Dissociation–equilibrium constant and bound conformation for weak antibiotic binding interaction with different bacterial ribosomes†

This paper characterises low-affinity antibiotic binding interactions by the T2 (CPMG) method. Three different compounds, a ketolide ‘telithromycin’ (HMR 3647), a macrolide ‘roxithromycin’ and a lincosamide ‘clindamycin’ belonging to the macrolide–lincosamide–streptogramin B (MLSB) class of antimicrobial agents were examined against Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus strains. The Carr–Purcell–Meiboom– Gill spin-echo decay rate allows determination of the H-NMR transverse relaxation (T2). T2 (CPMG) measurements represent a sensitive method and they can be used to characterise equilibrium binding of low-affinity antibiotics to bacterial ribosomes. This analysis revealed weak binding of telithromycin, roxithromycin and clindamycin with respectively, KD = 1.2 × 10 , 1.3 × 10 3 and 1.3 × 10 2 to E. coli, 3.4 × 10 , 3.6 × 10 3 and 1.4 × 10 3 to S. pneumoniae and 3.1 × 10 , 7.9 × 10 3 and 2.9 × 10 3 to S. aureus. Additionally, this paper demonstrates by a study using 2D transferred nuclear Overhauser effect spectroscopy (TRNOESY), that the three MLSB antibiotics exist in these ribosome binding sites in certain conformations. Analysis of the TRNOE experiments resulted in a set of constraints for all proton pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain the conformation of the antibiotic in its bound state. This study allowed us to compare the bound structure at the bacterial ribosomes for the active ketolide and MLSB antibiotics and to bring to the fore an interesting relationship between the conformation of the antibiotic when interacting and its activity. The weak interaction responses to S. pneumoniae and S. aureus strains require a ‘B’ conformation which is especially favoured for the ketolide telithromycin.