Biology subjects

Synowsky, S.

Publications and source records attributed to Synowsky, S..

3 recordsLinked to original sources

bioRxiv2026

Bacterial Aminopeptidase-Activated Peptide Prodrug Enables Species-Selective Targeting of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an adaptable organism, frequently found in chronic infections, and for which antimicrobial resistance is a growing concern. Therefore, there is an urgent need for alternative therapeutic strategies. Cationic antimicrobial peptides (AMPs) offer potent bactericidal activity but suffer from limited selectivity and potential host toxicity. To enhance species-specific targeting, we designed two prodrug variants of the AMP D-Bac8CLeu2,5 - EEEE{square}-Bac8CLeu2,5 and ELE{square} D-Bac8CLeu2,5 --engineered for activation by the P. aeruginosa extracellular aminopeptidase PaAP. While both prodrug motifs effectively neutralized the positive charge of D-Bac8CLeu2,5 and prevented DNA-peptide complex formation, EEEE{square}-Bac8CLeu2,5 showed negligible antimicrobial activity due to slow and incomplete activation. In contrast, ELEG{square}-Bac8CLeu2,5 underwent rapid PaAP{square}mediated activation, restoring bactericidal activity in planktonic cultures and biofilms. PaAP contributed significantly to complete prodrug activation, particularly within biofilms, where the accumulation of partially activated intermediates correlated with biphasic killing kinetics. The prodrug showed reduced activity against other ESKAPEE pathogens, demonstrating selective activation by P. aeruginosa. Experiments selecting resistant bacteria revealed distinct mutations in lipopolysaccharide biosynthesis pathways for D-Bac8CLeu2,5 and the prodrug, with limited cross{square}resistance. These findings establish aminopeptidase{square}activated AMP prodrugs as a promising approach for species{square}selective antimicrobial therapy and highlight the feasibility of exploiting bacterial enzymes for controlled antimicrobial peptide activation.

microbiology↗

bioRxiv2025

Investigating the Interactomic Landscape of Survival Motor Neurons (SMN) and the SMN Δ7 truncated protein

This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyse protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows for the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilised to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimised variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes.

cell biology↗

bioRxiv2021

Structure, dynamics, and inhibition of Staphylococcus aureus m1A22-tRNA methyltransferase

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

biochemistry↗