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Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

ABSTRACT: Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.

SUBMITTER: Dallmann HG

PROVIDER: S-EPMC2849275 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

Dallmann H Garry HG Fackelmayer Oliver J OJ Tomer Guy G Chen Joe J Wiktor-Becker Anna A Ferrara Tracey T Pope Casey C Oliveira Marcos T MT Burgers Peter M J PM Kaguni Laurie S LS McHenry Charles S CS

Biochemistry 20100301 11

Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitab ...[more]

PMID: 20184361

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Project description:Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

Project description:BACKGROUND:With the emergence of new influenza virus strains that are resistant to current inhibitors such as oseltamivir (anti-neuraminidase (NA)) and amantadine (anti-M2 proton channel), influenza A viruses continue to be a serious threat to the public health worldwide. With this in view, there is a persistent need for the development of broader and more effective vaccines and therapeutics. Identification of broadly neutralizing antibodies (bNAbs) that recognize relatively invariant structures ?on influenza haemagglutinin (HA) stem has invigorated efforts to develop universal influenza vaccines. AIM:The current computational study is designed to identify potential flavonoid inhibitors that bind to the contact epitopes of HA stem that are targeted by broadly neutralizing antibodies (bNAb). METHOD:In this study, we utilized the three-dimensional crystallographic structure of different HA subtypes (H1, H2, H5, H3, and H7) in complex with bNAb to screen for potential broadly reactive influenza inhibitors. We performed Quantitative Structure-Activity and Relationship (QSAR) for 100 natural compounds known for their antiviral activity and performed molecular docking using AutoDock 4.2 suite. Furthermore, we conducted virtual screening of 1413 bioassay hit compounds by using virtual lab bench CLC Drug Discovery. RESULTS:The results showed 18 lead flavonoids with strong binding abilities to bNAb epitopes of various HA subtypes. These 18 broadly reactive compounds exhibited significant interactions with an average of seven Hbonds, docking energy of -22.43 kcal·mol-1, and minimum interaction ? energy of -4.65 kcal·mol-1, with functional contact residues. Procyanidin depicted strong interactions with group 1 HAs, whereas both sorbitol and procyanidin exhibited significant interactions with group 2 HAs. CONCLUSION:Using in silico docking analysis, we identified 18 bioactive flavonoids with potential strong binding cababilities to influenza HA-stems of various subtypes, which are the target for bNAb. The virtual screened bioassay hit compounds depicted a high number of Hbonds but low interaction and docking values compared to antiviral flavonoids. Using structure-based design and nanotechnology-based approaches, identified molecules could be modified to generate next generation anti-influenza drugs.

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Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

Publications

Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

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