ChemBioChem最新文献

Enhanced proteasome activity is known to confer resistance to cellular stress in vitro and in vivo, but such effects have largely been achieved through genetic upregulation of proteasome subunits and assembly factors. Here, we investigate whether small-molecule 20S proteasome activators can modulate XBP1 signaling during IRE1-driven unfolded protein response (UPR) activation. We show that pre-treatment with a 20S activator prior to IRE1 induction significantly attenuates XBP1 signaling, whereas treatment after chemical induction of IRE1 produces no detectable effect. These findings indicate that proteasome activators can bolster proteasome activity under endoplasmic reticulum (ER) stress, but their ability to modulate an ongoing UPR is limited. This work highlights a potential temporal window in which proteasome activation may influence stress-adaptive signaling.

Bacterial biofilms are structured microbial communities embedded in a self-produced extracellular matrix, displaying enhanced tolerance and, in many cases, resistance to biocides, antimicrobial agents, and host immune responses. These traits make biofilms a major driver of chronic and recurrent infections, which are increasingly difficult to eradicate and represent a significant global health challenge in the context of rising antimicrobial resistance (AMR). Biofilms are recognized as surface and nonsurface-attached aggregates in diverse clinical, industrial, and environmental settings, broadening our knowledge of their ecological and physiological diversity. Surfactants have emerged as promising antibiofilm agents due to their dual functionality: the capacity to disrupt the extracellular matrix and their inherent antimicrobial activity. Among them, amino acid-based surfactants, particularly cationic derivatives such as those based on arginine, combine potent biocidal effects with favorable biocompatibility and environmental sustainability. These compounds offer a persuasive alternative to conventional biocides, which often promote cross-resistance and environmental concerns. This review integrates current knowledge of biofilm formation and persistence with advances in the development and application of amino acid-based surfactants as antibiofilm agents. Sustainable synthesis of these compounds, as well as mechanistic insights, and the translational challenges of moving from in vitro assays to real-world scenarios in the AMR era.

Genetically encoded photo-cross-linkable amino acids (PAAs) are powerful tools for analyzing direct protein–protein interactions (PPIs) in mammalian cells. Cleavable PAAs are particularly useful, enabling covalent capture and subsequent release of interacting partners, which facilitates the characterization of interaction interfaces using mass spectrometry. However, the limited options for cleavable linker structures have restricted the design of PAAs. In this study, we genetically encoded a novel trifunctional PAA, DiZAAsu, which contains three distinct chemical groups: diazirine, alkyne, and alkaline-cleavable alkyl ester moieties. An archaeal pyrrolysyl-tRNA synthetase was engineered to incorporate DiZAAsu efficiently into proteins in mammalian cells. We demonstrated the in-cell photoreactive function of diazirine by cross-linking the DiZAAsu-introduced GRB2 protein to its binding partner, SHC. Using the alkyne group for biotinylation, we established a tandem affinity purification strategy that enabled efficient enrichment of the cross–linked complex, thereby reducing nonspecific protein contamination. The alkaline-based cleavage of the ester group in DiZAAsu was also demonstrated, confirming its potential for the dissociation of covalently linked complexes. This system thus expands the design space of multifunctional PAAs and adds alkaline-based dissociation to the limited repertoire of available cleavage strategies.

Polyketides constitute a large class of natural products with important biological activities and applications such as antibiotics, antitumor agents, pesticides, and pigments. Their biosynthesis is catalyzed by polyketide synthases (PKSs) which are multidomain enzymes evolutionarily related to fatty acid synthases (FASs). Despite their close homology in structure and the chemistry they perform, FASs and PKSs differ fundamentally in their catalytic programming: FASs run fully reducing elongation reactions to yield saturated fatty acids, while iterative PKSs execute reductions just in selected cycles, generating complex oxidized compounds. In this study, we aimed at engineering the metazoan FAS in its ketoreduction (KR) domain to switch from fully reducing to a nonreducing mode during chain elongation. Guided by recent insights into KR programming, we incorporated a helix into metazoan FAS, which is found in KRs from iterative PKSs and type II FASs with chain length programming. These FAS variants initially catalyze complete fatty acid cycles but lose the ability of β-keto reduction in later elongation rounds, producing intermediates that spontaneously cyclize to pyrone products. Finally, our study provides valuable insight into the mechanism of KR catalysis identifying another amino acid next to the active tyrosine which is capable for intermediate protonation.

The rippled β-sheet was predicted by Linus Pauling and Robert Corey in 1953. Unlike the closely related pleated β-sheet, which rapidly expanded to become common textbook knowledge, the rippled β-sheet remained obscure for decades. The critical body of biophysical evidence for the structural viability of this neglected motif only began to emerge in the 2000s and onwards. The first crystal structure of a rippled β-sheet was reported by our laboratory in 2022, that is, 69 years since its original prediction. From model tripeptides, we gradually expanded to longer, biologically relevant sequences. Subsequent rational molecular design led to the creation of chimeric mixed chirality peptides capable of forming rippled sheets from single components (i.e., “self-rippling” peptides), as well as rippled sheet macrocycles. Over half of the canonical amino acid alphabet has meanwhile been observed in the context of the rippled β-sheet. The number of ripple-genic amino acids keeps expanding as the field continues to mature. The rippled β-sheet is a platform that allows readily accessing a wide variety of aggregated peptidic folds, often with unique properties. The field is wide open for discovery.

Antibiotic resistance has become a critical global threat, creating an urgent need for new antibacterial agents. Among therapeutic modalities, small-molecule antibacterials offer significant advantages, including controllable metabolism and flexible structural design, making them an excellent platform for combating drug-resistant bacteria. This review highlights recent progress in organic small-molecule antibacterials and is organized into three sections: antibiotics, natural products and their derivatives, and other synthetic small-molecule agents (including heterocyclic, sulfonamide, and amphiphilic compounds). Each section summarizes recent advances in the field, and the review concludes by discussing future directions and challenges in small-molecule antibiotic development. By drawing on strategies across these categories, this overview provides researchers in the field with a fresh perspective to inspire novel approaches and accelerate the development of new antibacterial drugs.

Traditional biocatalytic cascades typically require discrete enzymes for each synthetic step. Here, we report unprecedented trifunctional imine reductases (IRED) that conduct three sequential transformations—alkene reduction, intramolecular reductive amination, and imine reduction—all within a single catalytic cycle. This elegant single-enzyme catalytic system directly transforms linear substrates into enantiomerically pure 2-aryl pyrrolidines via a concerted cascade without intermediate isolation. Combining density functional theory (DFT) calculations and mechanistic studies, we elucidate how the IRED achieves step-selective catalysis. Our findings establish a proof-of-concept for simplifying complex biocatalytic cascades using multifunctional enzymes, offering a powerful strategy to streamline synthetic pathways.

Aromatic rings bearing amino groups provide natural products with structural diversity and potent biological activities. Although aromatic amination is a useful reaction in organic synthesis, knowledge of biological aromatic amination remains limited. In this study, we identified an unprecedented nitrite-dependent aromatic amination in nybomycin biosynthesis. By comparing biosynthetic gene clusters whose products have a diamino phenol scaffold, we hypothesized that nine genes, including two nitrite biosynthetic genes, are responsible for the biosynthesis of this scaffold. Using heterologous expression in Streptomyces albus, we identified the minimum number of enzymes required for 2,4-diamino-3-hydroxybenzoic acid (2,4,3-DAHBA) biosynthesis. Further analysis revealed that three enzymes (NybN, NybO, and NybC) were responsible for converting 3-hydroxyanthranilic acid (3-HAA) into 2,4,3-DAHBA using nitrite. In vitro assays revealed that NybO, an ATP-dependent ligase, catalyzes the diazotization of 3-HAA to form 2-diazo-3-hydroxybenzoic acid (2,3-DHBA) and that NybC, an NADPH-dependent oxidoreductase, catalyzes the reduction of 2,3-DHBA to form 2-hydrazino-3-hydroxybenzoic acid. Taken together with other experimental results, we propose two possible biosynthetic pathways for 2,4,3-DAHBA synthesis from 3-HAA. This study provides important insights into nitrite-mediated aromatic amination, expanding the availability of nitrite for natural product biosynthesis.

The Bcl-2 protein Bcl-xL is an inhibitor of intrinsic apoptosis which either directly inhibits the pore-forming Bcl-2 proteins, like Bax or Bak, or indirectly inhibits pore formation by sequestering the pro-apoptotic BH3-only activators. The structural basis of the inhibition of pore formation in the outer mitochondrial membrane is still largely unknown due to the lack of atomic resolution structures of the relevant inhibitory complexes at the membrane. Herein, a protocol to obtain high-yield recombinant monomeric full-length Bcl-xL proteins is presented. The monomeric Bcl-xL retains the ability to shuttle between membrane and aqueous environments and can successfully inhibit Bcl-2-induced membrane permeabilization via both modes of action, as proven by in vitro and in organelle assays with a minimal Bcl-2 interactome constituted by Bcl-xL, cBid, and Bax.

Cu(II) complexes with monoalkylated oxacyclen ligands (C₁₂, C₁₆, and C₁₈) have been investigated regarding their interaction with DNA by different methods: circular dichroism, UV/VIS (ultraviolet-visible) and fluorescence spectroscopy as well as by gel electrophoresis. The results demonstrate that the complexes can cleave DNA through both hydrolytic and oxidative mechanisms, with hydroxyl radicals and hydrogen peroxide identified as the reactive oxygen species involved. The targeted incorporation of alkyl chains significantly enhances the DNA-binding affinity of the Cu(II) complexes, and the length of the alkyl substituents plays an important role, as they can interact with the major groove of the DNA. Alkylation is the determining structural factor responsible for the enhanced DNA interaction, since such an interaction is not observed with unsubstituted complexes. Moreover, the length of the alkyl chains significantly influences this behavior, as longer substituents induce a concentration-dependent DNA aggregation, a phenomenon absent in the nonalkylated analog. This aggregation and condensation behavior is examined using atomic force microscopy and dynamic light scattering. Moreover, DNA/small molecule interactions are also investigated using molecular dynamics simulations.