Ribosomal binding region for the antibiotic tiamulin: stoichiometry, subunit location, and affinity for various analogs

Abstract

Equilibrium dialysis experiments with a highly purified preparation of labeled tiamulin, a semisynthetic derivative of the antibiotic pleuromutilin, and Escherichia coli ribosomes allowed the determination of two binding sites for the drug. The binding reaction showed a cooperative effect. Of the two subunits, the 50S particle was able to bind the antibiotic in a 1:1 stoichiometry. Hence, the 50S subunit contributed predominantly to the binding energy which held the antibiotic to the ribosomes. The 30S subunit, showing no strong affinity for the drug, may be needed for the generation of the second binding site in the 70S particle. If depleted of ammonium ions, 70S ribosomes lost their binding capacity for the antibiotic. The attachment sites for tiamulin could be restored by heating the ribosomes to 40°C in the presence of either ammonium ions or the antibiotic. Other pleuromutilin derivatives displaced labeled tiamulin from its ribosomal binding sites. By quantifying this competition, the relative affinity of various pleuromutilin derivatives for E. coli ribosomes was determined. The binding correlated with the minimal inhibitory concentrations of these compounds against E. coli. When compared with the minimal inhibitory concentrations against Staphylococcus aureus, the correlation was less strict, but the same trend prevailed. These results suggest that the antibacterial activities of various pleuromutilin derivatives on a given test organism are mainly determined by the strength of binding to the ribosomes within the bacterial cell.

U2504 Determines the Species Specificity of the A-Site Cleft Antibiotics:

“…Either the system “pays” that cost and then benefits from stacking interactions with U2504 (2539), which is what happens when linezolid binds (Figure 4e), or it does not, which is the outcome when tiamulin binds (Figure 4a). In either case, both linezolid and tiamulin should bind more strongly to eubacterial ribosomes than they do to eukaryotic/archaeal ribosomes, 36,37 and hence preferentially inhibit the former. [Preliminary data obtained in the course of this work indicate that the MIC for H. ma for tiamulin is ~ 400 μg/ml (~0.66 mM), which is about 8 times its MIC for E. coli and 8000 times its MIC for S. aureus 7(Gurel, unpublished observations).…”

Strategies for the Design of Rna-Binding Small Molecules

“…Paromomycin II A-site 1j7t, 203w [206] 82 Pleuromutilin PTC 80% protection at 20 µM 2ogo,ogn,ogo [207] 83 Pristinamycin IIA PTC 0.14 µM [208] 84 Promazine HIV-1 TAR Not reported [209] 84 Protoporphyrin IX tRNA/M1 RNA 3.5 µM [210] 85 Puromycin 50S A-site 480 µM 1q82 [211][212][213] 86 Quenosine Riboswitch 20 nM [214] 87 Quinacridone HIV-1 TAR~1 µM [215] 88 Quinupristin PTC 0.32/2.5 µM 1sm1 [175] 89 Ralenova (mitoxantrone) HIV-1 psi RNA/hvg RNA~10 µM (hvg RNA) [216] 90 Rbt203 HIV-1 TAR RNA 2 µM 1uud [217] [191] 98 S-adenosyl methionine Riboswitch <100 nM 2gis [220] 99 Sisomicin HCV IRES IIId 260nM [221] 100 Spectinomycin Small subunit 260 nM 1fjg [222] 101 Spiramycin A Exit tunnel, 50S 1.8 µM from kinetic data 1kd1 [163,223] …”

Recent Developments in Pleuromutilin Derivatives: A Promising Class Against Bacterial Respiratory Disease

“…Studies utilizing tiamulin, valnemulin and others chemically related substances demonstrated that pleuromutilins act as bacterial protein biosynthesis inhibitors [29][30][31][32][33]. These substances bind to 70S ribosome which is composed of two subunits, a small (30S) and a large (50S) [34,35].…”

“…It is important to mention that the binding site of pleuromutilins is different from erythromycin ( Fig. 5) compete with pleuromutilins for binding to the ribosome [19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. However, other antibiotics such as chloramphenicol (Fig.…”