Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.
Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.
Dallmann
H Garry
HG
Department of Chemistry and Biochemistry, University of Colorado, Campus Box 215, Boulder, Colorado 80309, USA.
Fackelmayer
Oliver J
OJ
Tomer
Guy
G
Chen
Joe
J
Wiktor-Becker
Anna
A
Ferrara
Tracey
T
Pope
Casey
C
Oliveira
Marcos T
MT
Burgers
Peter M J
PM
Kaguni
Laurie S
LS
McHenry
Charles S
CS
D001419:Bacteria / Q000201:enzymology*; D004261:DNA Replication / Q000187:drug effects; D004269:DNA, Bacterial / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D054852:Small Molecule Libraries; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase