first injection, in addition 8 students got the anti-HBs. CONCLUSION: It is an effective method to put the first-year middle school students into the immune-enhancement program, so as to improve the immunization memory effect and avoid the loss of protective antibodies.

[Study on the report criteria and focus management mode of the virus hepatitis cases in Tianjin].

OBJECTIVE: To establish the focus management mode and the report criteria more perfectly for the virus hepatitis cases, especially for the hepatitis B. METHODS: One district was set as the research area, in which there was enough cases resource and relatively separated from the other districts, then a first or second-class hospital was appointed to take the cases focus diagnosis, report and management. RESULTS: The focus hospitals had reported 97% (323/331) of cases in the research area between June,2007 and June,2008; moreover,the rate in establishing case-card was 97.21% (314/323), as compared with that in period of 2007 (261), the cases reported declined 61.30% in the first half year of 2008 (101). CONCLUSION: It should be an imperative situation to establish a report criteria and management mode for virus hepatitis (hepatitis B), however it is necessary to have more supports from health administrations.