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 highthroughput 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 IC 50 = 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.antibiotic target | tmRNA | non-stop translation T he increasing prevalence of antibiotic-resistant bacterial pathogens has spurred a search for new pathways that can be targeted for antibiotic development (1, 2). One pathway that has not been exploited is the trans-translation pathway, which resolves nonstop translation complexes. The components of trans-translation have been identified in every sequenced bacterial genome, and mutations in these components affect viability or virulence in a wide range of bacteria (3, 4), suggesting that inhibitors of trans-translation might be effective broad-spectrum antibiotics. In addition, the trans-translation pathway is not found in animals, so specific inhibitors are expected to have few side effects on the host.The purpose of trans-translation is to remove nonstop translation complexes, i.e., translation reactions in which the ribosome has reached the 3′ end of the mRNA without terminating at a stop codon (4-6). These complexes are prevalent in bacteria because bacterial ribosomes do not require any information from the 3′ end of the mRNA to initiate translation, and bacteria lack most of the mechanisms for mRNA proofreading found in eukaryotes (7). Because hydrolysis of peptidyl-tRNA by release factors requires a stop codon in the A site, normal translation termination cannot occur when the ribosome reads to the 3′ end of the mRNA and there is no in-frame stop codon. Transtranslation resolves nonstop translation complexes using a ribonucleoprotein complex containing transfer-messenger RNA (tmRNA) and the small protein SmpB (4-6). tmRNA-SmpB recognizes nonstop translation complexes and enters the ribosomal A site mimicking a tRNA (8). The nascent polypeptide is transferred to tmRNA, and a specialized reading frame within tmRNA is inserted into the mRNA channel (4-6). Translation resumes using this sequence as a message and terminates at a stop codon at the end of the reading frame, releasing the ribosome and a tagged protein (4-6). The tag sequence is recognized by proteases, and the tagged protein is rapidly degraded (9-12). The net reaction of trans-translation is the removal of all components o...

Resolving Nonstop Translation Complexes Is a Matter of Life or Death

“…The results of one high-throughput screening (HTS) investigation of inhibitors of trans-translation have been reported (36,91). Several small molecules identified by HTS inhibit trans-translation but not translation in vitro.…”

“…The trans-translation pathway is an attractive target for development of new antibiotics because it is required for viability or vir- Several cell-based assays for trans-translation activity have been described, and they all have the same basic construction (91). A strong transcriptional terminator is inserted before the stop codon of a reporter gene, such as the luc gene encoding luciferase (Fig.…”

La synthèse des protéines par le ribosome

“…Cet antibiotique cible uniquement la trans-traduction et non la traduction canonique de M. tuberculosis [38]. Enfin, un criblage réalisé à partir de 663,000 molécules a récemment permis de révéler de nouvelles molécules capables d'inhiber cette voie, ayant un effet bactéricide et à spectre large [39]. Ces travaux valident l'inhibition de cette voie comme une piste majeure de recherche de futurs antibiotiques.…”

Novel Methods to Address Antimicrobial Resistance

“…This results in the bacterium being unable to replicate. By halting bacterial growth, infection is unable to spread [5].…”