ChemBioChem最新文献

Protein ubiquitination is a pivotal posttranslational modification that regulates diverse biological processes depending on the type of ubiquitin chain linkage. Recently, ester-linked ubiquitin chains have been identified, yet their inherent hydrolytic instability has posed a significant challenge for biochemical investigations. In this study, a stable and isosteric amide analog of an ester-linked ubiquitin dimer, is chemically synthesized in which serine (Ser) at position 20 of the proximal ubiquitin is replaced with 2,3-diaminopropionic acid (Dap). The desired amide analog is synthesized using a convergent approach involving the sequential chemoselective ligation of three peptide fragments generated through Fmoc-based solid-phase peptide synthesis. Employing this chemically robust ubiquitin probe, a previously unrecognized interaction is uncovered between Ser20-linked ubiquitin chains and spliceosome-associated factors, notably ubiquitin-specific protease 39. These findings highlight the potential of the ester-to-amide bioisosteric strategy to unlock mechanistic insights into atypical ubiquitin modifications. The approach not only circumvents the intrinsic instability of ester-linked ubiquitin chains but also provides a broadly applicable framework for dissecting their biological roles, paving the way for future discoveries in ubiquitin signaling.

Heavily sulfated clusters of maltose are designed as mimetics of heparan sulfate (HS), exhibiting diverse biological activity properties of HS. Herein, the synthesis of three function-spacer-lipid (FSL) constructs containing mono, di, and tri sulfated maltoses is reported. FSLs are a class of synthetic glycolipids, the key features of which are simplicity of synthesis and capacity to integrate into cell membranes with sustained retention. A copper-catalyzed azide–alkyne cycloaddition click reaction is employed for the conjugation of the sulfated maltose entities with the respective spacer-lipid block because acylation with activated esters failed to provide the desired products. One FSL construct, featuring a single sulfated maltose unit conjugated to the lipid, demonstrated dose-dependent inhibition of complement activation in a cellular system, with maximal efficacy observed at 15 µg mL⁻¹. These findings highlight the potential of FSL-based HS mimetics for modulating immune responses.

The cover illustrates the first isolable cysteine sulfenyl iodide (Cys-SI), stabilized by a nanosized molecular cradle installed at the N-terminus of cysteine. This stabilization enabled direct structural and reactivity studies of the long-hypothesized intermediate in cysteine thiol oxidation. The Cys-SI exhibits high electrophilicity toward nucleophiles, including an indole ring, providing chemical evidence supporting mechanistic proposals for iodine mediated protein modification. More details can be found in the Research Article by Kei Goto and co-workers (DOI: 10.1002/cbic.202500619).

G-quadruplexes (G4s) are noncanonical nucleic acid structures with biological and therapeutic significance, and they are found in many aptamer sequences. Using c-kit 1 G4 DNA as a target, systematic evolution of ligands by exponential enrichment (SELEX) has been carried out using an RNA library, resulting in G4-rich aptamers. Herein, this article investigates the relationship between predicted G4-forming potential and aptamer enrichment by analyzing high-throughput SELEX libraries using three G4 prediction tools: G4NN, G4Hunter, and QGRS Mapper. While the tools demonstrate strong internal consistency and overlap in identifying G4-prone sequences, their predictions show limited concordance with experimental abundance and enrichment trends across SELEX rounds. Only a small fraction of sequences display both high G4 scores and consistent enrichment. Experimental validation using electrophoretic mobility shift assays confirmed that strong predicted G4-forming sequences often lack strong binding activity, whereas highly enriched aptamers with strong binding activities may show weaker G4 signatures computationally. These findings suggest that current G4-prediction tools alone are insufficient for aptamer candidate selection and highlight the need for integrative evaluation strategies that combine structural prediction with empirical performance data.

Dual cyclooxygenase-2/5-lipoxygenase (COX-2/5-LOX) inhibitors constitute safer alternatives to classical nonsteroidal anti-inflammatory drugs, widely used to effectively manage inflammation. In this article, molecular docking and molecular dynamics simulations guide the synthesis of novel thymol derivatives that interact with both COX-2 and 5-LOX active sites. Ligands are designed with the aim of improving thymol bioactivity, selectivity, stability, as well as pharmacokinetic properties. Therefore, –Br, –F, and –CF₃ inclusion on thymol is here evaluated, screening COX-2 and 5-LOX interactions with thymol (T), 4-fluorothymol (FT), 4-bromothymol (BT), isopropyl thymyl succinate (T1), 1,1,1,3,3,3-hexafluoroisopropyl thymyl succinate (T2), and 1′,1′,1′,3′,3′,3′-hexafluoroisopropyl 4-(4′’-thymyl)-4-oxobutanoate (T3). Molecular modeling reveals that the estimated ligands can establish favorable interactions with both COX-2 and 5-LOX active pockets, highlighting T1–T3 as the most promising compounds. In vitro assays identify T3 as the most active COX-2 inhibitor, while T2 results as the most effective ligand for 5-LOX. Interestingly, cavity analysis of the COX-2 entry site reveals that T3 insertion is favored over T1 and T2 due to its greater polarity, conferred by the presence of a free phenolic group (OH) able to establish H-bonds with surrounding residues.

A novel strategy based on genetic code expansion combined with click chemistry for the simultaneous visualization of distinct posttranslational modification (PTM) states of a single protein within living cells. As a model, it is focused on threonine 41 (T41) of β-catenin, a regulatory hotspot implicated in epithelial cancers and known to be phosphorylated, O-GlcNAcylated, or left unmodified. Using site-specific incorporation of the unnatural phenylselenocysteine, a β-catenin-EGFP fusion protein is engineered allowing selective installation of a S-GlcNAc moiety via oxidative elimination and thiol-Michael ligation. Additional β-catenin variants, phosphomimetic T41E-mCherry and wild-type-blue fluorescent protein fusions, are produced to represent other PTM states. All constructs are successfully introduced into Hep3B and HeLa cells by lipofection or TAT-mediated transduction. Fluorescence microscopy revealed distinct subcellular localization profiles for each PTM form. Notably, the S-GlcNAcylated β-catenin exhibited enhanced resistance to proteasomal degradation, consistent with known roles of O-GlcNAcylation in protein stability. This approach provides a versatile platform to functionally probe PTMs in a comparative, cell-based context.

Phosphoribosyl pyrophosphate (PRPP) functions as a central metabolic intermediate, supplying ribose-5-phosphate moieties for the biosynthesis of nucleotides, certain amino acids, and a range of essential cofactors. In this study, a thermostable phosphoribosyl pyrophosphate synthetase (PfPRS) was identified from the hyper thermophilic archaeon Pyrolobus fumarii 1A, a hyper thermophilic archaeon that grows optimally at 90–113 °C. The prs gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Peak catalytic activity of PfPRS was observed at approximately pH 7.5 and 55 °C and retained over 85% of its activity after 2 h of incubation across pH 4.0–10.5. PfPRS exhibited high thermal stability. The enzyme exhibited half-lives of 12 h at 90 °C, 5 h at 95 °C, and 3 h at 100 °C. Among the nucleotides tested as diphosphate donors, PfPRS showed a strong preference for ATP, whereas ADP served as an effective inhibitor. Kinetic analysis revealed K_m values of 35 µM for R5P and 46 µM for ATP, with turnover rates (k_cat) of 71 s⁻¹ and 56 s⁻¹. PfPRS was co-immobilized with polyphosphate kinase 2 (DrPPK2) from Deinococcus radiodurans using a cross-linked enzyme aggregate (CLEA) system to enable ATP regeneration and to explore the feasibility of using PfPRS for PRPP biosynthesis.

The rapid emergence of antibiotic resistance has rendered many conventional antibiotics ineffective, complicating the management of bacterial infections and leading to severe community- and hospital-acquired diseases. This growing resistance crisis poses a major threat to global public health and the stability of healthcare systems. Metal-based nanoparticles have recently gained significant attention as alternative antibacterial agents because of their broad-spectrum activity and low likelihood of inducing bacterial resistance. However, their clinical translation remains limited by nonspecific cytotoxicity and poor biocompatibility, which can adversely affect mammalian cells. To overcome these challenges, recent research has focused on engineering strategies that enhance the biocompatibility of metal nanoparticles (MNPs) without compromising their antibacterial efficacy. This review summarizes the latest advances in the rational design, surface modification, and functionalization of biocompatible metal-based nanoplatforms for infection treatment. Themechanisms of antibacterial action, approaches to minimize off-target toxicity, and potential clinical application. Together, these insights highlight the promise of engineered MNPs as next-generation antibacterial therapeutics capable of addressing the escalating threat of multidrug-resistant infections.

The prion concept fundamentally signifies the intrinsic cross-seeding potential of misfolded protein-generated amyloid entities to efficiently induce amyloid aggregation in normally folded proteins leading to formation of cytotoxic amyloid structures. A conformational crosstalk between the prion particle and the interacting protein appears critical for the molecular origin of seeded-aggregation. However, the intricacies of protein specificity, as a prerequisite for the onset of cross-seeding, hold negligible relevance to the pathobiology of amyloid-linked diseases because the amyloid-deposits are heteroprotein assemblies, and there is adequate evidence that substantiates the occurrence of sequence-independent amyloid-cross-seeding/co-aggregation reactions between diverse protein types. Importantly, extensive research on the self-assembly of single metabolites into cytotoxic amyloid-like entities containing cross-seeding competent conformers has certainly widened the boundary of prion concept much beyond the territory of proteins and peptides. Three important observations: 1) sequence-independent cross-seeding and co-aggregation among proteins; 2) efficient amyloid-cross-seeding of proteins triggered by self-assembled metabolite-nanostructures, and 3) molecular self-assembly of metabolites induced by pre-formed protein amyloid-seeds, propose a synergetic interplay between the amyloidogenic proteins and self-assembly-prone metabolites that can act as a key regulator for the overall amyloidogenesis mechanism. This review on the self-assembly of biologically relevant metabolites into amyloid-mimicking nanostructures mainly highlights their cytotoxic properties and cross-seeding potential, particularly focusing on the significance of the metabolite-aggregation in the etiology of amyloid hypothesis.

Hypoxia bioimaging attracts tremendous attention due to its profound implications in diagnosis of a range of pathological conditions. Nitroaromatics-based fluorescent probes are the most popular approach to tackle this problem. Despite intensive efforts of the field over the past 15 years and the development of a range of such probes, three challenges are still not addressed, i.e., highly sensitive probes necessitating a one-electron reduction, real-time monitoring probes with reversible sensing capability, and near-infrared probes with in vivo imaging potentials. Three groups have recently reported notable progresses regarding these challenges, which may spur another wave of development along this line of research and meet the need of real-world applications from researchers and doctors.