Unraveling new features of clindamycin interaction with functional ribosomes and dependence of the drug potency on polyamines.
The effect of spermine on the inhibition of peptide-bond formation by clindamycin, an antibiotic of the Macrolide-Lincosamide-StreptograminsB family, was investigated in a cell-free system derived from Escherichia coli. In this system peptide bond is formed between puromycin, a pseudo-substrate of the A-site, and acetylphenylalanyl-tRNA, bound at the P-site of poly(U)-programmed 70 S ribosomes. Biphasic kinetics revealed that one molecule of clindamycin, after a transient interference with the A-site of ribosomes, is slowly accommodated near the P-site and perturbs the 70 S/acetylphenylalanyl-tRNA complex so that a peptide bond is still formed but with a lower velocity compared with that observed in the absence of the drug. The above mechanism requires a high temperature (25 degrees C as opposed to 5 degrees C). If this is not met, the inhibition is simple competitive. It was found that at 25 degrees C spermine favors the clindamycin binding to ribosomes; the affinity of clindamycin for the A-site becomes 5 times higher, whereas the overall inhibition constant undergoes a 3-fold decrease. Similar results were obtained when ribosomes labeled with N1-azidobenzamidinospermine, a photo-reactive analogue of spermine, were used or when a mixture of spermine and spermidine was added in the reaction mixture instead of spermine alone. Polyamines cannot compensate for the loss of biphasic kinetics at 5 degrees C nor can they stimulate the clindamycin binding to ribosomes. Our kinetic results correlate well with photoaffinity labeling data, suggesting that at 25 degrees C polyamines bound at the vicinity of the drug binding pocket affect the tertiary structure of ribosomes and influence their interaction with clindamycin.
Kouvela
Ekaterini C
EC
Laboratory of Biochemistry, School of Medicine, University of Patras, 26500 Patras, Greece.
Petropoulos
Alexandros D
AD
Kalpaxis
Dimitrios L
DL
D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001386:Azides / Q000737:chemistry; D002981:Clindamycin / Q000737:chemistry*; D004347:Drug Interactions; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D012338:RNA, Ribosomal, 23S / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism; D013096:Spermine / Q000031:analogs & derivatives / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D001386:Azides; D010455:Peptides; D011073:Polyamines; D012338:RNA, Ribosomal, 23S; C068638:N(1)-(azidobenzamidino)spermine; D013096:Spermine; D002981:Clindamycin