Integrated omics reveal a unique antibacterial mechanism of action for the small molecule HSI#6

The continuous risk of antibiotic resistance development underscores the demand for new agents with mechanisms distinct from existing antibacterial drugs. Here, we investigated HSI#6, a small-molecule antibacterial previously identified as a SecA activator, using integrated omics and functional assays. HSI#6 exhibits a rapid, broad-spectrum bacteriostatic activity, and induces a distinct cell envelope-homeostasis stress signature accompanied by global stress reprogramming. Time-resolved transcriptomics and proteomics revealed early activation of envelope stress regulons and oxidative stress pathways, followed by suppression of ribosome biogenesis and central metabolism. Comparative analysis and biomarker-based principal component analysis (PCA) positioned HSI#6 within the envelope stress mechanistic space, closely aligned with membrane-active antibiotics yet displaying a distinct signature. Adaptive laboratory evolution (ALE) combined with whole-genome sequencing (WGS) revealed compensatory mutations in topoisomerase 1A gene (topA) and transcriptional regulators, without adaptive resistance emerged even under prolonged selection pressure. These findings establish HSI#6 as a mechanistically unique antibacterial agent with low resistance potential.