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.
Highlights
Article pyrazinamide or its active metabolite, pyrazinoic acid, in vitro or in vivo.[14]
To assess the importance of trans-translation in Mycobacterium tuberculosis (MTB), we first attempted to delete the genes encoding tmRNA and SmpB from the MTB chromosome using allelic exchange, but we could not obtain a deletion of either gene
To rigorously determine if trans-translation is essential in MTB, we engineered a strain (TetpsmpB:rTetR) in which the expression of smpB at its chromosomal locus is controlled by the tet repressor (TetR), such that addition of anhydrotetracycline (ATc) shuts off SmpB production (Figure 1a)
Summary
Pyrazinamide or its active metabolite, pyrazinoic acid, in vitro or in vivo.[14]. there are currently no antibiotics that target this pathway. KKL-35 (Figure 2a) and related 1,3,4-oxadiazole benzamides were identified by cell-based screening for inhibitors of trans-translation and were found to have broad-spectrum antibacterial activity.[17,18] To assess the ability of KKL-35 to inhibit growth of MTB, MIC and plating assays were performed. The data presented here indicate that KKL-35 bind to a highly conserved region of the ribosome, and previous results showed that KKL-35 inhibits transtranslation but not translation initiation, elongation, or termination.[17] Binding of a drug near the PTC might be expected to interfere with translation and mutation of nucleotides that form the KKL-35 binding site (for example, U2460, U2492, and U2493) (Figure 5b), leading to significantly decreased PTC activity and impaired cell growth.[24−27] KKL-35 did not inhibit translation of mRNAs containing an in-frame stop codon when tested at concentrations >100-fold above the IC50 for inhibiting transtranslation in vitro.[17] KKL-2098 cross-linked to 23S rRNA during in vitro translation of mRNAs containing an in-frame stop codon as well as translation of nonstop mRNAs (Figure S2), indicating that the binding site may be accessible during normal translation.
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