Small molecule inhibitors of trans-translation have broad-spectrum antibiotic activity

Abstract

The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a high-throughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC50 = 0.9 µM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.

Keywords: antibiotic target; non-stop translation; tmRNA.

Fig. 1.

Identification of trans-translation inhibitors by HTS. A cell-based assay with positive readout for trans-translation activity was used for HTS to identify inhibitors. (A) A schematic diagram of the HTS-compatible assay. Luciferase was made from a nonstop mRNA that has a strong transcriptional terminator (stem-loop sequence) before the stop codon. Translation of this mRNA when trans-translation is inhibited results in active luciferase and luminescence in the HTS assay. Conversely, when trans-translation is active, the luciferase is tagged and degraded, and there is no luminescence in the HTS assay. (B) Chemical structures of five compounds identified by HTS and characterized using secondary assays.

Fig. 2.

Characterization of inhibitors of tagging and proteolysis. Two mCherry-based reporters were used to determine which compounds inhibited tagging of nascent polypeptides and which inhibited proteolysis of tagged proteins. Schematic diagrams of the mCherry-trpAt reporter (A), and mCherry-tag reporter (D) indicate the conditions that produce fluorescent cells. Epifluorescence (mCherry panels) and DIC micrographs were used to measure the fluorescence in ΔssrA cells, ΔclpX cells, or in WT cells treated with DMSO only or with one of the compounds. Representative micrographs for mCherry-trpAt (B) and mCherry-tag (E) are shown. The fluorescence intensity in >330 individual cells was measured, and the cells were scored as positive if the intensity was >2 SDs higher than the average intensity for the DMSO-treated control. The percentage of positive cells for mCherry-trpAt (C) and mCherry-tag experiments (F) is shown. Compounds were added at 100 μM, with the exception of KKL-52 in C, which was added at 10 μM. Error bars indicate SDs.

Fig. 3.

In vitro assays for inhibition of trans-translation and translation. (A) DHFR genes with or without a stop codon were expressed in vitro in the absence or presence of additional tmRNA and SmpB. The locations of DHFR, as determined in control reactions, and of the lower-mobility tagged DHFR protein are indicated. (B) In vitro trans-translation reactions were performed after addition of 1.67% DMSO or 10 μM compound. Representative reactions are shown. The intensity of the DHFR and tagged DHFR bands were measured, and the tagging efficiency was calculated as the percentage of total DHFR protein in the tagged DHFR band. The average tagging efficiency with SD for at least three repeats is shown. To determine the dose–response behavior, in vitro trans-translation reactions performed after addition of KKL-35 at different concentrations, and the tagging efficiencies were calculated. (C) A representative experiment is shown. (D) Data from three repeats were averaged, graphed, and fit with a sigmoidal function to determine the IC₅₀. Whiskers indicate SD for each point. (E) In vitro translation reactions were performed after addition of DMSO, 100 μM chloramphenicol (chlor), or 100 μM compound, and a representative experiment is shown. The amount of DHFR in each lane was measured as a percentage of the amount in the DMSO-treated control, and the average value for three experiments was graphed with whiskers indicating the SD. (F) Inhibitor-treated reactions did not result in significantly different translation activity relative to the DMSO-treated control; P values from one-way ANOVA tests are indicated.

Fig. 4.

KKL-35 inhibits cell growth by preventing resolution of nonstop translation complexes. (A) Growth curves of S. flexneri with (●) and without (○) addition of KKL-35. The arrow indicates when KKL-35 was added. (B) ArfA suppresses the effect of KKL-35 on cell growth. Growth curves of S. flexneri ssrA::kan pCA24N-His₆-ArfA cells were similar when ArfA expression was induced before addition of KKL-35 (□), when ArfA expression was induced and no KKL-35 was added (+), and when ArfA expression was not induced and no KKL-35 was added (△). Growth was slower when ArfA expression was not induced before addition of KKL-35 (▲). The arrow indicates when KKL-35 was added. (C) Puromycin antagonizes the effect of KKL-35 on culture growth. Growth curves of untreated E. coli ΔtolC cells (no drug), cells treated with 3.3 µM KKL-35 (10× MIC), and cells treated with 3.3 µM KKL-35 and 0.1 µg/mL puromycin. Error bars indicate SDs.