Studies on the mode of action of the antibiotic bicyclomyci

The structurally unique antibiotic bicyclomycin (1) possesses a broad spectrum of antimicrobial activity and targets the rho transcription termination factor in Escherichia coli and Micrococcus luteus. We found that bicyclomycin inhibited wild-type rho poly(C)-stimulated ATPase activity $rm(Isb{50} = 60 mu M)$ by a reversible, noncompetitive pathway with respect to ATP $rm(Ksb{i} = 25 mu M).$ Bicyclomycin inhibited poly(dC)-ribo(C)$sb{10}$-stimulated ATPase activity of wild-type rho by a mixed kinetic pathway with respect to ribo(C)$sb{10},$ and 1 did not affect poly(C) binding to rho in the absence and presence of ATP. These data showed that bicyclomycin affected the secondary RNA binding site of rho. Furthermore, we found that the reversible inhibitor, dihydrobicyclomycin (24), displayed similar inhibitory properties to 1 in the transcription termination $rm(Isb{50} = 20 mu M)$ and poly(C) binding assays. These results indicated that bicyclomycin expresses its inhibitory activity under the in vitro assay conditions by a reversible, noncovalent pathway. Our mechanistic investigations mirrored previous studies conducted with E155K mutant rho showing that E155 does not play a key role in rho function.;Structure-activity relationship studies employing the poly(C)-stimulated ATPase and in vitro transcription termination assays demonstrated that both the C(1) triol and the (4.2.2) bicyclic ring units in 1 were critical structural elements for drug function, while the C(5)-C(5a) exomethylene unit was not. Accordingly, we selected the C(5)-C(5a) modified analogues bicyclomycin-5-norketone (58), C(5a)-(acetoxy)methylbicyclomycin (69), C(5a)- (2,6-bis-(trifluoromethyl)benz-oxy) methylbicyclomycin (70), and C(5a)-(chloro)methylbicyclomycin (72) as potential irreversible inactivators to help identify the bicyclomycin binding domain in rho. Compounds 58, 69 and 70 did not inactivate rho, while 72 irreversibly modified rho upon incubation. Rho adduction was found to be competitive with 1, and mass spectral analyses showed that 72-adduction occurred within amino acids 128-283 in rho.;The His-tag S266Y mutant rho was generated by oligonucleotide site-directed mutagenesis. This point mutation did not appreciably affect RNA and ATP binding and bicyclomycin inhibition of the poly(C)-stimulated ATPase activity of rho. We found that the bicyclomycin inhibition kinetics of S266Y mutant rho in the poly(dC)-ribo(C)$sb{10}$-stimulated ATPase assay was near noncompetitive with respect to ribo(C)$sb{10}$ suggesting that bicyclomycin did not compete with ribo(C)$sb{10}$ binding but rather interfered with ATP hydrolysis.