Bicyclomycin fluorescent probes: synthesis and biochemical, biophysical, and biological properties.
Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. Key aspects of the 1.rho interaction-K(d), stoichiometry for 1.rho binding, and whether 1 and ATP binding induce conformational changes in rho-remain unknown. In this study, the design, synthesis, and characterization of a series of bicyclomycin fluorescent probes (BFP) constructed to sense the 1.rho interaction are described and their use documented. We show that dihydrobicyclomycins with medium-to-large C(5a)-substituents afforded excellent inhibitory activities exceeding those of 1 in the poly(C)-dependent ATPase assay. The utility of BFP in bicyclomycin-rho binding studies was documented through the use of 5a-(phenazin-2-ylmethylsulfanyl)dihydrobicyclomycin (15). Excitation (290 nm) of W381 in wild-type rho in the presence of 15 and ATP led to fluorescence resonance energy transfer (FRET) and gave a K(d) (15) of 9.9 microM. Using ADP in place of ATP or excluding nucleotide did not result in energy transfer, which suggests that ATP binding induced a conformational change in rho. FRET measurements provided an approximate weighted average distance (23 A) between W381 and 15 in the presence of bound ATP. The K(d) value for 15.rho was correlated with ATP binding at the 3 tight ATP binding (K(d)(ATP) = 95 nM) sites in wild-type rho.
Brogan
Andrew P
AP
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, USA.
Widger
William R
WR
Kohn
Harold
H
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D004735:Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012234:Rho Factor / Q000037:antagonists & inhibitors / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005456:Fluorescent Dyes; D012234:Rho Factor; D000255:Adenosine Triphosphate; C024504:bicozamycin