Streptococcus pneumononiae gyrase ATPase: development and validation of an assay for inhibitor discovery and characterization.
The rise in bacterial resistance to antibiotics demonstrates the medical need for new antibacterial agents. One approach to this problem is to identify new antibacterials that act through validated drug targets such as bacterial DNA gyrase. DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils into plasmid and chromosomal DNA and is essential for DNA replication. Inhibition of the ATPase activity of DNA gyrase is the mechanism by which coumarin-class antibiotics such as novobiocin inhibit bacterial growth. Although ATPase inhibitors exhibit potent antibacterial activity against gram-positive pathogens, no gyrase ATPase activity from a gram-positive organism is described in the literature. To address this, we developed and optimized an enzyme-coupled phosphate assay and used this assay to characterize the ATPase kinetics of Streptococcus pneumoniae gyrase. The S. pneumoniae enzyme exhibits cooperativity with ATP and requires organic potassium salts. We also studied inhibition of the enzyme by novobiocin. Apparent inhibition constants for novobiocin increased linearly with ATP concentration, indicative of an ATP-competitive mechanism. Similar binding affinities were measured by isothermal titration calorimetry. These results reveal unique features of the S. pneumoniae DNA gyrase ATPase and demonstrate the utility of the assay for screening and kinetic characterization of ATPase inhibitors.
Miller
J Richard
JR
Department of Antibacterial Biology, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA. richard.miller2@pfizer.com
Herberg
John T
JT
Tomilo
Mark
M
McCroskey
Mark C
MC
Feilmeier
Bradley J
BJ
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000302:isolation & purification; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D027081:DNA Gyrase / Q000187:drug effects / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D015394:Molecular Structure; D009675:Novobiocin / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D059005:Topoisomerase II Inhibitors
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D003374:Coumarins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D000255:Adenosine Triphosphate; C030123:coumarin; D000251:Adenosine Triphosphatases; D027081:DNA Gyrase