ChemBioChem最新文献

The transfusion of blood products requires ABO compatibility due to A and B antigens on blood cells and their corresponding antibodies in plasma. The supply of red cell concentrates (RCCs) and platelet concentrates (PCs) is challenged by rising demand and declining numbers of donors. To counteract the supply limitations, a method is developed to generate universal RCCs and PCs. For this, the A antigen is enzymatically removed by two enzymes from Flavonifractor plautii working in concert and the B antigen is removed by galactosidases from Pedobacter panaciterrae (PpaGal_WT or the variant PpaGal_W260Y) and Akkermansia muciniphila (AmGH110A or AmGH110B). The glycosidases are immobilized on polymethacrylate microparticles and are removed by the 200 µm filter of the mandatory transfusion set before the transfusion to avoid a possible immune reaction by the enzymes. B-positive red cells in RCCs are reduced below a 4% background signal within 48 h at 2–6 °C. Pooled PCs showed a 34 ± 14% reduction in B-positive cells and a residual A-positive platelet population of 4 ± 1% after 4 h of treatment at 20–24 °C. This approach efficiently generates ABO-universal RCCs and PCs while preserving blood quality and reducing incompatibility risks.

Adenosine deaminases acting on RNA (ADAR) catalyze the deamination of adenosine to inosine in double-stranded RNA. Because inosine is read as guanosine during translation, this process enables programmable A-to-G recoding at the transcript level. ADARs can be harnessed for therapeutic correction of pathogenic mutations through site-directed RNA editing with guide RNAs. To expand the design space of editing-enabling guides, we applied EMERGe, a high-throughput screening platform, to identify motifs targeting a premature termination codon in the MeCP2 transcript associated with Rett syndrome. This uncovered a guide RNA motif that supported efficient ADAR2-mediated editing in vitro, featuring a 5′-GUG-3' sequence predicted to form an asymmetric loop. To enable therapeutic application, structure–activity relationship studies and chemical optimization were performed, yielding a fully modified guide RNA with 2′-O-methyl, 2′-fluoro, and phosphorothioate linkages. This stabilized guide retained the activity of unmodified RNA and showed enhanced nuclease resistance. The optimized guide induces dose-dependent editing at two MECP2 loci in reporter assays in HEK293T cells, demonstrating that EMERGe-selected motifs can be rendered viable in cells through targeted chemical modification. These findings highlight the utility of EMERGe as a discovery platform and establish a pipeline for identifying and optimizing editing-enabling guide RNA features beyond traditional design rules.

This cover highlights the dominant negative effects of Li–Fraumeni syndrome–associated germline TP53 variants at Arg337. Using a novel single-cell assay to visualize p53 heterotetramers and their transcriptional activities, the study reveals that R337H and R337C variants, despite forming heterotetramers with wild-type p53, drastically reduce its activity– especially at apoptosis-related response elements–uncovering a new mechanism underlying p53 dysfunction in cancer predisposition. More details can be found in the Research Article by Kazuyasu Sakaguchi and co-workers (DOI: 10.1002/cbic.202500330)

Granzyme A (GzmA), a serine protease highly expressed in cytotoxic immune cells, plays complex roles in antitumor immunity and inflammation. While elevated GzmA levels correlate with favorable outcomes in certain cancers, extracellular GzmA has been implicated in promoting tumorigenesis via inflammatory pathways. These contrasting functions highlight the need for selective tools that can detect GzmA activity with high spatiotemporal resolution in native biological contexts. A series of quenched activity-based probes that fluoresce only upon covalent binding to active GzmA have been developed. The lead probe, featuring the Ile–Gly–Asn–Arg recognition sequence, a phenyl phosphinate ester warhead, and a near-infrared sulfo-Cy5/QSY21 fluorophore/quencher pair, exhibits high reactivity, selectivity, and cell permeability. It enables robust detection of GzmA in vitro and in cells, with an excellent signal-to-noise ratio. These activatable probes will support downstream activity-based protein profiling and enable noninvasive imaging of the enzyme activity, offering a powerful means for dissecting the multifaceted biology of GzmA within the tumor immune microenvironment.

PDE6D is a trafficking chaperone of prenylated proteins, such as small GTPases. Several small molecule inhibitors have been developed against it, given that the oncogene K-Ras is one of the cargo proteins. Inhibitor development suffers from the fact that inhibitors against the hydrophobic pocket of PDE6D are typically poorly water-soluble. Herein, the development of genetically encoded inhibitors that are inspired by high-affinity natural cargo of PDE6D is described. The most potent inhibitor, SNAP-STI, encodes merely a farnesylated tetra-peptide, which efficiently blocks PDE6D binding of farnesylated cargo. Direct comparison with small molecule PDE6D inhibitors suggests its higher potency. It is shown that inhibition of K-Ras membrane anchorage and K-RasG12C-dependent MAPK signaling by SNAP-STI is weak, consistent with what is observed after PDE6D knockdown. The data therefore further support that PDE6D is not a suitable surrogate target for efficient inhibition of K-Ras membrane anchorage and MAPK-activity. Nonetheless, by exploiting contacts at the pocket entry, a generalizable strategy to design high-affinity PDE6D inhibitors is established, providing powerful tools for PDE6D biology and target validation.

The Michaelis constant (K_m) is central to enzyme kinetics, guiding variant selection, inhibitor screening, and metabolic modeling. However, K_m obtained by nonlinear regression can be substantially inaccurate even when the reported standard error (SE) appears small. Common software reports SE but provides no accuracy metric. This gap is addressed by extending the accuracy confidence interval (ACI) framework to K_m (ACI-K_m) through a binding-isotherm formulation of the velocity–substrate fit. Given confidence intervals for concentration accuracy, the method quantifies how residual systematic uncertainties in enzyme and substrate concentrations ( E₀ and S₀) propagate into the determined K_m values and provides a probabilistic interval expected to enclose the accurate value. The approach requires no additional kinetic experiments and is directly applicable to existing datasets. Concentration-accuracy intervals can be estimated from calibration data, reagent specifications, or quality-control records. ACI-K_m is valid across a wide range of E₀/K_m conditions, including relatively high E₀. A free web application (https://aci.sci.yorku.ca) implements ACI-K_m. Tests on synthetic and experimental datasets show that K_m ± SE can severely underestimate uncertainty, whereas ACI provides more reliable accuracy bounds for decision-making, complementing rather than replacing traditional precision metrics by providing quantitative diagnostic bounds for concentration-related uncertainties in K_m determination.

Abiraterone (Abi) acetate, an anticancer drug, produces metabolites in the serum of patients with prostate cancer. Three unknown metabolites of Abi are previously discovered and the structures of two of these compounds are determined by comparing their LC-based retention times with those of synthesized compounds. However, the structure of the third metabolite is not identified. Herein, two new Abi derivatives, 5α-Δ¹-abiraterone (5α-D1A) and 5β-Δ¹-abiraterone (5β-D1A), with a molecular weight of 348, are successfully synthesized as candidates for the third unknown metabolite. 5β-D1A is confirmed as this third metabolite based on its retention time in the extracted ion chromatogram. The metabolic pathway may have formed 5β-D1A rather than 5α-D1A because of the higher activity of steroid 5β-reductase compared with that of 5α-reductase. These findings are expected to lead to the discovery of new metabolic pathways.

DNA-metal complexes provide new dimensions in synthesizing DNA-based conducting biomaterials such as aptamers, DNAzymes, and their metal base pairing duplex structure. Structural modifications in the native DNA generate metal-specific binding sites in addition to phosphate backbone and nucleobase sites. One of the natural nonbenzenoid aromatic molecules, Tropolone, exhibits ion-specific binding with Cu(II) ion. In the repertoire of DNA metal sensors, troponyl-DNA can be explored for developing DNA-based Cu-sensors owing to the excellent Cu²⁺ binding affinity of Tropolone ligand. This report describes the synthesis of DNA penta-oligomer containing the repeated sequence of tr-dTnucleoside (T^*) as (5′-T^*T^*T^*T^*T-3′) and its binding affinity with Cu(II) ions. Its Cu(II) mediated duplex-like dimer structure shows promising electrical conductivity at pico-molar [Cu²⁺] in acetonitrile. Hence, troponyl-DNA can be applied for making Cu(II) ion sensing devices.

Gold nanoparticles (AuNPs) have emerged as powerful tools in the molecular sensing field for developing highly sensitive and selective molecular detection methods due to their unique properties, such as optical features and ease of surface modification. The core principle of AuNP-based sensors involves target molecule-induced aggregation, dispersion, or assembly of AuNPs through specific interactions with target molecules. These interactions cause changes in optical or electrochemical properties, which can be monitored through visible color shifts, surface-enhanced Raman scattering (SERS) signals, electrochemical techniques, and changes in scattering intensity. This review aims to highlight molecular detection strategies focusing on surface modification and aggregation mechanisms of AuNPs. It also introduces representative applications using colorimetric methods, SERS, electrochemical assays, and dark-field microscopy.

A phenyl urea unit is proposed as an aglet for completely favoring trans-Pro peptide bond, which is further stabilized by n → π* interaction. The urea-tagged Pro–Ile peptide is crystallized from nonaqueous solvent, and the X-ray structure analysis reveals self-assembly by clustering of hydrophobic side chains of Ile and phenyl ring, reminiscent of zipper structure found in coiled-coil peptides.