ABSTRACT
With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.
Subject(s)
Anti-Bacterial Agents , Pharmacology , Antibiotics, Antitubercular , Pharmacology , Antitubercular Agents , Pharmacology , Bacterial Proteins , Genetics , Metabolism , Drug Resistance, Bacterial , Electroporation , Ethambutol , Pharmacology , Isoniazid , Pharmacology , Microbial Sensitivity Tests , Mycobacterium smegmatis , Genetics , Metabolism , Oxidoreductases , Genetics , Metabolism , Plasmids , Rifampin , Pharmacology , Streptomycin , PharmacologyABSTRACT
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.
Subject(s)
Animals , Humans , Anti-HIV Agents , Chemistry , Pharmacology , Antiviral Agents , Chemistry , Pharmacology , Benzoquinones , Chemistry , Pharmacology , Cell Line, Tumor , Chlorocebus aethiops , Enterovirus B, Human , Physiology , HIV-1 , Physiology , Hep G2 Cells , Hepatitis B virus , Physiology , Herpesvirus 1, Human , Physiology , Herpesvirus 2, Human , Physiology , Lactams, Macrocyclic , Chemistry , Pharmacology , Lamivudine , Chemistry , Pharmacology , Madin Darby Canine Kidney Cells , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Pathology , Virology , Vero Cells , Virus ReplicationABSTRACT
Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).