RSC Chemical Biology最新文献

D-Luciferin (D-LH₂) is the most used substrate for beetle luciferases in various bioluminescence applications. Here, we successfully synthesized six D-LH₂ analogues including 5′,7′-dimethoxy-D-LH₂ and 7′-methylnaphthol-D-LH₂ as novel compounds. We also developed a continuous one-pot green synthesis method to improve yields of luciferins from condensation of quinone and D-Cys (63-fold greater than the previous report). The novel D-LH₂ analogues were tested with five luciferases (Fluc, SLR, Eluc, Pmluc-WT, and Pmluc-N230S), and all the compounds emitted bioluminescence at wavelengths longer than that of D-LH₂ (>80 nm). The reaction of SLR with 5′,7′-dimethoxy-D-LH₂ gave the longest red-shifted bioluminescence at 663 nm. Remarkably, the reactions of 5′-methyl-D-LH₂ emit longer wavelengths and brighter light than those of D-LH₂ in all tested luciferases, except for Eluc. Interestingly, the novel red-shifted 5′,7′-dimethyl-D-LH₂ also provided prolonged bioluminescence with a rate of light decay slower than that of D-LH₂. We further demonstrated applications of 5′-methyl-D-LH₂ and 5′,7′-dimethyl-D-LH₂ in mammalian cell lines expressing Fluc, SLR, and Pmluc-N230S. 5′-Methyl-D-LH₂ provided about 11.2-fold greater sensitivity to detect Fluc in the HEK293T crude lysate than D-LH₂, achieving the detection with a lower number of cell lines. The red-shifted 5′,7′-dimethyl-D-LH₂ also exhibits high sensitivity when using a red light filter to monitor live cell bioluminescence. These D-LH₂ analogues, 5′-methyl-D-LH₂ and 5′,7′-dimethyl-D-LH₂, are promising substrates for future cell-based assays and real-time monitoring applications.

We report the use of 5-furyl-2′-deoxyuridine (5FU) as a fluorescent probe to distinguish ligand binding to distinct G-quadruplex tetrads. Site-specific incorporation of 5FU into T95-2T at the capping regions enabled discrimination of peptide-binding events via fluorescence turn-on, providing insights into tetrad preference and binding affinities using the 5FU-G4/RHAU peptide system.

RNA has emerged as an attractive target for drug discovery, increasing the importance of methods for identifying RNA-binding small molecules. Fluorescent indicator displacement (FID) assays are commonly used for screening such molecules. However, because fluorescent indicators detect hit compounds through competitive binding, developing a diverse range of indicators is essential to avoid missing potential hits. Here, we introduce novel RNA-binding fluorogenic molecular probes for FID assays by conjugating thiazole orange (TO) derivatives to the unique RNA-binding molecule G-clamp, resulting in TO-G-clamp. G-clamp was chosen for its distinct RNA-binding mode compared to TO derivatives, as demonstrated by their large-scale RNA-binding profiles. Four TO-G-clamp analogs were synthesized and evaluated, all retaining the broad RNA-binding selectivity of G-clamp. Among them, TO-G-clamp-Bn, which features a benzyl substituent on the TO moiety, exhibited the highest consistency with the RNA-binding selectivity of G-clamp. FID assays using TO-G-clamp-Bn identified unique hit compounds that were insensitive to the well-known indicator TO-PRO-1. Furthermore, SHAPE-MaP analysis revealed the RNA-binding sites of TO-G-clamp, TO-PRO-1, and one of the hit compounds, AZ191, showing that the binding sites of TO-G-clamp were in close proximity to those of AZ191.

The human islet amyloid polypeptide (hIAPP) aggregates into amyloid fibrils that contribute to β-cell failure in type 2 diabetes. hIAPP is produced from a 67-residue precursor, proIAPP, but incomplete cleavage by prohormone convertase 2 (PC2) produces the 48-residue intermediate proIAPP(1–48), which accelerates amyloid formation in vivo. Here we show that proIAPP(1–48) assembles almost exclusively into a single fibril polymorph. Using cryo-electron microscopy we solved its structure at 3.5 Å resolution and uncovered a P-shaped, C2-symmetric dimer whose backbone and side-chain packing are nearly identical to the disease-associated TW2 polymorph propagated from pancreatic tissue, although with different helical symmetry. All eleven extra N-terminal residues remain disordered but create a weak density around His29. Based on time-averaged density derived from molecular dynamics (MD) simulations, we identified multiple hydrogen(H)-bonding interactions, which may contribute to stabilising the TW2-like fold and explain the peripheral cryo-EM density. These data establish a structural link between defective proIAPP processing and the polymorphic spectrum of islet amyloid and suggest a seeding pathway by which proIAPP(1–48) templates pathogenic architectures that fully processed hIAPP rarely adopts in vitro.

Sialic acid mimetics (SAMs) are chemically modified derivatives of sialic acids that can act as metabolic inhibitors or as sugar donors for sialyltransferases. This makes SAMs highly useful research tools to study and manipulate the biosynthesis of sialic acid-carrying glycans (sialoglycans). Moreover, SAMs that inhibit aberrant sialylation in cancer cells are emerging as potential therapeutics. Despite the wide use of SAMs, many aspects regarding their cellular uptake and metabolic fate are unknown. Here, we investigated the metabolic fate of an inhibitory SAM (P-SiaFNEtoc) and an incorporative SAM (P-SiaNPoc) in various mammalian cell lines. Using kinetic experiments and read-outs based on sialic acid-binding lectins, click chemistry, and nucleotide sugar analysis, we monitored the key steps of cellular SAM utilization. We found differences in the metabolism of SAMs that determine their potency in different mammalian cell lines. By identifying a murine macrophage cell line that is insensitive to SAMs, we have identified esterase activity as a bottleneck for the cellular utilization of SAMs. This study contributes to the understanding of the mechanisms underlying SAMs utilization in mammalian cell lines and provide relevant considerations for the future chemical design of SAMs and their application in mammalian systems.

Proteolysis targeting chimeras (PROTACs) have become a new modality for drug development of particular importance for cancer chemotherapy. PROTACs are composed of a ligand that binds to the protein of interest (POI) tethered by a linker to a ubiquitin E3 ligase-binding motif. These molecules can degrade the POI by ubiquitination and subsequent digestion using the ubiquitin-proteasome system (UPS). Although more than six hundred E3 ligases are encoded in human genome, only a small number are currently utilized by PROTACs. Because the expression levels and activities of E3 ligases vary among the cell lines, it can be advantageous to develop PROTACs that utilize new E3 ligase-binding components. In our current work we report new E3 ligase-binding ligands that employ viral protein R (Vpr), an accessory protein of the human immunodeficiency virus type-1 (HIV-1). Vpr can bind to both the E3 ligase complex, Cul4A-DDB1-DCAF1 and host proteins, such as UNG2 and facilitate host protein degradation via the UPS. We envisioned that Vpr fragments may function in PROTACs as new E3 ligase-binding ligands. Herein, we designed, synthesized and evaluated bromodomain 4 (BRD4)-targeting PROTACs (BRD4-PROTACs) that employ a well-known BRD4 inhibitor (JQ1) as a warhead and Vpr-derived peptides as the E3 ligase-binding ligands. We successfully demonstrate that the Vpr-derived peptides can function as E3 ligase-targeting ligands for PROTAC development. We also evaluated PROTACs based on the HIV-1 latency-reversing activity of JQ-1. The chemical degraders are less effective than the parent inhibitor as a latency-reversing agent (LRA). However, the low cytotoxicity of the new peptidic PROTACs allowed the compounds to be tolerated at high does, leading to potent LRA activity.

As the global fight against antimicrobial resistance in bacteria becomes increasingly pressing, new tool compounds are needed to study and evaluate novel therapeutic targets. Here, cysteine-directed fragment-based drug discovery is coupled with high throughput chemistry direct-to-biology screening to target the catalytic cysteine of a family of bacterial effector proteins, the novel E3 ligases (NELs) from Salmonella and Shigella. These effector E3 ligases are attractive as potential drug targets because they are delivered into host cells during infection, have no human homologues and disrupt host immune response to infection. We successfully identify hit compounds against the SspH subfamily of NELs from Salmonella and show that these proteins are inhibited by compound treatment, representing an exciting starting point for development into specific and potent tool compounds.

Bifunctional DNA glycosylases employ an active site lysine or the N-terminus to form a Schiff base with an abasic (AP) site base excision repair intermediate. For 8-oxoguanine DNA glycosylase 1 (OGG1), cleaving this reversible structure is the rate-determining step in the initiation of 8-oxoguanine (8-oxoG) repair in DNA. Evolution has led OGG1 to use a product-assisted catalysis approach, where the excised 8-oxoG acts as a Brønsted base for cleavage of a Schiff base intermediate. However, the physicochemical properties of 8-oxoG significantly limit the inherent enzymatic turnover leading to a weak, cellularly absent, AP lyase activity. We hypothesized that chemical synthesis of purine analogues enables access to complex structures that are suitable as product-like catalysts. Herein, the nucleobase landscape is profiled for its potential to increase OGG1 Schiff base cleavage. 8-Substituted 6-thioguanines emerge as potent and selective scaffolds enabling OGG1 to cleave AP sites opposite any canonical nucleobase by β-elimination. This effectively broadens the enzymatic substrate scope of OGG1, shaping a complete, artificial AP-lyase function. In addition, a second class of compounds, 6-substituted pyrazolo-[3,4-d]-pyrimidines, stimulate OGG1 function at high pH, while thioguanines govern enzymatic control at acidic pH. This enables up to 20-fold increased enzyme turnover and a de novo OGG1 β-elimination in conditions commonly not tolerated. The tool compounds employed here are non-toxic in cells and stimulate the repair of AP sites through a natural, APE1 dependent pathway, as opposed to previously reported β,δ-lyase stimulator TH10785.

Bacteriophages have emerged as important factors in human health and disease, with elevated phage levels associated with exacerbated inflammatory bowel disease, type 2 diabetes and poor outcomes in skin and lung infections. The mechanisms linking phages to these pathologies remain largely unknown, partly because specific chemical tools inhibiting bacteriophage replication (phage blockers) are lacking. Here, we identify benzimidazylpyrazoles as novel bacteriophage antivirals. Unlike existing synthetic antiphage compounds benzimidazylpyrazoles do not intercalate DNA and target an early stage of phage infection after adsorption. An optimized derivative reduced phage titer up to 10⁵-fold and demonstrated activity against different phage morphotypes and bacterial hosts, establishing it as a valuable chemical tool for the study of disease-related phage–host interactions.

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.