The Chemical Biology of Aptamers

Taking a strand : Aptamers are small single‐stranded oligonucleotides that fold into a well‐defined 3D structure and interact with high affinity and specificity with their target molecules, thereby inhibiting their biological functions. Aptamers can be synthesized by either chemical and/or enzymatic procedures and can thus be considered as both chemical and biological substances. The current status and new developments in this area are described.

     

Lost in Transcription—Inhibition of RNA Polymerase

Form and function : The natural product myxopyronin A provides the key to understanding the inhibition of bacterial RNA polymerase and should spark new ideas for the design of new antibiotics against tuberculosis and other infectious diseases.

     

From differentiating metabolites to biomarkers

The current developments in metabolomics and metabolic profiling technologies have led to the discovery of several new metabolic biomarkers. Finding metabolites present in significantly different levels between sample sets, however, does not necessarily make these metabolites useful biomarkers. The route to valid and applicable biomarkers (biomarker qualification) is long and demands a significant amount of work. In this overview, we critically discuss the current state-of-the-art of metabolic biomarker discovery, with highlights and shortcomings, and suggest a pathway to clinical usefulness.

Membrane Sensor Histidine Kinases: Insights from Structural,Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled “Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents” edited by Mary K. Phillips-Jones.     

Synthesis and NMR spectroscopic assignment of chlorinated benzimidazole-2-thione derivatives

The benzimidazole-2-thione scaffold is present in many drugs encompassing various therapeutic areas. Due to the broad spectrum of bioactivities it also represents an important starting point in drug discovery campaigns, especially those based on fragment-based design. Despite simple structures the tautomerism and regioisomerism of substituted benzimidazole-2-thiones makes unambiguous structural analysis difficult. Tautomeric duplicates are present in commercial libraries resulting in two tautomers being sold as different products. To showcase an example of appropriate structural determination, we synthesized and characterized a set of benzimidazole-2-thiones with different positions of a chlorine atom on the ring. Using NOESY and ¹³C NMR spectroscopy, we determined that the thione tautomer predominates in the thione-thiol equilibrium. Furthermore, NOESY and HMBC experiments confirmed the position of the substituents on the benzimidazole-2-thione ring.     

中国新三板市场阶段性集合竞价制度的价格发现研究

市场微观结构理论表明交易机制对资产价格的形成过程具有重要影响。本文以中国新三板交易机制改革为背景,从理论上分析了阶段性集合竞价制度的市场出清过程。阶段性集合竞价制度的核心在于市场出清时间间隔的设定。本文构建了一个存在信息摩擦和知情交易者学习机制的集合竞价市场出清模型,讨论了市场出清时间间隔对价格发现效率、资产价值不确定性和流动性风险的影响。研究发现:(1)在完美信息条件下,如果对市场规模较大和价值波动率较高的资产设定较短的市场出清时间间隔,将会降低投资者的流动性风险,提升市场质量;(2)在不完美信息条件下,除市场规模和资产价值波动率之外,信息不对称程度和知情交易者比例也是影响最优市场出清频率的重要因素;(3)在不完美信息条件下,对价值波动率较低的资产缩短市场出清时间间隔才能降低流动性风险,这与完美信息条件下的结论相反。     

Batch‐injection Amperometric Analysis on Screen‐printed Electrodes: Analytical System for High‐throughput Determination of Pharmaceutical Molecules

This work presents a single analytical system able to perform high‐throughput determinations of different pharmaceutical molecules on screen‐printed electrodes (SPEs) assembled on a batch‐injection analysis (BIA) cell. Two types of SPEs, both containing a carbon conductive ink as working electrode, were selected for the determination of levamisole (LVM) in aqueous and sodium levothyroxine (NaLVT) in hydroethanolic media. The main analytical characteristics of the proposed system for both examples are high precision (RSD <3.8 %, n=10), low detection limits (submicromolar range), and high sample‐throughput (>150 h^?1) using a single SPE, demonstrating the extended lifetime of such sensors, which are adequate for routine pharmaceutical analysis. The proposed analytical system requires battery‐powered portable devices, including potentiostat and reader, electronic micropipette, BIA cell and SPEs, and can be applied for a wide range of pharmaceutical molecules. In case of analyte adsorption on electrode surface, fast electrode cleaning can be supplied by external stirring easily adapted to the cell, which is demonstrated in this work for NaLVT determination.     

Stalis: A Computational Method for Template-Based Ab Initio Ligand Design

Proteins interact with small molecules through specific molecular recognition, which is central to essential biological functions in living systems. Therefore, understanding such interactions is crucial for basic sciences and drug discovery. Here, we present S tructure t emplate-based a b initio li gand design s olution (Stalis), a knowledge-based approach that uses structure templates from the Protein Data Bank libraries of whole ligands and their fragments and generates a set of molecules (virtual ligands) whose structures represent the pocket shape and chemical features of a given target binding site. Our benchmark performance evaluation shows that ligand structure-based virtual screening using virtual ligands from Stalis outperforms a receptor structure-based virtual screening using AutoDock Vina, demonstrating reliable overall screening performance applicable to computational high-throughput screening. However, virtual ligands from Stalis are worse in recognizing active compounds at the small fraction of a rank-ordered list of screened library compounds than crystal ligands, due to the low resolution of the virtual ligand structures. In conclusion, Stalis can facilitate drug discovery research by designing virtual ligands that can be used for fast ligand structure-based virtual screening. Moreover, Stalis provides actual three-dimensional ligand structures that likely bind to a target protein, enabling to gain structural insight into potential ligands. Stalis can be an efficient computational platform for high-throughput ligand design for fundamental biological study and drug discovery research at the proteomic level. © 2019 Wiley Periodicals, Inc.     

Chemical Kinetic Strategies for High‐Throughput Screening of Protein Aggregation Modulators

Insoluble aggregates staining positive to amyloid dyes are known histological hallmarks of different neurodegenerative disorders and of type II diabetes. Soluble oligomers are smaller assemblies whose formation prior to or concomitant with amyloid deposition has been associated to the processes of disease propagation and cell death. While the pathogenic mechanisms are complex and differ from disease to disease, both types of aggregates are important biological targets subject to intense investigation in academia and industry. Here we review recent advances in the fundamental understanding of protein aggregation that can be used on the development of anti‐amyloid and anti‐oligomerization drugs. Specifically, we pinpoint the chemical kinetic aspects that should be attended during the development of high‐throughput screening assays and in the hit validation phase. The strategies here devised are expected to establish a connection between basic research and pharmaceutical innovation.     

Determination of flurbiprofen in pharmaceutical preparations by GC–MS

This article describes a gas chromatography–mass spectrometry (GC–MS) method for the determination of flurbiprofen in pharmaceutical preparations. The method is based on the derivatization of flurbiprofen with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). For GC–MS, electron ionization mode (EI = 70 eV) and selected ion monitoring (SIM) mode were used for quantitative analysis (m/z 180 for flurbiprofen). Calibration curve was linear between the concentration range of 0.25–5.0 μg/mL. Intra- and inter-day precision values for flurbiprofen were less than 3.64, and accuracy (relative error) was better than 2.67%. The mean recovery of flurbiprofen was 99.4% for pharmaceutical preparations. The limits of detection and quantification of flurbiprofen were 0.05 and 0.15 μg/mL, respectively. No interference was found from tablet excipients at the selected assay conditions. Also, the method was applied for the quality control of five commercial flurbiprofen dosage forms to quantify the drug and to check the formulation content uniformity.