Discovery of Small-Molecule Antibiotics against a Unique tRNA-Mediated Regulation of Transcription in Gram-Positive Bacteria

Graphical Abstract

The Achilles′ heel of bacteria: An RNA function has been explored as a target for small-molecule intervention in Gram-positive bacterial infections, that foils the emergence of drug resistance. A small molecule (light blue) selected in silico disrupts tRNA-dependent control of transcription by binding to the nascent mRNA 5′-untranslated region (olive, RNA stem; purple, RNA loop) at a “codon” (red) complementary to the tRNA′s anticodon binding site. The compound thwarts the emergence of drug resistance.

Abstract

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 (K_d≈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.