A high-throughput fluorescence polarization assay for inhibitors of gyrase B.
DNA gyrase, a type II topoisomerase that introduces negative supercoils into DNA, is a validated antibacterial drug target. The holoenzyme is composed of 2 subunits, gyrase A (GyrA) and gyrase B (GyrB), which form a functional A(2)B(2) heterotetramer required for bacterial viability. A novel fluorescence polarization (FP) assay has been developed and optimized to detect inhibitors that bind to the adenosine triphosphate (ATP) binding domain of GyrB. Guided by the crystal structure of the natural product novobiocin bound to GyrB, a novel novobiocin-Texas Red probe (Novo-TRX) was designed and synthesized for use in a high-throughput FP assay. The binding kinetics of the interaction of Novo-TRX with GyrB from Francisella tularensis has been characterized, as well as the effect of common buffer additives on the interaction. The assay was developed into a 21-µL, 384-well assay format and has been validated for use in high-throughput screening against a collection of Food and Drug Administration-approved compounds. The assay performed with an average Z' factor of 0.80 and was able to identify GyrB inhibitors from a screening library.
SRI International, Menlo Park, CA 94025, USA.
Bryan T
BT
Glaser
Jeremiah P
JP
Malerich
Sarah J
SJ
Duellman
Julie
J
Fong
Christopher
C
Hutson
Richard M
RM
Fine
Boris
B
Keblansky
Mary J
MJ
Tang
Peter B
PB
Madrid
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D027081:DNA Gyrase / Q000737:chemistry / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005454:Fluorescence Polarization / Q000379:methods; D057166:High-Throughput Screening Assays; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009675:Novobiocin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054852:Small Molecule Libraries; D059005:Topoisomerase II Inhibitors
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D000255:Adenosine Triphosphate; D027081:DNA Gyrase
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