ChemBioChem最新文献

I-motifs are non-canonical DNA structures with recognized biological significance and a proven utility in material engineering. Consequently, understanding and control of i-motif properties is essential to sustain progress across both disciplines. In this work, we systematically investigate how proximity to the most common form of DNA, a double-stranded duplex, influences the thermodynamic and kinetic properties of adjacent i-motifs. We demonstrate that double-stranded stems in i-motif loops promote kinetic trapping of very stable and persistent partially folded conformations. Further, we investigate pathways toward rational control over a folding topology makeup.

Highly selective C-H functionalization remains an ongoing challenge in organic synthetic methodologies. Biocatalysts are robust tools for achieving these difficult chemical transformations. Biocatalyst engineering has often required directed evolution or structure-based rational design campaigns to improve their activities. In recent years, machine learning has been integrated into these workflows to improve the discovery of beneficial enzyme variants. In this work, we combine a structure-based machine-learning algorithm with classical molecular dynamics simulations to down select mutations for rational design of a non-heme iron-dependent lysine dioxygenase, LDO. This approach consistently resulted in functional LDO mutants and circumvents the need for extensive study of mutational activity before-hand. Our rationally designed single mutants purified with up to 2-fold higher yields than WT and displayed higher total turnover numbers (TTN). Combining five such single mutations into a pentamutant variant, LPNYI LDO, leads to a 40% improvement in the TTN (218±3) as compared to WT LDO (TTN = 160±2). Overall, this work offers a low-barrier approach for those seeking to synergize machine learning algorithms with pre-existing protein engineering strategies.

Aptamers are often employed as molecular recognition elements in the development of different types of biosensors. Many of these biosensors take advantage of the aptamer having a ligand-induced structure-formation binding mechanism. However, this binding mechanism is poorly understood. Here we use isothermal titration calorimetry, circular dichroism spectroscopy and NMR spectroscopy to study the binding and ligand-induced structural change exhibited by a dopamine-binding DNA aptamer. We analysed a series of aptamers where we shorten the terminal stem that contains the 5' and 3' termini of the aptamer sequence. All aptamers bind dopamine in an enthalpically driven process coupled with an unfavorable entropy. A general trend of the aptamer having a weaker binding affinity is observed as the terminal stem is shortened. For all aptamers studied, numerous signals appear in the imino region of the ¹H NMR spectrum indicating that new structure forms with ligand binding. However, it is only when this region of structure formation in the aptamer is brought close to the sensor surface that we obtain a functional electrochemical aptamer-based biosensor.

Enterococcus faecalis (E. faecalis) is commonly occurring pathogen                                                          associated with nosocomial infections. Infections are difficult to treat because of their multidrug-resistant (MDR) nature and their tendency to form biofilms. Therefore, it is essential to find alternative medicinal approaches of treatment. In this regard, targeting an important  protein for drug development can be an alternative approach. Sortase A (SrtA) is an important enzyme involved in anchoring cell surface-exposed proteins to the cell envelope. SrtA is present in Gram-positive bacteria which catalyses the attachment of several virulence factors and other proteins to the cell membrane. It is involved in bacterial pathogenesis, therefore, it's a promising drug target for the development of anti-microbial drugs targeting cell adhesion, evasion, and biofilm development. To identify SrtA potential inhibitors, we have purified E. faecalis Sortase A (EfSrtAΔN59).  Structural studies along with molecular docking of protein with selected ligand molecules were done and confirmed by MD simulation experiments. We have also performed functional validation of these compounds on bacterial growth, anti-biofilm assays and inhibition assay of selected ligands were also done against E. faecalis individually and in synergistic combinations.  Results indicated that both Eugenol and Ferulic acid bind to EfSrtAΔN59 with significant interactions and show promising results.

This study reports the synthesis of a new series of pyrazole-isoxazolines, at very good yields, from the cyclocondensation reaction of pyrazole-enaminones with hydroxylamine hydrochloride. Dehydration of the pyrazole-isoxazolines furnished another new series of the respective pyrazole-isoxazoles, at excellent yields. Both series of the obtained compounds were screened for antimycobacterial activity, and compounds 4 f and 5 c showed significant inhibition of bacterial growth with a time- and concentration-dependent bactericidal effect. Cytotoxicity tests in VERO cell line did not indicate toxicity of compounds 4 f and 5 c regarding cellular prediction, NO production or dsDNA release. However, both compounds were associated with an increase in total ROS levels, providing induction of oxidative stress, but without compromising cellular targets. These results highlight compounds 4 f and 5 c as promising candidates for antimycobacterial treatment with a favorable safety profile.

Proteolysis-targeting chimera (PROTAC) has emerged as an attractive therapeutic modality in drug discovery. PROTACs are bifunctional molecules that effectively bridge proteins of interest (POIs) with E3 ubiquitin ligases, such that, the target proteins are tagged with ubiquitin and subsequently degraded via the proteasome. Despite significant progress in the field of targeted protein degradation (TPD), the application of conventional PROTAC degraders still faces significant challenges, including systemic toxicity induced by non-tissue-specific targeting. To address this issue, a variety of smart PROTACs that can be activated by specific stimuli, have been developed for achieving conditional and spatiotemporal modulation of protein levels. Here, on the basis of our contributions, we overview recent advances of smart PROTACs, including tumor microenvironment-, photo-, and X-ray radiation-responsive PROTACs, that enable controllable TPD. The design strategy, case studies, potential applications and challenges will be focused on.

Streptococcus pneumoniae is a bacterial pathogen causing diseases as severe as pneumonia, sepsis and meningitis. Most commercial pneumococcal conjugate vaccines contain the 7F serotype, which is epidemiologically relevant and highly invasive. This serotype contains an O-acetyl group at the internal L-rhamnose of its polysaccharide repeating unit. Herein we report on the role of the O-acetyl moiety of 7F polysaccharide in both antigen recognition and the induction of a protective antibody response against 7F. Fully and partially de-O-acetylated 7F polysaccharides were chemically prepared and compared with the O-acetylated counterpart in their antigenicity and immunogenicity of their tetanus toxoid glycoconjugates. These comparative studies showed a slight but consistent decrease in the antigenicity for the fully de-O-acetylated polysaccharide, but not for the partly de-O-acetylated variant. The glycoconjugates derived from the O-acetylated and the fully de-O-acetylated polysaccharides had similar sizes and polysaccharide-to-protein ratio, and all proved both to be immunogenic and induce opsonophagocytic responses in mice. Nevertheless, the immune response elicited by the O-acetylated glycoconjugate was better in both quantity and quality, proving that the O-acetyl group is not strictly necessary but also not irrelevant for the antigenicity and immunogenicity of the 7F serotype polysaccharide and its glycoconjugates.

Over the last four decades, research on DNA as a functional material has primarily focused on its predictable conformation and programmable interaction. However, its low energy consumption, high responsiveness and sensitivity also make it ideal for designing specific signaling pathways, and enabling the development of molecular computers. This review mainly discusses recent advancements in the utilization of DNA nanotechnology for molecular computer, encompassing applications in storage, cryptography and logic circuits. It elucidates the challenges encountered in the application process and presents solutions exemplified by representative works. Lastly, it delineates the challenges and opportunities within this filed.

The immunoproteasome (iCP) has gained significant interest in recent years as it has been discovered to be significantly expressed under inflammatory conditions, as well as playing significant roles in several diseases, such as autoimmune disorders, viral infection, and cancer. Selective inhibitors have been generated as a method to overcome the off-target effects of current proteasome inhibitor therapeutics. However, selective probes that allow for monitoring this protein complex remain limited, hindering our understanding of the iCP. Current probes are non-selective, not commercially available, or require difficult synthesis. Here, we describe the modular synthesis and application of an iCP-selective probe. The modular nature of the synthetic strategy can enable the incorporation of different fluorophores and covalent warheads, demonstrating the versatility of this probe.

Modulation of membrane properties via photoswitchable lipids has attracted attention due to the unparalleled spatiotemporal resolution of their functional control. Beside lipids, detergents are another prominent class for selective membrane perturbations owing to their ease of handling and spontaneous insertion in lipid bilayers. Herein, we describe the synthesis and characterization of three classes of visible light-sensitive surfactants with various azobenzene tail chain lengths. The photoswitchable detergents show water-solubility and micellization as well as undergo reversible isomerization under blue-/green light illumination. We demonstrate that the light-induced structural change of azobenzene can lead to vesicle rupture, making them a tool for controlled cargo release from vehicles. Via spontaneous insertion into the plasma membrane of mammalian cells transiently transfected with MscL, we used the azobenzene-derived detergents to optically activate the transmembrane mechanosensitive channel. This led to the rapid controlled uptake of membrane-impermeable molecules. Since detergents are extensively used in biochemistry and biotechnology, we propose that the photoswitchable detergents will be useful tools for the spatiotemporal modulation of membrane properties. Additionally, our work provides a design strategy for new detergents in membrane (protein) research.