Mechanistic Studies on Trichoacorenol Synthase from Amycolatopsis benzoatilytica

Isotopic labeling experiments performed with a newly identified bacterial trichoacorenol synthase established a 1,5-hydride shift occurring in the cyclization mechanism. During EI-MS analysis, major fragments of the sesquiterpenoid were shown to arise via cryptic hydrogen movements. Therefore, the interpretation of earlier results regarding the cyclization mechanism obtained by feeding experiments in Trichoderma is revised.     

A Click-Chemistry-Based Enrichable Crosslinker for Structural and Protein Interaction Analysis by Mass Spectrometry

Mass spectrometry is the method of choice for the characterisation of proteomes. Most proteins operate in protein complexes, in which their close association modulates their function. However, with standard MS analysis, information on protein–protein interactions is lost and no structural information is retained. To gain structural and interactome data, new crosslinking reagents are needed that freeze inter- and intramolecular interactions. Herein, the development of a new reagent, which has several features that enable highly sensitive crosslinking MS, is reported. The reagent enables enrichment of crosslinked peptides from the majority of background peptides to facilitate efficient detection of low-abundant crosslinked peptides. Due to the special cleavable properties, the reagent can be used for MS² and potentially for MS³ experiments. Thus, the new crosslinking reagent, in combination with high-end MS, should enable sensitive analysis of interactomes, which will help researchers to obtain important insights into cellular states in health and diseases.     

Small-Molecule Inhibition of Glucose Transporters GLUT-1–4

Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose-dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform-selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.     

A Tetrahedral Boronic Acid Diester Formed by an Unnatural Amino Acid in the Ligand Pocket of an Engineered Lipocalin

Boronic acids have long been known to form cyclic diesters with cis-diol compounds, including many carbohydrates. This phenomenon was previously exploited to create an artificial lectin by incorporating p-borono-l -phenylalanine (Bpa) into the ligand pocket of an engineered lipocalin, resulting in a so-called Borocalin. Here we describe the X-ray analysis of its covalent complex with 4-nitrocatechol as a high-affinity model ligand. As expected, the crystal structure reveals the formation of a cyclic diester between the biosynthetic boronate side chain and the two ortho-hydroxy substituents of the benzene ring. Interestingly, the boron also has a hydroxide ion associated, despite an only moderately basic pH 8.5 in the crystallization buffer. The complex is stabilized by a polar contact to the side chain of Asn134 within the ligand pocket, thus validating the functional design of the Borocalin as an artificial sugar-binding protein. Our structural analysis demonstrates how a boronate can form a thermodynamically stable diester with a vicinal diol in a tetrahedral configuration in aqueous solution near physiological pH. Moreover, our data provide a basis for the further engineering of the Borocalin with the goal of specific recognition of biologically relevant glycans.     

Repurposing the Pummerer Rearrangement: Determination of Methionine Sulfoxides in Peptides

The reversible oxidation of methionine residues in proteins has emerged as a biologically important post-translational modification. However, detection and quantitation of methionine sulfoxide in proteins is difficult. Our aim is to develop a method for specifically derivatizing methionine sulfoxide residues. We report a Pummerer rearrangement of methionine sulfoxide treated sequentially with trimethylsilyl chloride and then 2-mercaptoimidazole or pyridine-2-thiol to produce a dithioacetal product. This derivative is stable to standard mass spectrometry conditions, and its formation identified oxidized methionine residues. The scope and requirements of dithioacetal formation are reported for methionine sulfoxide and model substrates. The reaction intermediates have been investigated by computational techniques and by ¹³C NMR spectroscopy. These provide evidence for an α-chlorinated intermediate. The derivatization allows for detection and quantitation of methionine sulfoxide in proteins by mass spectrometry and potentially by immunochemical methods.     

Influence of Methanol on Prion Reduction during High Temperature and High Pressure Oleochemical Processes

Prion‐reduction in standard biodiesel processes is caused by acidic and alkaline conditions. In the alternative RepCat biodiesel process, efficient prion‐reduction can be achieved by high‐temperature/pressure. However, a possible increase in thermostability of prions in the presence of methanol during these conditions has not previously been investigated, and is verified in this work. Samples are spiked with prions, treated with methanol, and incubated at 220 °C at 80 bar for 30 min. No traces of protease‐resistant prion protein (as proxy for prions) are detected in tallow or glycerine (as the final by‐product) after treatment. Serial dilutions of spiked prions show at least 6 log₁₀ prion reduction. More importantly, similar effects are detected using milder conditions of 200 °C at 70 bar for 15 min, representing the worst‐case conditions of the process. In conclusion, this study shows that methanol does not increase the thermostability of prions and the RepCat process can efficiently eliminate prions and is therefore safe for the usage of category 1 tallow. Practical Applications: The study further supports the applicability of RepCat process in reducing prion‐contamination in the presence of methanol. Furthermore, the conditions leads to a considerable reduction of prions in glycerine obtained as by‐product.     

Homogeneous and Universal Detection of Various Targets with a Dual-Step Transduced Toehold Switch Sensor

Toehold switch sensors represent a class of new advances that allow specific targets to trigger in situ expression of a protein reporter. Although they offer unique advantages of a label-free nature and high portability, they generally require repeated sequence design, high expenditure, and laborious optimization of toehold switch sequences according to different targets. To simplify the sensing process further and to improve its practicability, we innovatively introduce a dual-step pre-transduction upon traditional toehold switch sensor. Through two successive toehold-mediated strand-displacement reactions that are initiated, respectively, by a linear and an associative trigger, different DNAs, RNAs, or ligands of functional nucleic acids can be generally transduced into the input of one high-performance toehold switch sensor. This advance greatly increases the target range. Furthermore, the whole process is signal-on, homogeneous, and free of any requirements for complicated operations such as probe labeling, separation, and reconstruction of the toehold switch, being promising and practical even in portable or point-of-care assays.     

Nitroreductase-Mediated Release of Inhibitors of Lysine-Specific Demethylase 1 (LSD1) from Prodrugs in Transfected Acute Myeloid Leukaemia Cells

Lysine-specific demethylase 1 (LSD1) has evolved as a promising therapeutic target for cancer treatment, especially in acute myeloid leukaemia (AML). To approach the challenge of site-specific LSD1 inhibition, we developed an enzyme-prodrug system with the bacterial nitroreductase NfsB (NTR) that was expressed in the virally transfected AML cell line THP1-NTR⁺. The cellular activity of the NTR was proven with a new luminescent NTR probe. We synthesised a diverse set of nitroaromatic prodrugs that by design do not affect LSD1 and are reduced by the NTR to release an active LSD1 inhibitor. The emerging side products were differentially analysed using negative controls, thereby revealing cytotoxic effects. The 2-nitroimidazolyl prodrug of a potent LSD1 inhibitor emerged as one of the best prodrug candidates with a pronounced selectivity window between wild-type and transfected THP1 cells. Our prodrugs are selectively activated and release the LSD1 inhibitor locally, proving their suitability for future targeting approaches.     

Bridging the Gap: Plant-Endophyte Interactions as a Roadmap to Understanding Small-Molecule Communication in Marine Microbiomes

Probing the composition of the microbiome and its association with health and disease states is more accessible than ever due to the rise of affordable sequencing technology. Despite advances in our ability to identify members of symbiont communities, untangling the chemical signaling that they use to communicate with host organisms remains challenging. In order to gain a greater mechanistic understanding of how the microbiome impacts health, and how chemical ecology can be leveraged to advance small-molecule drug discovery from microorganisms, the principals governing communication between host and symbiont must be elucidated. Herein, we review common modes of interkingdom small-molecule communication in terrestrial and marine environments, describe the differences between these environments, and detail the advantages and disadvantages for studies focused on the marine environment. Finally, we propose the use of plant-endophyte interactions as a stepping stone to a greater understanding of similar interactions in marine invertebrates, and ultimately in humans.     

Metallic Nanoparticle-Enabled Sensing of a Drug-of-Abuse: An Attempt at Forensic Application

γ-Hydroxybutyric acid (GHB) functions as a depressant on the central nerve system and serves as a pharmaceutical agent in the treatment of narcolepsy and alcohol withdraw. In recent years, GHB has been misused as a recreational drug due to its ability to induce euphoric feelings. Moreover, it has gained increasing attention as a popular drug of abuse that is frequently related to drug-facilitated sexual assaults. At the moment, detection methods based on chromatography exhibit extraordinary sensitivity for GHB sensing. However, such techniques require complicated sample treatment prior to analysis. Optical sensors provide an alternative approach for rapid and simple analysis of GHB samples. Unfortunately, currently reported probes are mostly based on hydrogen bonding to recognize GHB, and this raises concerns about, for example, the lack of specificity. In this work, we report a bioinspired strategy for selective sensing of GHB. The method is based on specific enzyme recognition to allow highly selective detection of GHB with minimum interference, even in a complex sample matrix (e. g., simulated urine). In addition, the result can be obtained by either quantitative spectroscopy analysis or colorimetric change observed by the naked-eye, thus demonstrating its potential application in drug screening and forensic analysis.