Drug Discovery Today: Technologies最新文献

Bispecific antibodies combine the specificity of two antibodies into one molecule. During the past two decades, advancement in protein engineering enabled the development of more than 100 bispecific formats, three of which are approved by the FDA for clinical use. In parallel to protein engineering methods, advancement in conjugation chemistries have spurred the use of chemical engineering approaches to generate bispecific antibodies. Herein, we review selected chemical strategies employed to generate bispecific antibodies that cannot be made using protein engineering methods.

This review provides an overview of the various theoretical and practical aspects of biotech plant design. It covers engineering, quality, regulatory, safety, environmental and economical points to be considered. Current knowledge and future trends as well as their impact on the planning and design are also discussed.

X-ray crystallography has provided the vast majority of three-dimensional macromolecular structures. Most of these are high-resolution structures that enable a detailed understanding of the underlying molecular mechanisms. The standardized workflows and robust infrastructure of synchrotron-based macromolecular crystallography (MX) offer the high throughput essential to cost-conscious investigations in structure- and fragment-based drug discovery. Nonetheless conventional MX is limited by fundamental bottlenecks, in particular X-ray radiation damage, which limits the amount of data extractable from a crystal. While this limit can in principle be circumvented by using larger crystals, crystals of the requisite size often cannot be obtained in sufficient quality. That is especially true for membrane protein crystals, which constitute the majority of current drug targets. This conventional paradigm for MX-suitable samples changed dramatically with the advent of serial femtosecond crystallography (SFX) based on the ultra-short and extremely intense X-ray pulses of X-ray Free-Electron Lasers. SFX provides high-resolution structures from tiny crystals and does so with uniquely low levels of radiation damage. This has yielded a number of novel structures for G-protein coupled receptors, one of the most relevant membrane protein superfamilies for drug discovery, as well as tantalizing advances in time-resolved crystallography that elucidate protein dynamics. This article attempts to map the potential of SFX for drug discovery, while providing the reader with an overview of the yet remaining technical challenges associated with such a novel technique as SFX.

Mass spectrometry plays an essential role in qualitative and quantitative analysis of pharmaceutically relevant molecules. The present review summarizes some the most common applications of LC-MS for the characterization of therapeutic low-molecular-weight compounds, peptides and proteins, and oligonucleotides using low-resolution and high-resolution tandem mass spectrometry. In addition, the benefit of multistage MS, differential ion mobility, and data independent acquisition is emphasized. At last, the potential of coupling MS with novel interfaces for high-throughput analysis is discussed.

Membrane proteins mediate various biological processes. Most drugs commercially available target proteins on the cell surface. Therefore, proteomics of plasma membrane proteins provides useful information for drug discovery. However, membrane proteins are one of the most difficult biological groups to quantify by proteomics because of their hydrophobicity and low protein content. To obtain unbiased quantitative membrane proteomics data, specific strategies should be followed during sample preparation. This review explores the most recent advances in sample preparation for the quantitative analysis of the membrane proteome, including enrichment by subcellular fractionation and trypsin digestion.

The field of proteomics immensely depends on data generation and data analysis which are thoroughly supported by software and databases. There has been a massive advancement in mass spectrometry-based proteomics over the last 10 years which has compelled the scientific community to upgrade or develop algorithms, tools, and repository databases in the field of proteomics. Several standalone software, and comprehensive databases have aided the establishment of integrated omics pipeline and meta-analysis workflow which has contributed to understand the disease pathobiology, biomarker discovery and predicting new therapeutic modalities. For shotgun proteomics where Data Dependent Acquisition is performed, several user-friendly software are developed that can analyse the pre-processed data to provide mechanistic insights of the disease. Likewise, in Data Independent Acquisition, pipelines are emerged which can accomplish the task from building the spectral library to identify the therapeutic targets. Furthermore, in the age of big data analysis the implications of machine learning and cloud computing are appending robustness, rapidness and in-depth proteomics data analysis. The current review talks about the recent advancement, and development of software, tools, and database in the field of mass-spectrometry based proteomics.

Mass spectrometry imaging (MSI) has become a powerful method for mapping metabolite distribution in a tissue. Applied to bacterial colonies, MSI has a bright future, both for the discovery of new bioactive compounds and for a better understanding of bacterial antibiotic resistance mechanisms. Coupled with separation techniques such as ion mobility mass spectrometry (IM-MS), the identification of metabolites directly on the image is now possible and does not require additional analysis such as HPLC-MS/MS. In this article, we propose to apply a semi-targeted workflow for rapid IM-MSI data analysis focused on the search for bioactive compounds. First, chemically-related compounds showing a repetitive mass unit (i.e. lipids and lipopeptides) were targeted based on the Kendrick mass defect analysis. The detected groups of potentially bioactive compounds were then confirmed by fitting their measured ion moibilites to their measured m/z values. Using both their m/z and ion mobility values, the selected groups of compounds were identified using the available databases and finally their distribution was observed on the image. Using this workflow on a co-culture of bacteria, we were able to detect and localize bioactive compounds involved in the microbial interaction.

During the last years, X-ray free electron lasers (XFELs) have emerged as X-ray sources of unparalleled brightness, delivering extreme amounts of photons in femtosecond pulses. As such, they have opened up completely new possibilities in drug discovery and structural biology, including studying high resolution biomolecular structures and their functioning in a time resolved manner, and diffractive imaging of single particles without the need for their crystallization. In this perspective, we briefly review the operation of XFELs, their immediate uses for drug discovery and focus on the potentially revolutionary single particle diffractive imaging technique and the challenges which remain to be overcome to fully realize its potential to provide high resolution structures without the need for crystallization, freezing or the need to keep proteins stable at extreme concentrations for long periods of time. As the issues have been to a large extent sample delivery related, we outline a way for native mass spectrometry to overcome these and enable so far impossible research with a potentially huge impact on structural biology and drug discovery, such as studying structures of transient intermediate species in viral life cycles or during functioning of molecular machines.

As a starting point for drug discovery, affinity selection-mass spectrometry (AS-MS) is ideal for the discovery of lead compounds from chemically diverse sources such as botanical, fungal and microbial extracts. Based on binding interactions between macromolecular receptors and ligands of low molecular mass, AS-MS enables the rapid isolation of pharmacologically active small molecules from complex mixtures for mass spectrometric characterization and identification. Unlike conventional high-throughput screening, AS-MS requires no radiolabels, no UV or fluorescent chromophores, and is compatible with all classes of receptors, enzymes, incubation buffers, cofactors, and ligands. The most successful types of AS-MS include pulsed ultrafiltration (PUF) AS-MS, size exclusion chromatography (SEC) AS-MS, and magnetic microbead affinity selection screening (MagMASS), which differ in their approaches for separating the ligand-receptor complexes from the non-binding compounds in mixtures. After affinity isolation, the ligand(s) from the mixture are characterized using high resolution UHPLC-MS and tandem mass spectrometry. Based on these elemental composition and structural data, the identities of the lead compounds are determined by searching on-line databases for known natural products and by comparison with standards. The structures of novel natural products are determined using a combination of spectroscopic techniques including two-dimensional NMR and MS.