cb300018x_si_001.pdf (1.44 MB)
Small-Molecule Inhibitors of Bacterial AddAB and RecBCD Helicase-Nuclease DNA Repair Enzymes
journal contribution
posted on 2012-05-18, 00:00 authored by Susan
K. Amundsen, Timothy Spicer, Ahmet C. Karabulut, Luz Marina Londoño, Christina Eberhart, Virneliz Fernandez
Vega, Thomas D. Bannister, Peter Hodder, Gerald R. SmithThe AddAB and RecBCD helicase-nucleases are related enzymes
prevalent
among bacteria but not eukaryotes and are instrumental in the repair
of DNA double-strand breaks and in genetic recombination. Although
these enzymes have been extensively studied both genetically and biochemically,
inhibitors specific for this class of enzymes have not been reported.
We developed a high-throughput screen based on the ability of phage
T4 gene 2 mutants to grow in Escherichia
coli only if the host RecBCD enzyme, or a related helicase-nuclease,
is inhibited or genetically inactivated. We optimized this screen
for use in 1536-well plates and screened 326,100 small molecules in
the NIH molecular libraries sample collection for inhibitors of the Helicobacter pylori AddAB enzyme expressed in an E. coli recBCD deletion strain. Secondary screening used
assays with cells expressing AddAB or RecBCD and a viability assay
that measured the effect of compounds on cell growth without phage
infection. From this screening campaign, 12 compounds exhibiting efficacy
and selectivity were tested for inhibition of purified AddAB and RecBCD
helicase and nuclease activities and in cell-based assays for recombination;
seven were active in the 0.1–50 μM range in one or another
assay. Compounds structurally related to two of these were similarly
tested, and three were active in the 0.1–50 μM range.
These compounds should be useful in further enzymatic, genetic, and
physiological studies of these enzymes, both purified and in cells.
They may also lead to useful antibacterial agents, since this class
of enzymes is needed for successful bacterial infection of mammals.