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1.  Mannich reaction derivatives of novobiocin with modulated physiochemical properties and their antibacterial activities 
Synthetic derivatives of the natural product antibiotic novobiocin were synthesized in order to improve their physiochemical properties. A Mannich reaction was used to introduce new side chains at a solvent-exposed position of the molecule, and a diverse panel of functional groups was evaluated at this position. Novobiocin and the new derivatives were tested for their binding to gyrase B and their antibacterial activities against S. aureus, M. tuberculosis, F. tularensis and E. coli. While the new derivatives still bound the gyrase B protein potently (0.07 – 1.8 μM IC50), they had significantly less antibacterial activity. Two compounds were identified with increased antibacterial activity against M. tuberculosis, with a minimum inhibitory concentration of 2.5 μg/ml.
doi:10.1016/j.bmcl.2011.08.035
PMCID: PMC3178263  PMID: 21871799
Novobiocin; Gyrase B; Tuberculosis; Antibacterial; Aminocoumarin
2.  A High-throughput Fluorescence Polarization Assay for Inhibitors of Gyrase B 
Journal of biomolecular screening  2011;16(2):230-238.
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 A2B2 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.
doi:10.1177/1087057110392038
PMCID: PMC3176662  PMID: 21245469
fluorescence polarization; gyrase; assay development; high-throughput screen; anthracycline
3.  Studying the Salt Dependence of the Binding of σ70 and σ32 to Core RNA Polymerase Using Luminescence Resonance Energy Transfer 
PLoS ONE  2009;4(8):e6490.
The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of σ70 with theβ' coiled-coil fragment (amino acids 100–309). Here we describe an LRET binding assay used to monitor the interaction of E. coli σ70 and σ32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of σ70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the σ32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of σ32 with core RNAP is stronger than σ70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function.
doi:10.1371/journal.pone.0006490
PMCID: PMC2715106  PMID: 19649256

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