RSC Chemical Biology最新文献

The fidelity of biosynthesis by modular polyketide synthases (PKSs) depends on specific moderate affinity interactions between successive polypeptide subunits mediated by docking domains (DDs). These sequence elements are notably portable, allowing their transplantation into alternative biosynthetic and metabolic contexts. Herein, we use integrative structural biology to characterize a pair of DDs from the toblerol trans-AT PKS. Both are intrinsically disordered regions (IDRs) that fold into a 3 α-helix docking complex of unprecedented topology. The C-terminal docking domain (^CDD) resembles the 4 α-helix type (4HB) ^CDDs, which shows that the same type of DD can be redeployed to form complexes of distinct geometry. By carefully re-examining known DD structures, we further extend this observation to type 2 docking domains, establishing previously unsuspected structural relations between DD types. Taken together, these data illustrate the plasticity of α-helical DDs, which allow the formation of a diverse topological spectrum of docked complexes. The newly identified DDs should also find utility in modular PKS genetic engineering.

Pretargeted PET imaging using bioorthogonal chemistry is a leading strategy for the tracking of long-circulating agents such as antibodies and nanoparticle-drug delivery systems with short-lived isotopes. Here, we report the synthesis, characterisation and in vitro/vivo evaluation of a new ⁶⁸Ga-based radiotracer [⁶⁸Ga]Ga-THP-Tetrazine ([⁶⁸Ga]Ga-THP-Tz) for bioorthogonal click radiochemistry and in vivo labelling of agents with slow pharmacokinetics. THP-tetrazine (THP-Tz) can be radiolabelled to give [^68/67Ga]Ga-THP-Tz at room temperature in less than 15 minutes with excellent radiochemical stability in vitro and in vivo. [⁶⁸Ga]Ga-THP-Tz was tested in vitro and in vivo for pretargeted imaging of stealth PEGylated liposomes, chosen as a leading clinically-approved platform of nanoparticle-based drug delivery, and for their known long-circulating properties. To achieve this, PEGylated liposomes were functionalised with a synthesised transcyclooctene (TCO) modified phospholipid. Radiolabelling of TCO-PEG-liposomes with [^68/67Ga]Ga-THP-Tz was demonstrated in vitro in human serum, and in vivo using both healthy mice and in a syngeneic cancer murine model (WEHI-164 fibrosarcoma). Interestingly in vivo data revealed that [⁶⁸Ga]Ga-THP-Tz was able to in vivo radiolabel liposomes present in the liver and spleen, and not those in the blood pool or in the tumour. Overall, these results demonstrate the potential of [⁶⁸Ga]Ga-THP-Tz for pretargeted imaging/therapy but also some unexpected limitations of this system.

Hydrogen sulfide (H₂S) is an endogenous gasotransmitter that signals via persulfidation. There is evidence that the cysteine residues of certain zinc finger (ZF) proteins, a common type of cysteine rich protein, are modified to persulfides by H₂S. To determine how frequently ZF persulfidation occurs in cells and identify the types of ZFs that are persulfidated, persulfide specific proteomics data were evaluated. 22 datasets from 16 studies were analyzed via a meta-analysis approach. Persulfidated ZFs were identified in a range of eukaryotic species, including Homo sapiens, Mus musculus, Rattus norvegicus, Arabidopsis thaliana, and Emiliania huxley (single-celled phytoplankton). The types of ZFs identified for each species encompassed all three common ZF ligand sets (4-cysteine, 3-cysteine-1-histidine, and 2-cysteine-2-hisitidine), indicating that persulfidation of ZFs is broad. Overlap analysis between different species identified several common ZFs. GO and KEGG analysis identified pathway enrichment for ubiquitin-dependent protein catabolic process and viral carcinogenesis. These collective findings support ZF persulfidation as a wide-ranging PTM that impacts all classes of ZFs.

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Usnic acid is a natural product with versatile biological activities against various organisms. Here, we utilise a chemical proteomic strategy to gain insights into its target scope in bacterial and human cells. First, we excluded DNA binding as a major reason for its antibacterial activity, and second, we commenced with target profiling, which unravelled several metal cofactor-dependent enzymes in both species indicating a polypharmacological mode of action. Interestingly, our synthetic studies revealed a selectivity switch at usnic acid, which maintains antibacterial activity but lacks strong cytotoxic effects.

Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells that contain various cellular components such as proteins, nucleic acids, and lipids from the parent cell. EVs are abundant in body fluids and can serve as circulating biomarkers for a variety of diseases or as a regulator of various biological processes. Considering these characteristics of EVs, analysis of the EV cargo has been spotlighted for disease diagnosis or to understand biological processes in biomedical research. Over the past decade, technologies for rapid and sensitive analysis of EVs in biofluids have evolved, but detection and isolation of targeted EVs in complex body fluids is still challenging due to the unique physical and biological properties of EVs. Recent advances in chemical biology provide new opportunities for efficient profiling of the molecular contents of EVs. A myriad of chemical biology tools have been harnessed to enhance the analytical performance of conventional assays for better understanding of EV biology. In this review, we will discuss the improvements that have been achieved using chemical biology tools.

Cyclotides are a diverse class of plant-derived cyclic, disulfide-rich peptides with a unique cyclic cystine knot topology. Their remarkable structural stability and resistance to proteolytic degradation can lead to improved pharmacokinetics and oral activity as well as selectivity and high enzymatic stability. Thus, cyclotides have emerged as powerful scaffold molecules for designing peptide-based therapeutics. The chemical engineering of cyclotides has generated novel peptide ligands of G protein-coupled receptors (GPCRs), today's most exploited drug targets. However key challenges potentially limit the widespread use of cyclotides in molecular grafting applications. Folding of cyclotides containing bioactive epitopes remains a major bottleneck in cyclotide synthesis. Here we present a modular ‘plug and play’ approach that effectively bypasses problems associated with the oxidative folding of cyclotides. By grafting onto a pre-formed acyclic cyclotide-like scaffold we show that difficult-to-graft sequences can be easily obtained and can target GPCRs with nanomolar affinities and potencies. We further show the suitability of this new method to graft other complex epitopes including structures with additional disulfide bonds that are not readily available via currently employed chemical methods, thus fully unlocking cyclotides to be used in drug design applications.

Fluorescent reporters that visualize phosphatidylinositol 4-phosphate (PI4P) in living cells are indispensable to elucidate the roles of this fundamental lipid in cell physiology. However, currently available PI4P reporters have limitations, such as Golgi-biased localization and low detection sensitivity. Here, we present a series of fluorescent PI4P reporters based on the pleckstrin homology (PH) domain of oxysterol-binding protein-related protein 9 (ORP9). We show that the green fluorescent protein AcGFP1-tagged ORP9-PH domain can be used as a fluorescent PI4P reporter to detect cellular PI4P across its wide distribution at multiple cellular locations, including the plasma membrane (PM), Golgi, endosomes, and lysosomes with high specificity and contrast. We also developed blue, red, and near-infrared fluorescent PI4P reporters suitable for multicolor fluorescence imaging experiments. Finally, we demonstrate the utility of the ORP9-PH domain-based reporter to visualize dynamic changes in the PI4P distribution and level in living cells upon synthetic ER–PM membrane contact manipulation and GPCR stimulation. This work offers a new set of genetically encoded fluorescent PI4P reporters that are practically useful for the study of PI4P biology.

Unnatural base pairs (UBPs) augment the chemical diversity of artificial nucleic acids and can thus enable the generation of new aptamers and catalytic nucleic acids by in vitro selection. However, owing to a lack of methodologies, the reverse transcription of UBPs, a key step in RNA aptamer selection, has not been sufficiently characterized. Here, we present a series of versatile assays to investigate the reverse transcription of the TPT3:NaM base pair as a representative for hydrophobic unnatural base pairs. We determine the fidelity and retention of the UBP for four different reverse transcriptases (RT) in the context of RNA in vitro evolution. The retention of the TPT3:NaM pair during the RNA in vitro selection process was investigated using a novel click-chemistry based electromobility shift assay. Real-time monitoring of reverse transcription kinetics revealed considerable differences in polymerase activity processing the TPT3:NaM base pair. Our findings identified SuperScript IV RT as the most efficient RT for processing the TPT3:NaM pair. Our approach can be applied universally to study newly developed UBPs, not only at the reverse transcription level, but also during PCR and in vitro transcription.

The account attempts to substantiate the hypothesis that, from an evolutionary perspective, the coenzyme couple pyridoxal phosphate and pyridoxamine phosphate preceded the coenzyme thiamine pyrophosphate and acted as its less efficient chemical analogue in some form of early metabolism. The analysis combines mechanism-based chemical reactivity with biosynthetic arguments and provides evidence that vestiges of “TPP-like reactivity” are still found for PLP today. From these thoughts, conclusions can be drawn about the key elements of a primordial form of metabolism, which includes the citric acid cycle, amino acid biosynthesis and the pentose phosphate pathway.