Abstract Sactipeptides are a class of ribosomally synthesised and post-translationally modified peptides (RiPPs), which have a cyclic topology formed by unique thioether crosslinks called sactionines. Sactionines are introduced by radical S-adenosylmethionine (rSAM) enzymes between cysteine residues and the α-carbon of other amino acids. Genome sequencing has revealed many putative sactipeptide biosynthetic gene clusters; however, few sactipeptides have been isolated and characterised. Here, we use genome mining to identify a novel sactipeptide from Bacillus smithii. The novel sactipeptide, named smithiosin, was extracted from the industrially important thermophile B. smithii and characterised as a novel homologue of the previously characterised sactipeptide subtilosin A from B. subtilis. Furthermore, the smithiosin precursor peptide gene was heterologously expressed in the subtilosin A producer Bacillus subtilis 168 without knockout of the native precursor peptide, successfully leading to the production of both subtilosin A and smithiosin. This demonstrates for the first time that a non-native precursor peptide is able to outcompete the subtilosin A precursor for all of the native biosynthetic enzymes in B. subtilis, including the radical SAM enzyme AlbA. It also opens a potential new opportunity for screening non-native precursor peptides with the subtilosin A biosynthetic enzymes via heterologous expression in B. subtilis.
Covalent strategies offer a powerful route to expand the druggable proteome, yet selective lysine targeting remains difficult due to the high pKa and protonation of the amine side chain. The translation initiation factor eIF4E, an mRNA cap-binding protein that controls nuclear export and translation of oncogenic transcripts, has long been considered an undruggable RNA-binding protein. We apply an anionic electrophile strategy that converts a carboxylic acid within a reversible eIF4E ligand into a vinyl acylsulfonamide warhead, preserving salt-bridge and hydrogen bonding interactions in the cap-binding pocket and facilitating Lys162 deprotonation to enable site-specific engagement. This single-step transformation yields JL-03, a potent covalent eIF4E inhibitor with excellent stability toward biological nucleophiles, consistent with context-dependent activation of the warhead in the binding site. In cells, JL-03 achieves robust target engagement and suppresses translation of the oncogenic driver cyclin D. JL-03 reduces the viability of HOXA9-high acute myeloid leukemia cells with greater potency than reversible eIF4E inhibitors and synergizes with the menin inhibitor revumenib, demonstrating that coordinated transcriptional and translational blockade exhausts leukemic dependencies. These findings establish eIF4E as a tractable covalent target and highlight anionic electrophile installation as a generalizable strategy for lysine-directed covalent inhibitor design.
Abstract The glycocalyx – a complex meshwork of sugars, proteins, and lipids – coats all human cells and plays a crucial role in cell signaling and immune system regulation. While an understanding of its biochemical functions is emerging, its influence on cell mechanics remains comparably underexplored. Recent evidence suggests that the glycocalyx regulates membrane morphology and cell adhesion, yet its mechanical role has been difficult to study, primarily due to the absence of high-throughput analysis strategies that account for cell-to-cell variability. Here, we overcome this challenge by integrating real-time deformability cytometry (RT-DC) with genetic and chemical glycocalyx engineering to tune the glycocalyx state and assess how cell mechanics are affected. Using this approach, we uncovered mechanical adaptations that contribute to promoting cell migration and metastasis. These findings establish glycocalyx remodeling as a regulator of cancer-cell mechanics and a potential target for therapeutic intervention.
