ACS Chemical Biology最新文献

Glycans on glycoproteins play roles that range from quality control in protein folding, to mediation of interactions with other proteins, to stabilization of the protein to which they are attached. Computation can suggest structures that underlie these roles, but confidence is limited by the accuracy of energetic calculations and their applicability to the aqueous environment in which proteins function. Experimental validation of suggested structures is therefore of primary importance. Here we use NMR data, including long-range pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs), to screen structures produced by a version of accelerated molecular dynamics (Pep-GaMD). This version was designed to improve the search for peptide–protein interactions, but here it is successfully applied to glycans attached to a target protein. The target protein, the N-terminal domain of human CEACAM1, is expressed with homogeneous GlcNAc₂Man₅ glycans at its three N-glycosylation sites. One site (N104) is found to have preferred conformations that exploit hydrophobic interactions between its glycans and protein hydrophobic residues, potentially adding to protein stability and protection from adverse interactions.

Colibactin is a pseudo-C₂-symmetric gut microbiome metabolite that induces DNA interstrand cross-links and plays a causal role in colorectal cancer. Since efforts to isolate colibactin have not been successful, we developed colibactin 742 (3a/b) as a stable colibactin mimetic. However, colibactin 742 (3a/b) exists as a mixture of ring and chain isomers, which complicates analysis of its activity. We report here the discovery of colibactin 686 (9) as a superior colibactin mimetic. Colibactin 686 (9) is more potent than colibactin 742 (3a/b) and recapitulates the bacterial genotoxic phenotype. Colibactin 686 (9) possesses a C₂-symmetric structure, which will expedite its synthesis, and is incapable of ring–chain isomerization, which will simplify analysis of its biological activity. We additionally establish that colibactins do not passively diffuse into cells, and are substrates for monocarboxylate transporter pumps. These latter findings have implications for trafficking of natural colibactin, which remains poorly understood.

Activity-Based Probes (ABPs) are invaluable tools for investigating enzymatic activity but can suffer from onerous syntheses and low stability in complex proteomes. Herein, we present the first synthesis of a robust, vinyl methyl ester (VME), bearing amino acid, which is compatible with solid phase peptide synthesis (SPPS). Novel peptidic probes incorporating the VME motif were prepared, and their labeling activity was investigated against the deubiquitinating enzyme (DUB) Otubain 1 (OTUB1), a critical cysteine protease DUB with remarkable specificity for Lys48 linked polyubiquitin chains. OTUB1 is implicated in DNA repair and immune response mechanisms and is currently considered a biomarker for tumorigenesis. A probe featuring the VME warhead demonstrated high reactivity and selectivity toward OTUB1, highlighting the significant potential of this approach to create robust and selective covalent tools for interrogating cysteine isopeptidases.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is highly expressed in therapy-resistant and metastatic cancers and represents a clinically relevant biomarker for selective activation strategies. We report AAP, an OFF-ON photosensitizer activated through ALDH1A1-mediated oxidation that produces singlet oxygen upon light exposure. AAP uses a donor photoinduced electron transfer (d-PeT) mechanism to suppress intersystem crossing in its unreacted benzaldehyde form, which minimizes background activity. Oxidation by ALDH1A1 disrupts d-PeT and restores phototoxicity. AAP showed minimal off-target activation by other ALDH isoforms or oxidative stress. In vivo, AAP suppressed tumor growth in two non-small cell lung cancer (NSCLC) models. In the first, intratumoral delivery into established tumors confirmed efficacy and ALDH1A1 dependence. In the second, liposomal AAP enabled intravenous delivery to early stage lesions with limited vascularization where treatment remained effective. These findings establish d-PeT suppression of intersystem crossing as an effective chemical biology strategy for enzyme-activated photodynamic therapy.

Small molecules that bind specific sites in RNAs hold promise for altering RNA function, manipulating gene expression, and expanding the scope of druggable targets beyond proteins. Identifying binding sites in RNA that can engage ligands with good physicochemical properties remains a significant challenge. fpocketR is a software and framework for identifying, characterizing, and visualizing ligand-binding sites in RNA. fpocketR was optimized, through a comprehensive analysis of currently available RNA-ligand complexes, to identify pockets in RNAs able to bind small molecules possessing favorable properties, generally termed drug-like. Here, we demonstrate multiple, complex, uses of fpocketR to analyze RNA-ligand interactions and novel pockets in small and large RNAs, to assess ensembles of RNA structure models, to identify pockets in dynamic RNA systems, and to evaluate the shapes of RNA pockets. fpocketR performs best with RNA structures visualized at atomistic resolution but also provides useful information with lower resolution structures and computational models. fpocketR is a powerful, ligand-agnostic tool for discovery and analysis of targetable pockets in RNA molecules.

Several luciferases have been developed for imaging and biosensing, and the collection continues to grow as new applications are pursued. The current workflow for luciferase optimization, while successful, remains laborious and inefficient. Mutant libraries are generated in vitro and screened, “winning” mutants are picked by hand, and the isolated sequences are subjected to additional rounds of mutagenesis and screening. Here, we present a streamlined platform for luciferase engineering that removes the need for manual library generation during each cycle. We purposed an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of a well-known luciferase (GeNL). Short cycles of culturing and screening were sufficient to evolve the enzyme, with no repetitive manual library generation necessary. New GeNL variants were identified that exhibit improved light outputs with a noncognate and inexpensive luciferin. We further characterized the novel luciferases in cell models. Collectively this work establishes OrthoRep and continuous hypermutation as a viable method to engineer luciferases, and sets the stage for more rapid development of bioluminescent reporters.

Creating artificial organelles that sequester and release specific proteins in response to a small molecule in mammalian cells is an attractive approach for regulating protein function. In this work, by combining phase-separated condensates formed by the tandem fusion of two oligomeric proteins with a trimethoprim (TMP)-responsive nanobody switch for GFP (^GFPLAMA; ligand-modulated antibody fragment), we developed a synthetic condensate system that initially sequesters GFP-tagged proteins within condensates and rapidly releases them into the cytoplasm upon TMP treatment. The released proteins can then be resequestered by washing out the TMP. This system enabled user-defined, temporal, rapid, and reversible control of cellular processes, including membrane ruffling mediated by exogenously expressed GFP-Vav2 and modulation of the cellular localization of endogenous ERK2-GFP generated by genome knock-in. Our results highlight the utility of the ^GFPLAMA-based synthetic condensate platform as a novel, chemically switchable tool for regulating protein function through controlled protein sequestration and release in mammalian cells.

Protein core-fucosylation plays a crucial role in regulating cell surface protein functions and is involved in various biological processes, including cell signaling, immune response, and cancer progression. However, core-fucosylation (CF) poses particular challenges in identification and characterization due to its low abundance and the high heterogeneity of N-glycosylation. To overcome these challenges, this study systematically investigated the oxidation mechanisms of core-fucose and developed a selective enrichment strategy that combines chemical oxidation with glycan truncation for cell surface core-fucosylation characterization. Specifically, sodium periodate was employed to selectively oxidize cell surface glycans. In combination with the endoglycosidases Endo M and Endo F3, which possessed complementary substrate specificity and broad tolerance, this approach efficiently truncated N-glycans, leaving core-fucosylated glycopeptides bearing aldehyde tags. Utilizing reversible hydrazide chemistry, core-fucosylated glycopeptides were selectively enriched. The developed strategy was applied to profile cell surface core-fucosylated protein in HeLa cells, which yielded 74 core-fucosylated glycopeptides corresponding to 21 key cell surface drug targets, thereby validating the efficacy of this approach. Collectively, this study systematically investigated the mechanism of core-fucosylation oxidation and developed a new technical tool for studying cell surface protein core-fucosylation.

Most antibiotics used in human medicine inhibit the biosynthesis of peptidoglycan (PG), which is a vital component of the bacterial cell wall. However, the rapid rise in antimicrobial resistance (AMR) represents a major global health threat. Gaining deeper insight into PG metabolism is a crucial step in combating AMR, an objective achievable only through the use of well-defined molecular probes. In this study, we developed a chemo-enzymatic method to synthesize PG oligosaccharides using a lysozyme-derived glycosynthase. To prevent unwanted polymerization and enable precise control over the product size, a glycosyl fluoride donor bearing a terminal galactosyl unit was designed. The trisaccharide Gal-β-(1→4)-GlcNAc-β-(1→4)-1,6-anhydro-MurNAc was biosynthesized in metabolically engineered Escherichia coli cells and subsequently fluorinated at the reducing end via chemical modification. Successive glycosylation and degalactosylation steps using this donor, starting from two disaccharide acceptors, led to the synthesis of PG tetra-, hexa-, and octasaccharides, with a 60–70% yield at each cycle. These compounds were used to probe the specificity of E. coli DedD, a SPOR-domain-containing protein involved in bacterial cell division. NMR spectroscopy and NMR-restraint-driven molecular dynamics provide new insights into the role of this protein relative to those of other E. coli SPOR domain-containing proteins.

Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein-protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative strategies for efficient cellular delivery. In this study, we used the SPRY domain-containing SOCS box protein 2 (SPSB2) E3 ligase as a model system to develop TE strategies in vitro and in cellulo using polar degron-based peptides. By conjugating various polycationic cell-penetrating peptides (CPPs) to the degron sequence, we present a study demonstrating cellular delivery. We obtained a high-resolution crystal structure and used various biophysical techniques to assess the influence of each modification, while confocal microscopy and BRET-based assays confirmed successful cellular delivery as well as potent TE.