Ribosome Rescue Inhibitors Kill Actively Growing and Nonreplicating Persister Mycobacterium tuberculosis Cells

Abstract

The emergence of Mycobacterium tuberculosis (MTB) strains that are resistant to most or all available antibiotics has created a severe problem for treating tuberculosis and has spurred a quest for new antibiotic targets. Here, we demonstrate that trans-translation is essential for growth of MTB and is a viable target for development of antituberculosis drugs. We also show that an inhibitor of trans-translation, KKL-35, is bactericidal against MTB under both aerobic and anoxic conditions. Biochemical experiments show that this compound targets helix 89 of the 23S rRNA. In silico molecular docking predicts a binding pocket for KKL-35 adjacent to the peptidyl-transfer center in a region not targeted by conventional antibiotics. Computational solvent mapping suggests that this pocket is a druggable hot spot for small molecule binding. Collectively, our findings reveal a new target for antituberculosis drug development and provide critical insight on the mechanism of antibacterial action for KKL-35 and related 1,3,4-oxadiazole benzamides.

Keywords: 1,3,4-oxadiazoles; Mycobacterium tuberculosis; antibiotics; ribosome rescue.

Figure 1

SmpB is essential in MTB. (a) Schematic illustration for the design of the smpB depletion constructs in MTB. (b) Growth curves for the SmpB depletion and control strains. (c) Schematic diagram of the smpB locus in the parental H37Rv strain, the reported ΔsmpB::dif, and ΔsmpB::dif observed from whole genome sequencing showing that the ΔsmpB::dif strain has a copy of smpB. (d) qRT-PCR analysis showing that both ssrA and smpB are expressed in the ΔsmpB::dif strain. smpB (yellow) and ssrA (gray) mRNA levels in midexponential phase MTB cells were quantified by qRTPCR and normalized to the housekeeping gene sigA. Mean values from 3 technical replicates of one biological sample are shown with error bars indicating the standard deviation.

Figure 2

Compound structures and activity for KKL-35 against MTB. (a) Structures of the 1,3,4-oxadiazole benzamides KKL-35, KKL-40, the photolabile click probe KKL-2098, and the trifunctional fluorescent molecule, KKL-2107. (b) Growth inhibitory profiles for MTB cultures treated with KKL-35 and monitored by luminescence. (c) CFU counts for MTB liquid cultures treated with KKL-35. (d) CFUs recovered from MTB cells grown using the hypoxia model for nonreplicating persisters treated with KKL-35. The median from two replicates is shown with error bars indicating the standard deviation.

Figure 3

Target identification workflow. The photolabile probe KKL-2098 was added to a growing bacterial culture. Cells were irradiated with UV light to activate the probe and enable cross-linking. Cells were lysed, and protein was denatured and subjected to click chemistry with the fluorescent affinity compound KKL-2107 and analyzed by SDS-PAGE. Alternatively, total RNA was purified and used in click conjugation assays with KKL-2107 or primer extension assays to detect RNA modification. Agarose or polyacrylamide gel electrophoresis was used to visualize and identify the probe-linked macromolecule.

Scheme 1. Synthesis of the Dual Function Photo-Reactive Click Probe: 4-Azido-N-(5-(4-ethynylphenyl)-1,3,4-oxadiazol-2-yl)benzamide (KKL-2098)

Reagent conditions: (a) NaNO₂, HCl, H₂O, 0 °C, 1 h; (b) NaN₃, HCl, H₂O, 0 °C, 1 h; (c) SOCl₂, reflux 12 h; (d) NaOH, MeOH, rt, 4 h; (e) 1 N HCl, pH 2; (f) POCl₃, reflux, 12 h; (g) pyridine, 50 °C, 12 h.

Figure 4

KKL-2098 binds 23S rRNA. (a) Agarose gel analysis for the click conjugation and control reactions with total RNA preparations from M. smegmatis cells. (b) Autoradiogram showing primer extension results using RNA prepared from cells treated with KKL-35 or KKL-2098. The arrow indicates the extension product seen only in the cross-linked sample treated with KKL-2098 (see Figure S3 for the full gel and experiments using other primers). (c) Structure of the E. coli ribosome (PDB ID 4V69) showing the location of H89 (magenta) extending from the PTC (green) to the factor binding site (purple). (d) Cartoon and surface structures of H89 showing the location of Ψ2504 and C2452.

Figure 5

Nucleotides that cross-link to KKL-2098 are highly conserved. Comparison of the nucleotide sequence and secondary structures of H89. The KKL-2098 cross-link sites are indicated by asterisks. Mutation of the nucleotides highlighted in red is known to impair peptidyl-transferase activity, ribosome fidelity/integrity, or cell growth in bacteria.⁻

Scheme 2. Synthesis of the Tri-Functional Fluorescent Reporter N-(6-((6-Azidohexyl)(6-(5-(dimethyl amino) Naphthalene-1-sulfonamido)hexyl)amino)hexyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl) Pentanamide (KKL-2107)

Reagent conditions: (a) Et₃N, CH₂Cl₂, 0 °C, rt, 12 h; (b) DIPEA, CH₃CN, reflux 12 h; (c) DIPEA, CH₃CN, reflux 12 h; (d) TFA/CH₂Cl₂ (1:1), 0 °C, rt, 3 h; (e) Et₃N, MeOH, rt, 12 h.

Figure 6

KKL-35 docks to a predicted binding hot spot in H89. (a) Docked KKL-35 (cyan) in a pocket at the base of H89 adjacent to the PTC (PDB ID 4ABR). (b) Lateral view (from the loop region) of the docking site for KKL-35 illustrating potential polar interactions (dashed lines). (c) Reversed view (from the PTC side) of the binding site. (d) Solvent mapping results for H89 showing probe clustering. The dotted region indicates a hot spot located within the predicted binding site for KKL-35. (e) Close-up of the highlighted hot spot in “d” showing the docked KKL-35 and FTMap probe clusters. Probes are color-coded to distinguish between different consensus sites (CSs): CS1-(9), CS2-(12), CS3-(11), and CS4-(4) (number of probe clusters in each CS in parentheses).