ACS Chemical Biology最新文献

The orphan nuclear receptor NR2F6 (Nuclear Receptor subfamily 2 group F member 6) is an emerging therapeutic target for cancer immunotherapy. Upregulation of NR2F6 expression in tumor cells has been linked to proliferation and metastasis, while in immune cells NR2F6 inhibits antitumor T-cell responses. Small molecule modulation of NR2F6 activity might therefore be a novel strategy in cancer treatment, benefiting from this dual role of NR2F6. However, there are no molecular strategies available for targeting NR2F6, hampered among others by lack of structural insights and appropriate biochemical assays. To overcome these challenges, several noncanonical nuclear receptor coregulator peptide motifs were identified to be constitutively recruited to the NR2F6 ligand binding domain (LBD). Co-crystallization of the NR2F6 LBD with a peptide from the coregulator Nuclear Receptor Binding SET Domain Protein 1 (NSD1) enabled, for the first time, the structural elucidation of the unliganded (apo) form of NR2F6. This revealed an autorepressed, homodimeric LBD conformation in which helix 12 folds over the canonical coregulator binding site, generating an alternative contact surface for NSD1 binding. Screening of a focused library of covalent NR probes identified compounds that preferentially target a cysteine residue near the NSD1 binding site, inhibiting NR2F6 coregulator recruitment. Combined, these results provide structural insights into the ligand-independent transcriptional activity of NR2F6 and may serve as a starting point for the development of novel NR2F6 modulators.

Small-molecule metabolic chemical probes are tailored chemical biology tools that are designed to detect and visualize biological processes within a cell or an organism. Nucleoside analogues are a subset of metabolic probes that enable the study of DNA synthesis, proliferation kinetics, and cell cycle progression. However, most available nucleoside analogue probes have been designed for use in mammalian cells, limiting their use in other species, where there are metabolic pathway differences. The current gold-standard probe for studies of DNA synthesis in mammalian cells, 5-ethynyl-2′-deoxyuridine (EdU), can be detected via a copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with a fluorescent azide. However, the use of EdU in malaria parasites is not possible as Plasmodium lacks thymidine kinase, the first enzyme in the sequential phosphorylation of thymidine needed for eventual incorporation of this probe into DNA. We previously demonstrated that a pronucleotide approach with modified EdU analogues (designated DNADetect probes) can be used to study DNA proliferation in Plasmodium. However, as cytotoxicity can be associated with the use of copper(I) ions as a catalyst for CuAAC, and cells require fixation, negating the potential for live cell imaging, alternative probes would help overcome these challenges in Plasmodium and other species lacking thymidine kinase. Herein, we report the development and synthesis of novel 5-vinyl-2′-deoxyuridine (VdU)-based chemical probes, designated vinyl-DNADetect. These probes were designed not only to bypass thymidine kinase requirements but also to have the advantage of detection via a catalyst-free and spontaneous inverse-electron-demand Diels–Alder (IEDDA) click reaction with a fluorogenic tetrazine that does not require parasites to first be fixed. Using flow cytometry and fluorescence microscopy, we quantified and visualized incorporation into Plasmodium DNA, with several probes demonstrating high efficiency labeling. In addition, we demonstrated that these VdU-based probes could label DNA in live Plasmodium falciparum-infected erythrocytes and show the first use of two orthogonal metabolic chemical probes, VdU- and EdU-based, in a pulse-chase experiment for DNA double staining in wild-type Plasmodium.

High salt concentrations affect the electron transport chain of bacterial cells, leading to an oxidative stress response that encompasses the formation of reactive oxygen species (ROS). The salt 2,3,5-triphenyltetrazolium chloride (TTC) triggers antibacterial activity against the phytopathogen Ralstonia solanacearum in Bacillus species; however, the underlying mechanisms remain unknown. Here, we tested the hypothesis that TTC-inducible activity is related to the formation of ROS and its metabolites. We found that l-ascorbic acid, superoxide dismutase, and catalase counteracted TTC-inducible activity in various Bacillus species. Furthermore, R. solanacearum exhibited a higher susceptibility to H₂O₂ than Bacillus spp. Genomic analysis showed differences in stress-related genes, with Bacillus strains containing the ROS scavengers bacillithiol and bacillibactin, while glutathione inR. solanacearum. Multivariate analysis indicated that the Bacillus species and TTC influence Bacillus metabolome, resulting in higher levels of quinazoline alkaloids, with potential antibacterial activity against R. solanacearum. Results suggest that TTC induces the production of O₂^•– and H₂O₂ and metabolites that arrest R. solanacearum growth.

C-diazeniumdiolate siderophores are a small class of photoactive bacterial Fe(III) chelators. Driven by genome mining, we discovered a new C-type diazeniumdiolate siderophore, pandorachelin, produced by the rhizospheric bacterium, Pandoraea norimbergensis DSM 11628. The biosynthetic gene cluster encoding the production of pandorachelin is conserved across several Pandoraea species. Pandoraea spp. are environmentally widespread and are increasingly prevalent clinical pathogens, spurring new interest in their metabolites. UV irradiation photolytically cleaves the N–N bonds within the diazeniumdiolate-containing graminine constituents of pandorachelin. With EPR spin trapping, we directly detect nitric oxide released from the two C-diazeniumdiolate ligands of pandorachelin upon UV irradiation. Additionally, we show that nitric oxide can react with the intermediates during the photoreaction to reconstruct the diazeniumdiolate groups via exchange of the distal nitric oxide (NO) and thereby recover Fe(III)-binding capacity. The photochemistry of this class of siderophores points to a broader biological role, both in their propensity to release the biological signaling molecule, nitric oxide, and in their ability to undergo photoinduced NO exchange.

Targeted protein degradation (TPD) is a promising modality that leverages the endogenous cellular protein degradation machinery to degrade selected proteins. Recently, we validated CUL3^KLHL20 E3 ligase as a new actionable E3 ligase for TPD application by developing a synthetic macrocycle ligand to engage KLHL20. Linking the KLHL20 ligand to JQ1, we created the PROTAC molecule BTR2004, which exhibited potent degradation of BET family proteins BRD 2, 3, and 4. As CUL3^KLHL20 is new to the TPD field, here we report the first temporal and spatial characterization of CUL3^KLHL20-driven TPD with BTR2004. Our study revealed the target protein degradation kinetics, BTR2004 intracellular activity half-life, and the onset of BTR2004 cell permeabilization. Employing proximity ligation and confocal microscopy techniques, we also illustrate the subcellular location of the ternary complex assembly upon BTR2004 treatment. These characterizations provide further insight into the processes that govern TPD and features that could be incorporated into the design of future macrocyclic PROTAC molecules.

Glycoside phosphorylases (GPases) enable oligosaccharide assembly using sugar-1-phosphate donors, but ATP dependency for kinase-mediated phosphorylation limits practicality. Here, a polyphosphate kinase (PPK)-coupled ATP regeneration system is introduced, requiring only <0.05 equiv of AMP to synthesize diverse oligosaccharides (40–92% yields) from monosaccharides and polyphosphate. By integrating PPK with GPases and sugar 1-kinases, lacto-N-biose I, galacto-N-biose, N-glycan core trisaccharides, and β-1,2/3/4-mannosides were efficiently produced in one-pot reactions. This ATP-free strategy eliminates exogenous nucleotide costs, circumvents product inhibition, and demonstrates broad compatibility with GPases targeting galactosides, glucosaminides, and mannosides, offering a scalable and cost-efficient enzymatic platform.

Dynamic protein–protein interactions are key drivers of many cellular processes. Determining the relative sequence and precise timing of these interactions is crucial for elucidating the functional dynamics of biological processes. Here, we developed a time-resolved analysis of protein–protein ensembles using a destabilizing domain (TRAPPED) to study protein–protein interactions in a temporal manner. We have taken advantage of a dihydrofolate reductase-destabilizing domain (DHFR(DD)) that can be fused to a protein of interest and is constitutively degraded by the proteosome. Addition of the ligand trimethoprim (TMP) can stabilize DHFR(DD), preventing proteasomal degradation of the fusion protein and thereby inducing accumulation in cells. We synthesized and optimized TRimethoprim Analog Probes that maintain stabilization activity and contain a terminal alkyne for Click functionalization and a thiol reactive group to covalently tag DHFR(DD). Click reaction with a biotin tag and subsequent streptavidin enrichment enable time-resolved mass spectrometric identification of interacting partners. We evaluated the timing of protein interactions of SARS-CoV-2 and SARS-CoV nonstructural protein 15 (nsp15) over a 2 h period. We found interactors GEMIN5 and YBX3, known regulators of SARS-CoV-2 infection that bind viral RNA, as well as CACYBP and FHL1 that implicate nsp15 in the disruption of host ERK1/2 signaling. We further revealed that these interactions remain relatively steady from 0 to 2 h post translation of nsp15. TRAPPED methodology can be applied to determine the sequence and timing of protein–protein interactions of temporally regulated biological processes such as viral infection or signal transduction.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) with potent antimicrobial functions. Their biosynthesis is carried out by dedicated biosynthetic enzymes, including the recently described Class III-b LanKC enzymes, which represent a newly defined subclass of trifunctional synthetases. Here, we report the high-resolution cryo-EM structure and biochemical characterization of SalKC from Streptococcus salivarius, which catalyzes the maturation of the antimicrobial peptide salivaricin. SalKC adopts a conserved dimeric architecture stabilized by a His36 hotspot, mirroring that of the previously characterized PneKC. Cryo-EM structure resolved to sub-3.0 Å revealed the side chains of the bound leader peptide in atomic detail, allowing clear visualization of a conserved recognition motif and offering new structural insight into peptide engagement. Biochemical assays showed that SalKC prefers ATP over GTP, contrasting with the GTP-preferring PneKC. Structural comparison identified a single amino acid switch: Lys303 in SalKC versus His300 in PneKC, as the key determinant of this specificity. Mutation of Lys303 to histidine reverses nucleotide preference, confirming its functional role. Together, these findings revealed conserved principles and specialized adaptations within Class III-b LanKC enzymes and provided a molecular framework for understanding their substrate and cofactor selectivity.

Fragment-based drug discovery typically relies on specialized spectrometric methods to identify low-affinity compounds that bind to biomolecules. Here, we report a proof-of-concept study on the development of a streamlined fragment-based screening platform for small molecules targeting RNA. This method employs low molecular weight fragments appended with a diazirine reactive moiety and an alkyne tag. Upon photolysis and click chemistry with an azide-containing fluorophore, these compounds can be visualized for binding to the r(CUG) repeat expansion [r(CUG)^exp] implicated in myotonic dystrophy type 1 (DM1). Fragments were found to bind the 1 × 1 nucleotide U/U internal loops formed when r(CUG)^exp folds, guiding the design of homodimeric compounds capable of interacting with adjacent internal loops in a single molecule. One dimeric compound exhibited enhanced affinity and was converted into a proximity-induced covalent binder for prolonged target occupancy. This work establishes a versatile platform for targeting structured RNAs with potential applications across a variety of disease-relevant RNA targets.