ChemBioChem最新文献

Bispecific antibodies (BsAbs) are engineered immunoglobulins that can simultaneously recognize two distinct antigens or two distinct epitopes on the same antigen. They exhibit cooperative therapeutic effects surpassing those of natural monoclonal antibodies, such as bridging the immune cells and tumor cells to stimulate targeted immune response, or blocking codependent signaling pathways. These advantages make them attractive therapeutic reagents for various diseases such as cancers, immunodeficiency syndromes, and ophthalmic disorders. However, the unique structural characteristics of BsAbs pose various challenges to their preparation. In the past few decades, various types of BsAbs have been designed and prepared through genetic engineering or chemical conjugation strategies, many of which have been approved as drugs or entered clinical trials. This review provides a systematic summary of these strategies and their corresponding principles, and focuses on the application of modern genetic engineering and chemical conjugation methods in the generation of BsAbs.

Cysteine sulfenyl iodides (Cys-SIs) have long been recognized as important intermediates in the oxidative modification of cysteine thiols since 1950s, implicated in pathways such as hydrolysis to cysteine sulfenic acid (Cys-SOH) and electrophilic aromatic substitution with an indole ring of tryptophan to form Cys-Trp thioether linkages. Despite their proposed significance in both biological and chemical contexts, direct examination of Cys-SIs has been precluded by their intrinsic instability. Herein, the first isolation of a small-molecule Cys-SI, stabilized using a nanosized molecular cradle at the N-terminus of cysteine, is reported. The structure of the isolable Cys-SI is determined by X-ray crystallography, and its reactivity is investigated with a range of nucleophiles. Hydrolysis to Cys-SOH and reaction with an indole derivative provide direct chemical evidence for long-standing mechanistic proposals. Furthermore, rapid formation of sulfenamides with amines and high-yield adduct formation with dimedone, a canonical sulfenic-acid probe, reveal that Cys-SI possesses even greater electrophilicity than Cys-SOH. These results deliver a structurally defined reference for Cys-SI and inform mechanisms of iodine-mediated protein oxidation and peptide modification.

Biosensors and microdevices using living cells have gained significant attention in recent years, including methods for cell immobilization on antibody-coated surfaces. Random antibody fixation may lead to suboptimal exposure of recognition sites, reducing interaction efficiency. In contrast, aligned antibody immobilization enhances antigen recognition and binding capacity during cell immobilization. Site incorporation of azido-phenylalanine (AzF) into antibodies facilitates site-specific conjugation, enabling aligned antibody immobilization. In this study, we expressed and purified anti-mCherry variable domains of heavy-chain-only antibodies (VHHs), incorporating AzF at the C-terminus using an unnatural amino acid system to ensure its placement away from the antigen-binding site for mCherry. Anti-mCherry VHH-AzF was conjugated to dibenzylcyclooctyne (DBCO)-coated glass via click chemistry, followed by incubation with HeLa cells expressing mCherry on their surfaces (mCherry-coated cells). This resulted in increased immobilization of mCherry-coated cells on the VHH-coated glass, whereas normal HeLa cells did not adhere.

The spatiotemporal control of biomolecular functions via light-triggered bond cleavage has emerged as a powerful approach in chemical biology and cell biology. In this concept review, three major modalities of photo-cleavable systems—proteins, small molecules, and metal complexes—are classified and discussed, highlighting their design principles, biological applicability, and remaining challenges. Emphasis is placed on recent efforts to address key design challenges—such as balancing functional performance, biological compatibility, and optical responsiveness—across different molecular modalities, offering perspectives for the next generation of photo-responsive tools for biological research.

Genomic DNA stores genetic information and regulates functions of various biological processes. Genetic diseases can be caused by abnormal gene expression. Genetic manipulation requires sequence-selective recognition of double-stranded DNAs, various chemical approaches for double-duplex invasion have been developed. In the previous research, a photo-induced double-duplex invasion (pDDI) is introduced that uses artificial nucleotides, 3-cyanovinylcarbazole nucleoside (^CNVK) as photo-crosslinker and 5-cyanouridine (^CU) as inter-probe photo-crosslinking inhibitor. In this study, while investigating the invasion mechanism of the pDDI, the invasion independence of pDDI probes is discovered and proposed a photo-induced duplex invasion (pDI) that achieves fast invasion using only ^CNVK while still maintaining a high invasion efficiency. This rapid pDI approach provides a powerful new tool for site-specific manipulation of genomic DNA.

The effects of the substitution of a guanine (G) base by the oxidative lesion 8-oxo-7,8-dihydroguanine (OG) on the affinity of the DNA aptamer selected against L-argininamide (L-Rm) are studied. Results indicate that, depending on the position of the modified base located in the recognition site, the substitution of only one G by OG could either reduce, not affect, or increase such an affinity. In addition, attempts are carried out to promote chemical crosslinks between the aptamer and its target following selective oxidation of OG. Results show that such crosslinks could be produced with a high efficacy through nucleophilic addition of L-argininamide, presumably onto the C5 position of the oxidized OG base inserted into the aptamer in any position, and that no crosslink is generated for the original aptamer (not containing any OG). However, such reaction being very efficient, crosslinks are also produced between L-argininamide and oligonucleotides (including scramble sequences) that are not supposed to bind to that amino acid. Such an effect could be explained by the electrostatic interactions between the negatively charged oligonucleotides and the protonated amino acid, which favor the formation of crosslinks upon OG oxidation. Efforts to decrease formation of such nonspecific crosslinks are only partly successful.

Transcription factors (TFs) are important gene regulators whose abnormal expression or function is involved in the occurrence of various diseases. The emergence of transcription factor-targeted proteolysis-targeting chimeras (TF-PROTACs) using DNA with a specific sequence as the targeting ligand of TFs provides a promising strategy to overcome the difficulties of using small molecules to inhibit TFs without well-defined ligand-binding pockets. A smart nanomachine is reported to realize near-infrared (NIR) light-triggered release and activation of TF-PROTAC using upconversion nanoparticles loaded with caged TF-PROTAC targeting the transcriptional factor NF-κB. The release of the active TF degrader (dNF-κB) under 980 nm NIR irradiation is demonstrated in a controlled manner, enabling precise control of the degradation of the transcriptional factor NF-κB p65 in live cells. The construction of the NIR-responsive nanomachine makes it possible to load and release various TF-PROTACs to degrade different transcriptional factors on demand with spatial and temporal resolution.

Antimicrobial peptides (AMPs) represent a promising alternative to conventional antibiotics in combating multidrug-resistant pathogens, yet their clinical translation is hindered by proteolytic instability, cytotoxicity, and poor bioavailability. Herein, it is demonstrated that glycosylphosphatidylinositol-mediated membrane tethering of honeybee defensin1 (Def1) in Drosophila melanogasterenhances its antimicrobial efficacy by $$100-fold compared to secreted or untethered forms, while preserving physiological and behavioral integrity under baseline conditions. Using a genetically engineered Drosophila model, three Def1 variants are expressed: native (Def1), secreted (s-Def1), and membrane-tethered (t-Def1). Flies expressing t-Def1 exhibit superior bacterial clearance of Pseudomonas aeruginosa and show improved survival postinfection, with no adverse effects on locomotion, courtship, or sleep architecture. However, under stress paradigms—including sleep deprivation and dextran sulfate sodium (DSS)-induced gut injury—t-Def1 exacerbates intestinal barrier dysfunction, as evidenced by elevated Smurf phenotype incidence, highlighting a trade-off between antimicrobial potency and epithelial vulnerability. This work establishes Drosophila as a powerful platform for dissecting AMP mechanisms and engineering spatially targeted therapies, offering translational insights for pollinator health and human infectious disease management. These results advocate for iterative refinement of membrane-anchoring strategies to balance therapeutic efficacy with host safety, advancing the development of next-generation AMPs with minimized off-target effects.

Frataxin is a 23 kDa mitochondrial iron-binding protein involved in the biogenesis of iron–sulfur (Fe–S) clusters. Deficiency in frataxin is associated with Friedreich's ataxia, a progressive neurodegenerative disorder. CyaY, the bacterial ortholog of eukaryotic frataxin, is believed to function as an iron donor in Fe–S cluster assembly, making it a key target for structural and functional studies. In this work, a comprehensive structural analysis of the Escherichia coli CyaY protein is presented, comparing its structure at room temperature and cryogenic conditions. Notably, the first room-temperature structures are obtained using the Turkish Light Source “Turkish DeLight” X-ray diffractometer and serial synchrotron X-ray crystallography, marking a significant step forward in understanding CyaY under near-physiological conditions.

A key challenge in tissue engineering is developing scaffolds that balance mechanical strength and bioactivity. Segmented poly(esterurethanes) (SPEU) are versatile polymers widely used in biomedical applications, particularly in the fabrication of elastomeric scaffolds for tissue engineering. Their mechanical properties and degradation rates can be tailored by modifying their chemical composition and morphology. However, the inherent hydrophobicity of SPEU often limits cell adhesion and proliferation, affecting their biocompatibility. To address this issue, surface modification, such as controlled dip-coating in gelatin methacrylate (GelMA), was explored in this work to enhance cell-material interactions. 3D-printed SPEU60 structures (with 60% hard segment content) are fabricated, surface-modified, and characterized using scanning electron microscopy, infrared spectroscopy, and goniometry. The ability of these scaffolds to support cell adhesion, proliferation, and viability, is evaluated in vitro using lentivirus transfected green fluorescent 3T3-L1 murine preadipocyte cells. Results from these biological activity assays demonstrate that the GelMA coating significantly enhances the cellular response. In conclusion, these GelMA-SPEU60 structures can be considered extracellular matrices suitable for tissue engineering applications.