Kinase experiment:

ATPase activity assays are carried out with rho (100 ng) except that either bicyclomycin (0–60 μM) or dihydrobicyclomycin (0–90 μM) is added to the reaction solution. The samples are preheated to 32℃ (2.5 min), and the reaction is initiated by the addition of ATP (9.1–100 μM) and [g-32P]ATP (0.5mCi) to the side of the tube, briefly vortexed, centrifuged (2 s), and returns to the water bath. Aliquots (1 mL) are removed at five time points (15, 30, 45, 60, and 75 s), and spotted on Baker-Flex cellulosePEI TLC plates. The rates of reaction are determined by measuring the relative amount of radiolabeled inorganic phosphate and ATP and then plotting the amount of ATP hydrolyzed versus time. The initial rates for each ATP concentration plus or minus inhibitors are plotted as double reciprocal plots[3].

Animal experiment:

Rats: A total of 25 SD male rats receives intramuscular administration of bicyclomycin at a single dose of 50mg/kg. Blood samples are taken by heart-puncture from 5 rats each at 0.5, 1, 2, 3, and 5 hours after the administration[4]. Mice: A total of 40 ICR male mice receives intramuscular administration of bicyclomycin at a single dose of 50mg/kg. Eight mice each are bled at 5, 10, 20, 30 and 60 minutes after administration by heart-puncture with heparin[4]. Rabbits: Five rabbits and 5 dogs are given bicyclomycin intramuscularly at a single dose of 50 mg/kg and blood samples are withdrawn from these animals at similar intervals. The samples are allowed to clot and sera are separated for assay by the cylinder plate method[4].

Bicyclomycin benzoate is a novel antibiotic produced by S. sapporonensi [1]. Bicyclomycin shows an inhibitory effect on Gram-negative bacteria, such as E. coli, Klebsiella, Shigella, Salmonella, Citrobacter, E. cloacae, and the pathogenic group of Neisseria [2]. Bicyclomycin benzoate is inactive against Proteus, P. aeruginosa, and Gram-positive bacteria [1,2].

In vitro: The MIG of bicyclomycin for E.coli NIHJ JC-2 was 25-50 μg/ml. Bicyclomycin (25-50 μg/ml) completely inhibited formation of visible colonies or turbidity of this organism on agar plates, in nutrient broth, and in heart infusion broth [2]. Bicyclomycin inhibited ATPase activity in the presence of poly(dC) and ribo(C)10. The approximate IC50 value for inhibition of transcription termination at Rho-dependent sites was 5 μM. The inhibitory effect of bicyclomycin on Rho-dependent transcripts was accompanied by the appearance of a new set of transcripts. In the presence of poly(dC), bicyclomycin reversibly inhibited the ribo(C)10-stimulated ATPase activity. The extrapolated Ki for bicyclomycin was 2.8 μM without ribo(C)10, which was increased to 26 μM in the presence of ribo(C)10 [3].

In vivo: Bicyclomycin showed therapeutic activity for infections with several strains of E. coli which were resistant to the control antibiotics. The ED50 of bicyclomycin for infection with GP-resistant E.coli 312 was 3.05 (1.47-7.66) mg/mouse [2].

References:
[1] Miyoshi, T. ,Miyairi, N.,Aoki, H., et al. Bicyclomycin, a new antibiotic. I. Taxonomy, isolation and characterization. J.Antibiot.(Tokyo) 25(10), 569-575(1972).
[2] Nishida, M. ,Mine, Y.,Matsubara, T., et al. Bicyclomycin, a new antibiotic. III. In vitro and in vivo antimicrobial activity. J.Antibiot.(Tokyo) 25(10), 582-593(1972).
[3] Magyar A, Zhang X, Kohn H, et al.  The antibiotic bicyclomycin affects the secondary RNA binding site of Escherichia coli transcription termination factor Rho[J]. Journal of Biological Chemistry, 1996, 271(41): 25369-25374.