RSC Chemical Biology最新文献

Transcriptional regulatory proteins are frequent drivers of oncogenesis and common targets for drug discovery. The transcriptional co-activator, ENL, which is localized to chromatin through its acetyllysine-binding YEATS domain, is preferentially required for the survival and pathogenesis of acute leukemia. Small molecules that inhibit the ENL/AF9 YEATS domain show anti-leukemia effects in preclinical models, which is thought to be caused by the downregulation of pro-leukemic ENL target genes. However, the transcriptional effects of ENL/AF9 YEATS domain inhibitors have not been studied in models of intrinsic or acquired resistance and, therefore, the connection between proximal transcriptional effects and downstream anti-proliferative response is poorly understood. To address this, we identified models of intrinsic and acquired resistance and used them to study the effects of ENL/AF9 YEATS domain inhibitors. We first discovered that ENL/AF9 YEATS domain inhibition produces similar transcriptional responses in naive models of sensitive and resistant leukemia. We then performed a CRISPR/Cas9-based genetic modifier screen and identified in-frame deletions of the essential transcriptional regulator, PAF1, that confer resistance to ENL/AF9 YEATS domain inhibitors. Using these drug-resistance alleles of PAF1 to construct isogenic models, we again found that the downregulation of ENL target genes is shared in both sensitive and resistant leukemia. Altogether, these data support the conclusion that the suppression of ENL target genes is not sufficient to explain the anti-leukemia effects of ENL/AF9 antagonists.

We previously reported that the nascent SKIK peptide enhances translation and alleviates ribosomal stalling caused by arrest peptides (APs) such as SecM and polyproline when positioned immediately upstream of the APs in both Escherichia coli in vivo and in vitro translation systems. In this study, we conducted a comprehensive screening of translation-enhancing peptides (TEPs) using a randomized artificial tetrapeptide library. The screening focused on the ability of the peptides to suppress SecM AP-induced translational stalling in E. coli cells. We identified TEPs exhibiting a range of translation-enhancing activities. In vitro translation analysis suggested that the fourth amino acid in the tetrapeptide influences the reduction of SecM AP-mediated stalling. Additionally, we developed a machine learning model using a random forest algorithm to predict TEP activity, which showed a strong correlation with experimentally measured activities. These findings provide a compact peptide toolkit and a data-driven approach for alleviating AP-induced ribosome stalling, with potential applications in synthetic biology.

Membrane proteins and lipids are essential for a wide range of cellular processes, making their structural characterisation essential for understanding biological function. However, the amphipathic nature of membrane proteins poses a significant challenge for traditional structural biology techniques. Membrane mimetics offer an alternative approach to studying membrane proteins in more native-like environments. Among them, peptidiscs have emerged as a promising tool for stabilising membrane proteins, allowing reconstitution from detergent micelles into a detergent-free, native-like environment that preserves structural integrity. While peptidiscs have shown utility in techniques such as mass photometry and cryo-EM, their compatibility with native mass spectrometry (MS) remains largely unexplored. In this study, we evaluate the feasibility of using peptidiscs for native MS analysis of membrane proteins and their complexes, focusing on the antibiotic resistance efflux pump AceI and the β-barrel assembly machinery (BAM complex). We reconstituted these proteins into peptidiscs using both ‘on-column’ and ‘on-bead’ assembly methods and assessed complex integrity and stability post-reconstitution using native MS. Our findings highlight the potential of peptidiscs as a tool for native MS-based structural characterisation of membrane protein and their assemblies.

The growing need for effective HBV treatments and lead compounds with novel mechanisms prompted us to explore synthetic strategies for generating skeletally diverse alkaloidal Michael acceptors. Our approach uniquely embeds Michael acceptors directly within multicyclic alkaloid-inspired frameworks, exploiting the azepinoindole scaffold—a privileged structure in indole alkaloids. A single-step assembly between the versatile intermediate 13 with methyl propiolate 14 or its derivatives enabled the rapid and divergent synthesis of six alkaloidal Michael acceptors (15–20). This strategy facilitated systematic diversification of three-dimensional functional group arrangements and precise tuning of the electronic and steric properties of the embedded α,β-unsaturated carbonyl moieties. The optimal hit 15 inhibited hepatitis B surface antigen (HBsAg) production with an IC₅₀ of 2.48 μM and significantly reduced levels of covalently closed circular DNA (cccDNA), the master template of HBV. Unlike existing nucleoside/nucleotide-based anti-HBV drugs that primarily inhibit reverse transcription, the alkaloidal Michael acceptor 15 suppressed both cccDNA and relaxed circular DNA (rcDNA) levels, suggesting a potential pathway toward a functional HBV cure. Our study also streamlined the target identification by leveraging the covalent binding properties of the Michael acceptors and the operational simplicity of biotin- or fluorescent-tag attachment via a pre-installed alkyne moiety. Competitive pull-down experiments identified several potential target proteins, involving DNA polymerase epsilon subunit 3 (POLE3). Notably, the alkaloidal Michael acceptor 15 was demonstrated to covalently modify Cys51 in POLE3, providing new insights into virus–host interactions and opening novel avenues for targeted anti-HBV therapies. This approach represents a significant advance beyond traditional screening methods and underscores the potential of skeletally diverse alkaloidal Michael acceptors in antiviral drug development.

Nanchangmycin is a natural product with broad-spectrum activity against various organisms, exhibiting antibiotic, antiviral, anticancer, and antifibrotic effects. Nanchangmycin belongs to the family of polyether ionophores and is proposed to exert its therapeutic effects by altering ion gradients across biological membranes. Although this therapeutic mechanism has been well characterised in cancer models, it does not fully explain how nanchangmycin inhibits Zika virus infection, as recently reported. The specific molecular targets responsible for mediating nanchangmycin's antiviral activity remain unknown. Here, we designed a photoreactive clickable nanchangmycin probe and employed chemical proteomics to identify protein targets of nanchangmycin related to Zika virus infection in human cells. Among the most prominent targets was the protein SEC11A, a key component of the signal peptidase complex, which is essential for cleaving and processing Zika virus proteins. We showed that nanchangmycin blocks the cleavage of a Zika virus polyprotein, suggesting a novel mechanism for nanchangmycin-mediated inhibition of Zika virus infection.

Riboswitches are widespread regulatory RNA modules in bacteria, with many different classes already identified and even more yet to be discovered. Traditionally, the identification of riboswitches has relied on bioinformatic analyses and genetic screens. In this work, we explored the possibility of identifying and characterizing predicted and novel riboswitches using an affinity purification-based approach with a functionalized preQ₁ ligand. We successfully enriched a predicted preQ₁ riboswitch from L. monocytogenes total RNA. Biophysical characterization revealed that this riboswitch can simultaneously bind two ligand molecules and functions as a regulator of translation in vivo. Furthermore, a transcriptome-wide pull-down experiment resulted in strong preQ₁-dependent enrichment of several candidate sequences. Characterization of the lmo2684 candidate mRNA revealed a preQ₁ riboswitch-like sequence in its 5′ untranslated region. Notably, preQ₁ allowed translation of an upstream open reading frame in this region by promoting stop codon readthrough. Our findings highlight the utility of ligand-based pull-down strategies for enriching mRNAs with aptamers that elude computational detection and may possess undiscovered functions.

DNA methylation is a key epigenetic modification involved in genomic imprinting, X-chromosome inactivation, and repression of repetitive element transcription and transposition. Despite its biological significance, the impact of epigenetic modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) on the structural and UV-induced dynamics of DNA remains poorly understood. Here, we employed the fluorescent nucleobase analogue 2-aminopurine (2Ap) in combination with steady-state and time-resolved spectroscopy, molecular dynamics, and quantum mechanical calculations to investigate these effects. Our findings reveal distinct differences in base stacking and helical stability between mC and hmC-modified DNA. mC-modified DNA predominantly adopts a stacked conformation, promoting efficient fluorescence quenching of 2Ap. In contrast, hmC-modified DNA displays both stacked and non-stacked conformations, leading to reduced base stacking and a more hydrophobic local environment, as indicated by blue-shifted emission spectra. Furthermore, although charge-transfer quenching occurs in all systems, hmC shows weaker charge-transfer character, resulting in higher fluorescence quantum yields and longer lifetimes. These results highlight the subtle but crucial role of hmC in modulating local DNA conformation and stability. Moreover, they demonstrate the effectiveness of 2Ap as a sensitive probe for detecting epigenetic modifications, offering deeper insights into the molecular mechanisms of DNA methylation and demethylation pathways.

The glycoprotein α-dystroglycan is essential in establishing cell-matrix interactions and is implicated in the pathology of muscular dystrophies. Novel tools are needed to study its rare and intriguing O-mannosyl glycans. This report describes the synthesis and evaluation of alkyne-tagged ribitol-5-phosphate derivatives for the metabolic labelling of α-dystroglycan in mammalian cells.

Cell-specific control of the function of antisense oligonucleotides (ASOs) is highly desirable for precise gene therapy while minimizing adverse effects in normal cells. Herein, we report a novel class of chemically inducible ASOs (iASOs) that achieve tumor-cell-selective gene silencing through hydrogen peroxide (H₂O₂)-triggered activation. Through post-synthetic incorporation of phenylboronic acid (BO) caging groups at the backbone positions, we developed iASOs that remain functionally inactive until the H₂O₂-triggered removal of the BO groups caused activation. Using EGFP as a reporter system, we demonstrated that the optimal BO-modified iASO exhibited slight gene silencing activity in normal cells but achieved >80% knockdown of the target mRNA in tumor cells. The BO-modified iASO was further applied to target the endogenous Bcl₂ gene, demonstrating its ability for controlling gene silencing and inducing cell death. This study establishes a simple and effective platform for conditional gene regulation and the development of cell-specific ASO therapeutics.

The synthesis of ester bonds using lipases is one of the most frequently performed reactions in biocatalysis, yet examples of the enzymatic synthesis of phenyl benzoate esters are comparatively rare. In this report we show that the ligase ClxA, from Clostridium cavendishii, initially reported to have roles in amide bond formation in the biosynthesis of benzoxazole antibiotics, is an effective catalyst for the formation of phenyl benzoate esters from acid and phenol substrates using ATP in an aqueous medium. The structure of ClxA in a complex with both AMP and 3,4-aminohydroxybenzoic acid was determined by X-ray crystallography to 2.15 Å resolution and used as a platform to engineer the enzyme to create variants N226L and K140A possessing broader substrate specificity for ester formation, and also the ability to enable the synthesis of native amide product oligomers.