A multi-stage computational pipeline and in vitro validation for the discovery of small-molecule translation inhibitors targeting the bacterial ribosome

Figures

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  Fig. 1. Cartoon representation of E. coli 70S ribosome (PDB ID 4v7v), where small (30S) and large (50S) subunits are displayed with ribosomal proteins on the left. The investigated region, including the peptidyl transferase center on 23S rRNA, is indicated on 50S on the right. The native inhibitor Clindamycin (CLY) is shown in sticks. Mg2+ ions in the crystal structure are depicted as magenta spheres.

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  Fig. 2. Computational workflow for the identification of novel PTC inhibitors, which starts with a sequential filtering of FDA-approved, experimental, and investigational drug libraries using Glide (SP and XP modes) and AutoDock Vina. The number of compounds prioritized at each stage is indicated in parentheses. Threshold scores and binding energies (kcal mol−1), established based on the benchmark inhibitor Clindamycin, are displayed alongside the corresponding filtering steps. The final selection for molecular dynamics (MD) simulations was based on consensus scoring, ligand-RNA interaction patterns common with known inhibitors, and structural similarity analysis for the compounds.

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  Fig. 3. RMSD profile of (a) the truncated ribosomal structure and (b) Clindamycin in three independent MD simulations. (c) Clindamycin interactions with 23S rRNA, which are observed in at least 10% of the trajectory in each replica, are displayed as a map. The nucleotides with known interactions with the antibiotic are highlighted in yellow at the top. The types of non-bonded interactions are color-coded as hydrogen bond (green), π-cation (orange), hydrophobic (slate), and salt-bridge (light orange). Involvement of the nucleotides in non-bonded interactions is indicated by 1, whereas their absence is indicated by 0. The critical interactions between Clindamycin and 23S rRNA are shown for (d) the crystal structure and (e) the MD simulations.

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  Fig. 4. Time-kill curve of Mitoxantrone. Error bars represent standard error of the mean. Any error bars that are not visible are smaller than the data symbols.

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  Fig. 5. In vitro inhibition of protein synthesis by Clindamycin (control) and the drug candidates in the E. coli cell-free translation system. Error bars represent standard error of the mean. Any error bars that are not visible are smaller than the data symbols.

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  Fig. 6. 23S rRNA-ligand interaction fingerprint based on Clindamycin (CLY, in yellow), Chloramphenicol (CLM, in blue), Linezolid (ZLD, in cyan), Hygromycin A (HGR, in red) and A201A (4M2, in green) binding sites is shown on the 23S rRNA secondary structure. The inhibitor Mitoxantrone (purple), the lead compounds Plerixafor (orange), Olcegepant (slate), and Ziritaxestat (pink), and their favorable interactions (hydrogen bond, π–cation, π–π, salt-bridges) are also indicated on the secondary structure (on the left panel) and the three-dimensional structures (on the right panel).