trans-Translation inhibitors bind to a novel site on the ribosome and clear Neisseria gonorrhoeae in vivo

Abstract

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.

Fig. 1. Optimized acylaminooxadiazoles inhibit trans-translation to kill N. gonorrhoeae.

A Zones used to guide synthetic strategy with characteristics that govern activity are indicated, and the structures of KKL-35 and MBX-4132 are shown. B Properties of the initial hit, KKL-35, and optimized inhibitor MBX-4132 (CC₅₀ – half-maximal cytotoxic concentration against HeLa cells; MMS – murine liver microsome stability). C Inhibition of trans-translation in E. coli cells was monitored using a non-stop luciferase reporter. The average of two biological repeats is shown. D Inhibition of trans-translation in vitro was assayed using an E. coli S12 extract to express a truncated, non-stop nano-luciferase gene in the presence of a mutant tmRNA that added the remainder of the nano-luciferase protein. Trans-translation activity resulted in luminescence, and addition of MBX-4132 inhibited the reaction (black). As a control, a full-length nano-luciferase gene was used to demonstrate that MBX-4132 does not inhibit translation (blue). The percentage of activity compared to activity in absence of MBX-4132 is shown from the average of at least two repeats. E Time-kill assays using N. gonorrhoeae show that MBX-4132 is bactericidal at ≥4X MIC. Ceftriaxone (CRO) was used as a control. Counts below the detection limit (100 cfu/ml) were plotted at 100 cfu/ml. Source data are provided as a Source Data file.

Fig. 2. MBX-4132 clears infection by a multiple-antibiotic resistant N. gonorrhoeae strain in a murine infection model.

Mice were infected with N. gonorrhoeae H041 for two days and treated with a single oral dose of 10 mg/kg MBX-4132 or vehicle on day 0 (green arrow), (n = 20 mice for MBX-4132 and n = 21 mice for vehicle). As a positive control, 48 mg/kg gentamicin (GEN) was administered by intraperitoneal injection beginning on day 0 (5QD, black arrows). A The percentage of infected mice over 8 days post-treatment. Mice that were culture-negative for at least 3 consecutive days were considered to have cleared infection. MBX-4132 and GEN significantly reduced the percent of infected mice compared to vehicle (Mantel-Cox, p < 0.0001). B Mean bacterial burden (cfu/ml) recovered daily following treatment on day 0. MBX-4132 and GEN significantly reduced the bacterial burden compared to vehicle (2-way ANOVA with Bonferroni for multiple comparisons, p < 0.0001). Limit of detection (20 cfu/ml) is denoted by the horizontal dashed line. Error bars indicate standard error of the mean. Source data are provided as a Source Data file.

Fig. 3. KKL-2098 binds near the peptidyl transferase center.

A The chemical structure of KKL-2098. B The cryo-EM structure of the E. coli 70S non-stop ribosome with P-site tRNA, mRNA, ribosomal protein bL27 and KKL-2098 indicated. C KKL-2098 is positioned close to the peptidyl transferase center adjacent to 23S rRNA A2602, C2452, U2506, and U2585 and the CCA end of the P-site tRNA. D The N terminus of bL27 (purple) moves 180° to pack against ribosomal protein uL16 and the acceptor arm of the P-site tRNA. The normal position of bL27 in translating ribosomes is shown in green (PDB 6ENU).

Fig. 4. Truncation of bL27 causes hypersensitivity to acylaminooxadiazoles.

A Growth of E. coli ∆tolC expressing full-length bL27 (black) or variants missing residues 2–4 (-3, blue) or 2–7 (-6, red) was monitored in broth microdilution experiments and the IC₅₀ for MBX-4132 was determined. At least two technical replicates were performed for each biological replicate and the average of at least three biological replicates is shown with error bars indicating standard deviations. B Violin plot showing normalized mean IC₅₀ values from at least 3 biological replicates of experiments as in (A) show that truncation of bL27 increases sensitivity to KKL-35 and MBX-4132 but has no effect on erythromycin (ERM), linezolid (LZ), spectinomycin (SPEC) or tetracycline (TET). Source data are provided as a Source Data file.

Fig. 5. Comparison of the position of KKL-2098 to other PTC inhibitors.

A Overview of the bacterial ribosome showing the position of sparsomycin (PDB 1NJM), chloramphenicol (PDB code 6ND5) and KKL-2098. B Inset view of the interactions of PTC nucleotides with sparsomycin. The nucleobase of 23S rRNA A2602 moves ~180° upon sparsomycin binding. KKL-2098 and A2602 in the absence of sparsomycin binding are shown in red. C Inset view of the interactions of PTC nucleotides with chloramphenicol. The nucleobase of 23S rRNA A2062 also moves ~180° upon chloramphenicol binding. KKL-2098 and A2062 in the absence of chloramphenicol binding are shown in red.