A novel pleuromutilin antibacterial compound, its binding mode and selectivity mechanism

Abstract

The increasing appearance of pathogenic bacteria with antibiotic resistance is a global threat. Consequently, clinically available potent antibiotics that are active against multidrug resistant pathogens are becoming exceedingly scarce. Ribosomes are a main target for antibiotics, and hence are an objective for novel drug development. Lefamulin, a semi-synthetic pleuromutilin compound highly active against multi-resistant pathogens, is a promising antibiotic currently in phase III trials for the treatment of community-acquired bacterial pneumonia in adults. The crystal structure of the Staphylococcus aureus large ribosomal subunit in complex with lefamulin reveals its protein synthesis inhibition mechanism and the rationale for its potency. In addition, analysis of the bacterial and eukaryotes ribosome structures around the pleuromutilin binding pocket has elucidated the key for the drug's selectivity.

Figure 1

(A) The chemical structure of lefamulin. (B) The weighted 2F_o-F_c electron density map of the complex S50S-lefamulin around the lefamulin’s binding site contoured at 1.0σ. (C) The binding site of lefamulin (green) within the SA50S-lef structure at the PTC (D) Zoom into the binding pocket where lefamulin (green) is held within the PTC 23S rRNA nucleotides (orange) with hydrogen bonds (dashed lines). The U:U interactions between U2585 and U2506 stabilize the lefamulin binding pocket.

Figure 2. Comparative analysis of the lefamulin binding pocket.

(A) A superposition of the PTC in apo SA50S structure (blue) and in the SA50S-lef complex (lefamulin in green 23S rRNA in orange) reveals relocations of nucleotides U2585 and U2506 in the bound vs. apo structure. (B) A superposition of the PTC in the two S. aureus pleuromutilins complexes [SA50SBC3205 (teal and pink) (4WFB) and SA50S-lef (orange and green) - this work. The dash line represents the unique hydrogen bond BC-3205 forms with the 23S rRNA. (C) A superposition of pleuromutilins within their binding pockets of S. aureus and D. radiodurans [SA50S-lef (orange and green) - this work, D50S-retapamulin (dark grey) (2OGO), D50S-tiamulin (yellow) (1XBP)9].

Figure 3. Inhibition of bacterial protein synthesis.

The inhibitory effect of BC-3205, lefamulin and tiamulin on protein expression in S. aureus in vitro transcription-translation cell-free system. The activity of the reporter protein (luciferase) in the presence of various concentrations of BC-3205, lefamulin and tiamulin is shown as arbitrary unit of luminescence [a.u.]. The IC₅₀ values calculated by the plotted data showed better inhibition of protein synthesis by lefamulin than by BC-3205 and tiamulin.

Figure 4. The binding pocket of lefamulin.

(A) SA50S-lef (lefamulin in green and 23S rRNA in orange) superimposed on the structure of human 80S (grey) (4UG0). In S. aureus nucleotides C2452 and U2504 interact with each other (marked with dashed lines) while the same nucleotides in eukaryotes’ structures have the same identity but different orientations, so they cannot interact with each other. Nucleotides A2543 and U2500 are paired (marked with dashed lines) and stacked to C2452 and U2504 in the bacterial ribosomes. In eukaryotes’ structures, nucleotides 2453 and 2500 are both uridines and no interaction between them occurs owing to their orientation. In eukaryotes, U2504 has different orientation than in bacteria which allows pi stacking to A2055 (C in bacteria, A in eukaryotes). This interaction stabilizes the “open” conformation of the PTC which is the key for the pleuromutilins’ selectivity. (B) A superposition of the rRNA H50S-tiamulin structure (light blue) (3G4S) on that of SA50S-lef (orange). In archaea, nucleotides A2543 and U2500 cannot form Watson-Crick base-pair due to the stacking between U2504 and A2055. (C) A superposition of the rRNA apo SA50S structure (blue) (4WCE) on the SA50S-lef (orange) shows that the position of these nucleotides is the same in the bound and the unbound 50S. (D) Multiple sequence alignment of part of the pleuromutilin binding site, comparing the bacterial, archaeal and eukaryotes large ribosomal subunit rRNA. Helix 89 nucleotides, which are part of the PTC, are marked in a box. Arrows are indicating on the nucleotides C2452, A/U2453, U2504 and U2500 which are 1^st and 2^nd shell from the mutilin moiety. The lines represent Watson-crick base pairs, dashed lines are base pairs only in bacteria.