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

Highlights

• HSI#6 is a rapid, micromolar, broad-spectrum bacteriostatic agent.

• Triggers immediate, multi-pathway cell envelope stress responses.

• Drives temporal omics reprogramming toward metabolic shutdown.

• Shows transcriptomic similarity to ionophores yet a distinct MOA.

• Exhibits low resistance evolution with compensatory topA mutations.

Abstract

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.

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https://www.sciencedirect.com/science/article/pii/S2666517426000684