The emergence of multidrug‐resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram‐positive bacteria use a well‐conserved tRNA‐responsive transcriptional regulatory element in mRNAs, known as the T‐box, to regulate the transcription of multiple operons that control amino acid metabolism. T‐box regulatory elements are found only in the 5′‐untranslated region (UTR) of mRNAs of Gram‐positive bacteria, not Gram‐negative bacteria or the human host. Using the structure of the 5′UTR sequence of the Bacillus subtilis tyrosyl‐tRNA synthetase mRNA T‐box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram‐positive bacteria, but not Gram‐negative bacteria, including drug‐resistant clinical isolates at minimum inhibitory concentrations (MIC 16–64 μg mL−1). Resistance developed at an extremely low mutational frequency (1.21×10−10). At 4 μg mL−1, the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl‐tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl‐tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (Kd≈24 μm). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T‐box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.

The Cover Feature shows 20 of 200 compounds that were bound strongly in silico within an RNA grove. The 200 compounds were selected from 305,000. They were used to target an RNA function, the tRNA‐dependent regulatory mechanism unique to Gram‐positive pathogens, in search of a novel compound that inhibits bacterial growth and reduces emergent resistance. The green RNA helix (top and bottom) is part of Stem 1 of the 5’‐untranslated region of nascent mRNAs, the transcription of which is regulated by unacylated tRNA. The blue is a loop that consists of an adenosine‐rich stack and a red “codon” that binds the tRNA anticodon. More information can be found in the Full Paper by Paul F. Agris et al. on page 758 in Issue 7, 2019 (DOI: 10.1002/cmdc.201800744).