RSC Chemical Biology最新文献

The immunoproteasome (iCP) can be expressed under inflammatory conditions, such as exposure to interferon-gamma (IFN-γ), that alerts the cell to begin generating iCP preferentially over the standard proteasome (sCP). With the iCP becoming a widely targeted isoform in a variety of diseases, there is a need to understand its activity and expression in cells and in vivo. Activity-based probes for the iCP have been developed but their application has been limited due to their difficult synthesis and cannot be used in tissues or whole animals. Our lab has previously demonstrated we can monitor iCP activity using a 4-mer peptide linked to a fluorophore and a peptoid. This was utilized in the development of the first cell-permeable iCP activity-based probe that did not include a covalent reactive moiety. Here, we demonstrate that this same peptide recognition sequence can be appended to aminoluciferin, caging it, until its interaction with the iCP. This probe should be applicable to monitor iCP activity in animal models where tumor or other tissue has been engineered to produce luciferase. We anticipate it could also be applied to observe iCP activity as tumors are formed in vivo.

The pressing climate change issues have intensified the need for a rapid transition towards a bio-based circular carbon economy. Harnessing acetogenic bacteria as biocatalysts to convert C1 compounds such as CO₂, CO, formate, or methanol into value-added multicarbon chemicals is a promising solution for both carbon capture and utilization, enabling sustainable and green chemical production. Recent advances in the metabolic engineering of acetogens have expanded the range of commodity chemicals and biofuels produced from C1 compounds. However, producing energy-demanding high-value chemicals on an industrial scale from C1 substrates remains challenging because of the inherent energetic limitations of acetogenic bacteria. Therefore, overcoming this hurdle is necessary to scale up the acetogenic C1 conversion process and realize a circular carbon economy. This review overviews the acetogenic bacteria and their potential as sustainable and green chemical production platforms. Recent efforts to address these challenges have focused on enhancing the ATP and redox availability of acetogens to improve their energetics and conversion performances. Furthermore, promising technologies that leverage low-cost, sustainable energy sources such as electricity and light are discussed to improve the sustainability of the overall process. Finally, we review emerging technologies that accelerate the development of high-performance acetogenic bacteria suitable for industrial-scale production and address the economic sustainability of acetogenic C1 conversion. Overall, harnessing acetogenic bacteria for C1 valorization offers a promising route toward sustainable and green chemical production, aligning with the circular economy concept.

Photodynamic therapy (PDT) is an approved cancer treatment modality. Despite its high efficiency, PDT is limited in terms of specificity and by the poor solubility of the rather lipophilic photosensitizers (PSs). In order to alleviate these limitations, PSs can be conjugated to oligonucleotides. However, most conjugation methods often involve complex organic synthesis and result in the appendage of single modifications at the 3′/5′ termini of oligonucleotides. Here, we have investigated the possibility of bioconjugating a range of known PSs by polymerase-mediated synthesis. We have prepared a range of modified nucleoside triphosphates by different conjugation methods and investigated the substrate tolerance of these nucleotides for template-dependent and -independent DNA polymerases. This method represents a mild and versatile approach for the conjugation of single or multiple PSs onto oligonucleotides and can be useful to further improve the efficiency of the PDT treatment.

Correction for ‘Virtual screening, identification and in vitro validation of small molecule GDP-mannose dehydrogenase inhibitors’ by Jonathan P. Dolan et al., RSC Chem. Biol., 2023, 4, 865–870, https://doi.org/10.1039/D3CB00126A.

Miltefosine (MLT) is an alkylphosphocholine with clinical success as an anticancer and antiparasitic drug. Although the mechanism of action of MLT is highly debated, the interaction of MLT with the membrane, specifically lipid rafts of eukaryotes, is well-documented. Recent reports suggest MLT impacts the functional membrane microdomains in bacteria – regions of the membrane structurally and functionally similar to lipid rafts. There have been conflicting reports, however, as to whether MLT impacts the overall fluidity of cellular plasma membranes. Here, we apply steady-state fluorescence techniques, generalized polarization of laurdan and anisotropy of diphenylhexatriene, to discern how MLT impacts the global ordering and lipid packing of Staphylococcus aureus membranes. Additionally, we investigate how the transport of a range of small molecules is impacted by MLT for S. aureus and Bacillus subtilis by employing time-resolved second harmonic scattering. Overall, we observe MLT does not have an influence on the overall ordering and packing of S. aureus membranes. Additionally, we show that the transport of small molecules across the membrane can be significantly altered by MLT – although this is not the case for all molecules studied. The results presented here illustrate the potential use of MLT as an adjuvant to assist in the delivery of drug molecules in bacteria.

The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization and synthesis of deletion mutants revealed a critical cycle size of cL3 essential for C1 binding and efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of “Peptactins” – peptide-based activators of complement – and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration.

Affinity-based probes are valuable tools for detecting binding interactions between small molecules and proteins in complex biological environments. Metalloproteins are a class of therapeutically significant biomolecules which bind metal ions as part of key structural or catalytic domains and are compelling targets for study. However, there is currently a limited range of chemical tools suitable for profiling the metalloproteome. Here, we describe the preparation and application of a novel, photoactivatable affinity-based probe for detection of a subset of previously challenging to engage metalloproteins. The probe, bearing an 8-mercaptoquinoline metal chelator, was anticipated to engage several zinc metalloproteins, including the 26S-proteasome subunit Rpn11. Upon translation of the labelling experiment to mammalian cell lysate and live cell experiments, proteomic analysis revealed that several metalloproteins were competitively enriched. The diazirine probe SMK-24 was found to effectively enrich multiple components of the minichromosome maintenance complex, a zinc metalloprotein assembly with helicase activity essential to DNA replication. Cell cycle analysis experiments revealed that HEK293 cells treated with SMK-24 experienced stalling in G0/G1 phase, consistent with inactivation of the DNA helicase complex. This work represents an important contribution to the library of cell-permeable chemical tools for studying a collection of metalloproteins for which no previous probe existed.

Arabinogalactan proteins (AGPs) are plant-specific glycoproteins involved in cellular mechanics and signal transduction. There has been major progress in understanding the structure, synthesis, and molecular functions of their carbohydrate chains; however, the mechanisms by which they function as signalling molecules remain unclear. Here, methyl-glucuronosyl arabinogalactan (AMOR; Me-GlcA-β(1,6)-Gal), a disaccharide structure at the end of AGP carbohydrate chains, was oligomerised via chemical synthesis. The biological activity of AMOR oligomers was enhanced via clustering of the carbohydrate chains. Furthermore, AMOR oligomers yielded a pollen tube morphology (i.e., callose plug formation) similar to that when cultured with native AMOR, suggesting it may be functionally similar to native AMOR.

Pharmacokinetic properties and duration of therapeutic action of a pharmaceutical agent can be significantly extended through the combination of two distinct strategies aimed at increasing plasma half-life: fatty acid acylation and Fc-conjugation. Using insulin as a case study, we demonstrate that a doubly protracted insulin analog produces a substantial prolongation of pharmacodynamic effect to lower blood glucose in STZ-treated mice when compared to the Fc-only counterparts. This enhancement is further corroborated by direct pharmacokinetic measurements in rat and dog models, demonstrating the potential for once-monthly insulin therapy. The results suggest that this approach might have broad application across a diverse spectrum of peptide- and protein-based therapeutics.

Covalent protease inhibitors serve as valuable tools for modulating protease activity and are essential for investigating the functions of protease targets. These inhibitors typically consist of a recognition motif and a covalently reactive electrophile. Substrate peptides, featuring residues capable of fitting into the substrate pockets of proteases, undergo chemical modification at the carbonyl carbon of the P1 residue with an electrophile and have been widely applied in the development of covalent inhibitors. In this study, we utilized a DNA-encoded peptide library to replicate peptide binder sequences and introduced a vinyl sulfone warhead at the C-termini to construct the DNA-encoded peptide covalent inhibitor library (DEPCIL) for targeting cysteine proteases. Screening results toward 3CL protease demonstrated the efficacy of this library, not only in identifying protease inhibitors, but also in discovering amino acids that can conform to aligned protease pockets. The identified peptide sequences provide valuable insight into the amino acid preferences within substrate binding pockets, and our novel technology is indicative of the potential for similar strategies to discover covalent inhibitors and profile binding preferences of other proteases.