Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

Inhibition of HIV-1 reverse transcriptase-catalyzed DNA strand transfer reactions by 4-chlorophenylhydrazone of mesoxalic acid.

DNA strand transfer reactions occur twice during retroviral reverse transcription catalyzed by HIV-1 reverse transcriptase. The 4-chlorophenylhydrazone of mesoxalic acid (CPHM) was found to be an inhibitor of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. Using a model strand transfer assay system described previously [Davis, W. R., et al. (1998) Biochemistry 37, 14213-14221], the mechanism of CPHM inhibition of DNA strand transfer has been characterized. CPHM was found to target the RNase H activity of HIV-1 reverse transcriptase. DNA polymerase activity was not significantly affected by CPHM; however, it did inhibit the polymerase-independent RNase H activity with an IC(50) of 2.2 microM. In the absence of DNA synthesis, CPHM appears to interfere with the translocation, or repositioning, of RT on the RNA.DNA template duplex, a step required for efficient RNA hydrolysis by RNase H. Enzyme inhibition by CPHM was found to be highly specific for HIV-1 reverse transcriptase; little or no inhibition of DNA strand transfer or DNA polymerase activity was observed with MLV or AMV reverse transcriptase, T7 DNA polymerase, or DNA polymerase I. Examination of additional 4-chlorophenylhydrazones showed that the dicarboxylic acid moiety of CPHM is essential for activity, suggesting its important role for enzyme binding. Consistent with the role of the dicarboxylic acid in inhibitor function, Mg(2+) was found to chelate directly to CPHM with a K(d) of 2.4 mM. Together, these studies suggest that the inhibitor may function by binding to enzyme-bound divalent metal cofactors.