RSC Chemical Biology最新文献

Identifying cell type-specific molecular markers on the cancer cell surface is essential for understanding cancer progression and for discovering critical neoantigens relevant to immunotherapy. Nucleic acid aptamers serve as powerful tools for probing complex and dynamic cell surface characteristics. Here, we introduce a streamlined comparative aptamer profiling methodology that enables side-by-side analysis of cell surface remodeling. Using cell-SELEX (systematic evolution of ligands by exponential enrichment), we generated a modified base-incorporated aptamer library directly from cells, which was then employed to explore the surface states of normal and mutant protein-expressing cells. Differential analysis of aptamer enrichment using next-generation sequencing revealed distinct aptamer signatures that correlated with cell types. Our analysis demonstrated that mutant K-Ras expression dynamically altered cell surface composition. Individual aptamers showed specific binding to mutant K-Ras-expressing cells without requiring sequence optimization. Moreover, target identification of one aptamer revealed abnormal translocation of a mitochondrial matrix protein to the cell surface without detectable changes in mRNA or protein levels upon altered cellular signaling. These findings highlight the dynamic modulation of cell surface states by aberrant cellular signaling. Overall, we present a useful comparative strategy to investigate cell surface alterations. This approach may help uncover previously unrecognized cell surface markers associated with oncogenic signaling.

Heterobifunctional molecules that induce targeted degradation have emerged as powerful tools in chemical biology, target validation, and drug discovery. Despite their promise, the field is constrained by the relative paucity of ligands available for E3 ligases. Expanding the ligand repertoire for E3 ligases and other components of ubiquitin-proteasome system could significantly broaden the scope of the targeted degradation field. In this study, we report the identification of ligands for non-essential E3 ligases that are preferentially expressed in cancer tissues relative to normal tissues. Using a protein-observed NMR-based fragment screen, an ideal technique for this purpose, we identified fragment ligands and characterized their binding modes by X-ray crystallography. These ligands represent promising starting points for further optimization toward the discovery of tumor-selective degraders that may enhance the therapeutic window targeting proteins for which inhibition or degradation is associated with systemic toxicity.

KRAS is one of the most frequently mutated oncogenes in human cancers and remains a challenging target for therapeutic intervention, often labeled “undruggable.” We herein synthesized triazole-containing peptidomimetics TTh1 and TTh2, to explore their selective interactions with DNA quadruplexes. Biophysical studies reveal that TTh2 with a prolinamide motif selectively binds to and stabilizes the KRAS G-quadruplex structure, resulting in marked suppression of the KRAS mRNA and protein levels in HeLa cells. This downregulation correlates with the inhibition of key downstream signaling pathways, including MAPK and Akt/mTOR, which are critical for cancer cell proliferation and survival. These results highlight the potential of G4-binding peptidomimetics as chemical tools for modulating oncogene expression through selective stabilization of promoter G-quadruplex structures.

Neuraminidases (NAs) are critical virulence factors in pathogens. In viruses such as influenza A, neuraminidase facilitates the release of virions, thereby enabling infection propagation. In pathogenic bacteria, NA activity has been linked to the pathogenicity of species such as S. pneumoniae, P. aeruginosa, and V. cholerae. Studies suggest that bacterial NAs play roles in mucus degradation, exposing host epitopes to enhance bacterial adhesion, biofilm formation, and bacterial survival. However, the specific mechanisms by which bacterial NAs contribute to pathogenesis remain poorly understood and largely unknown. To gain a deeper understanding of the molecular mechanisms underlying this class of enzymes, highly selective and sensitive strategies are needed for screening, detecting, and studying active NAs in complex biological samples. Specifically, chemical tools that can covalently label NAs without interfering with their enzymatic activity offer a powerful approach to precisely label and visualize these enzymes in their native environments. In this work, we present the development of novel ortho-quinone methide-based probes featuring an azide and biotin tags for the labeling and detection of NAs. These probes exhibit high selectivity in labeling recombinantly expressed NAs from influenza A virus and pathogenic Gram-negative Prevotella strains at nanomolar concentrations. Moreover, we developed a strategy that significantly improves labeling specificity of NAs when using our probes in complex samples, addressing the common issue of nonspecific labeling associated with quinone methide-based probes. Additionally, we demonstrate the potential of these probes for imaging extracellular NAs on bacterial surfaces, highlighting their utility for studying NAs in their native environments.

Small molecule- and antibody-based approaches have shown tremendous success in both therapeutic and diagnostic applications. Aptamers, which are engineered nucleic acid ligands for proteins, have not found similar broad applicability, potentially due to their susceptibility to nuclease-mediated degradation and short engagement times of their targets. One approach to mitigate these issues is the use of covalent aptamers. Here, the aptamer sequence is functionalized with an electrophilic motif, combining the high specificity of aptamer–protein binding with the ability to form a permanent covalent bond at nucleophilic residues on the target protein. These electrophilic motifs can be either non-cleavable, allowing for the formation of aptamer–protein conjugates, or cleavable, allowing for transfer of a payload onto the target protein. The chemical structures of these motifs define their functions which range from protein detection to targeted protein degradation. The covalent bond formed between the electrophile and a nucleophilic amino acid sidechain at the protein surface dramatically increases the engagement time and duration of action of the functional moiety. In this review, we summarize efforts in establishing, understanding, and applying the chemistry of covalent aptamers.

The growth and division of the Gram-positive cell requires the coordinated action of enzymes involved in the synthesis and degradation of the heteropolymer peptidoglycan. Herein, we present the use of the diamide masarimycin, an inhibitor of the exo-N-acetylglucosaminidase (GlcNAcase) LytG from Bacillus subtilis, as a chemical biology probe to elucidate the biological role of this cell wall degrading enzyme. Using a combination of chemical biology and genetic approaches we provide the first evidence that LytG activity influences the elongation and division complexes in B. subtilis. Chemical inhibition of LytG resulted in dysregulated cell elongation and localization of the division plane and the induction of the cell wall stress response. In the presence of masarimycin, cells show asymmetrical thickening of the cell wall and dysregulation of division plane localization. The use of genetic and synergy/antagonism screens established connections to late-stage peptidoglycan synthesis, particularly related to cross-linking function. These results stand in stark contrast to those observed for the ΔlytG knockout, which does not exhibit these phenotypes. Cell-wall labelling with a fluorescent D-amino acid and muropeptide analysis has highlighted a functional connection between LytG, the carboxypeptidase DacA, and D,D-endopeptidases. These results highlight the use of chemical probes such as masarimycin to inform on the biological function of autolysins by providing insight into the role LytG plays in cell growth and division.

The immense microbial diversity on Earth represents a vast genomic resource, yet discovering novel enzymes from complex environments remains challenging. Here, we combine a microbial enrichment with metagenomics and metaproteomics to facilitate the identification of microbial glycoside hydrolases that operate under defined conditions. We enriched microbial communities on the carbohydrate polymer pullulan at elevated temperatures under acidic conditions. Pullulan is a natural polysaccharide composed of maltotriose units linked by α-1,6-glycosidic bonds. Pullulan, along with its hydrolyzing enzymes, has broad applications across various industries. The enrichment inocula were sampled from thermophilic compost and from soil from the bank of a pond. In both cases, Alicyclobacillus was identified as the dominant microorganism. Metaproteomic analysis of the enriched biomass and secretome enabled the identification of several pullulan-degrading enzyme candidates from this organism. These enzymes were absent in the metagenomic analysis of the initial inoculum, which is highly complex with a wide diversity of species. This underscores the effectiveness of combining microbial enrichment with multi-omics for uncovering novel enzymes and sequence variants that operate under defined conditions from complex microbial environments.

Dicamba, although a potent and useful herbicide in weed management, is notorious for its off-target movement due to volatility. Here, we describe carbohydrate esters of dicamba as an unexplored pro-herbicide approach that addresses the volatility of dicamba, while maintaining its herbicidal qualities. Varying the carbohydrate and dicamba attachment point led to changes in potency and hydrolysis, potentially allowing for reactivity tuning of molecules for future weed management practices.

Polyamines are essential metabolites that play a crucial role in regulating key cellular processes. While previous studies have shown that polyamines modulate protein function through non-covalent interactions, the lack of robust analytical methods has limited the systematic identification of these interactions in living cells. To address this challenge, we synthesized a series of novel photoaffinity probes and applied them to a model cell line, identifying over 400 putative protein interactors with remarkable polyamine analog structure-dependent specificity. Analysis of probe-modified peptides revealed photocrosslinking sites for dozens of protein binders and demonstrated that all but one of the probes, the spermine analog, were intracellularly stable. The interaction profiles of these probes were visualized through in-gel fluorescence scanning, and their subcellular localization was examined using fluorescence microscopy. Spermidine analogs interacted with proteins in the nucleoplasm, colocalizing with nucleolar and nuclear-speckle proteins, as well as in the cytoplasm. By contrast, diamine analogs localized to vesicle-like structures near the Golgi apparatus, implying that different polyamine types exhibit a proclivity for specific cellular compartments. Notably, spermidine analogs bound preferentially to proteins containing acidic stretches, often located within intrinsically disordered regions. Focusing on one such case, we provide in-cellulo evidence of direct interactions between G3BP1/2 and spermidine analogs and advance the hypothesis that such interactions influence stress-granule dynamics. Overall, this study provides a comprehensive profile of polyamine analogs–protein interactions in live cells, offering valuable insights into their roles in cellular physiology.

Enzymes are powerful catalysts that perform chemical reactions with remarkable speed and specificity. Their intrinsic dynamics often play a crucial role in determining their catalytic properties. To achieve a comprehensive understanding of enzymes, a diverse and sophisticated experimental toolbox capable of studying enzyme dynamics at the single-molecule level is necessary. In this review, we discuss nanopore technology as an emerging and powerful platform in single-molecule enzymology. We demonstrate how nanopores can be employed to probe enzyme dynamics in real-time, and we highlight how these studies have contributed to fundamentally and quantitatively elucidating enzymological concepts, such as allostery and hysteresis. Finally, we explore the potentials and limitations of nanopores in advancing single-molecule enzymology. By presenting the unique possibilities offered by nanopores, we aim to inspire the integration of this technology into future enzymology research.