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Methods in Enzymology

Charles A Lesburg
No abstract text is available yet for this article.
2018: Methods in Enzymology
Ashley Taylor, Bradley C Doak, Martin J Scanlon
Herein we describe a method for the design, purchase, and assembly of a fragment-screening library from a list of commercially available compounds. The computational tools used in assessment of compound properties as well as the workflow for compound selection are provided for reference as implemented in commercially available software that is free and accessible to most academic users. The workflow can be modified as necessary to generate a fit-for-purpose fragment library with the desired compound property profiles...
2018: Methods in Enzymology
Stephanie Kay Ashenden
Thanks to technological advances and a greater understanding of the biological and chemical natures of targets and related diseases, high-throughput screening (HTS) has been allowed to be faster, cheaper, and more accessible. Yet, despite these increased technologies and understandings, the frequency of novel and drugs are being approved each year has not being increasing over the years. 2017 was considered a "bumper" year with a total of 46 approved drugs, over double that of the previous year. However, it is thought that part of the problem that HTS has not lived up to expectations is because of the contents of current chemical libraries...
2018: Methods in Enzymology
Qi Wang, Fangming Yang, Huijue Jia
The human microbiome is our "other genome." Implicated in a growing list of complex diseases, for which genomic studies typically explain a portion of the disease susceptibility, the human gut microbiome has been at the spotlight for our understanding of human diseases. As the microbiome is intrinsically more variable than the human genome, it is important to take careful considerations at each step of a study. Here, we put forward our recommendations, which we envision would facilitate identification of true drug targets in the human microbiome in the colon as well as at other body sites...
2018: Methods in Enzymology
Daniel R McMasters
Selective binding of a drug for its target vs other proteins is an important consideration in designing compounds with minimal risk of toxicity and therefore a key aspect of lead optimization. Screening all compounds against all known off-target proteins would be prohibitively expensive, so project teams must decide which compounds to screen in which assays. This chapter describes informatics-based methods that help prioritize testing, including screening minipanels and prioritization using predictive models (e-counterscreening)...
2018: Methods in Enzymology
Moses Moustakim, Suet Ling Felce, Nancy Zaarour, Gillian Farnie, Fiona E McCann, Paul E Brennan
Chemical probes are small molecules with potency and selectivity for a single or small number of protein targets. A good chemical probe engages its target intracellularly and is accompanied by a chemically similar, but inactive molecule to be used as a negative control in cellular phenotypic screening. The utility of these chemical probes is ultimately governed by how well they are developed and characterized. Chemical probes either as single entities, or in chemical probes sets are being increasingly used to interrogate the biological relevance of a target in a disease model...
2018: Methods in Enzymology
Kirk E Hevener, Russell Pesavento, JinHong Ren, Hyun Lee, Kiira Ratia, Michael E Johnson
High-throughput screening assays have become nearly ubiquitous in the search for small compounds or peptides that can modulate biological processes for therapeutic purposes. While many assays have become quite robust, with well-established protocols, the subsequent steps of validating the hits and choosing the best ones to take forward into leads for further chemical development are less established. In this chapter, we describe a variety of approaches, including chemical assessment, the use of various computational approaches, a variety of counter-screens, and "orthogonal" biophysical assays using nuclear magnetic resonance, surface plasmon resonance, isothermal titration calorimetry or thermal shift assays as methods for validating and assessing the quality of hits...
2018: Methods in Enzymology
Patrick M Collins, Alice Douangamath, Romain Talon, Alexandre Dias, Jose Brandao-Neto, Tobias Krojer, Frank von Delft
The XChem facility at Diamond Light Source offers fragment screening by X-ray crystallography as a general access user program. The main advantage of X-ray crystallography as a primary fragment screen is that it yields directly the location and pose of the fragment hits, whether within pockets of interest or merely on surface sites: this is the key information for structure-based design and for enabling synthesis of follow-up molecules. Extensive streamlining of the screening experiment at XChem has engendered a very active user program that is generating large amounts of data: in 2017, 36 academic and industry groups generated 35,000 datasets of uniquely soaked crystals...
2018: Methods in Enzymology
Miklos Kepiro, Boglarka H Varkuti, Ronald L Davis
High content, phenotypic screens offer a powerful approach to systems biology at the cellular level. The approach employs cells carrying fluorescently labeled molecules or organelles in 384- or 1536-well microplates, and an automated confocal screening microscope for capturing images from each well. Although some specifics vary according to the assay type, each will apply some degree of image processing and feature extraction followed by a data analysis pipeline to identify the perturbations (small molecules, etc...
2018: Methods in Enzymology
Becky S Leifer, Shelby K Doyle, André Richters, Helen L Evans, Angela N Koehler
Many promising therapeutic protein targets were previously considered "undruggable" due to a deficit in structural information to guide drug design and/or a lack of an obvious binding pocket. Fortunately, array-based methods for evaluating protein binding against large chemical libraries, such as small-molecule microarray screening, have provided one of several emerging inroads to ligand discovery for these elusive targets. Despite the advance in the area of ligand discovery for poorly structured and intrinsically disordered proteins provided by array-based technologies involving cell lysates, the extension of this technology for screening proteins with short half-lives in physiologically relevant conformations has been technically challenging...
2018: Methods in Enzymology
Tracy A Young, Ben Moree, Margaret T Butko, Bason Clancy, Mary Geck Do, Tarun Gheyi, John Strelow, Juan J Carrillo, Joshua Salafsky
Second-harmonic generation (SHG) has recently emerged as a biophysical tool for conformational sensing of a target biomolecule upon binding to ligands such as small molecules, fragments, proteins, peptides, and oligonucleotides. To date, SHG has been used to measure conformational changes of targets such as soluble proteins, protein complexes, intrinsically disordered proteins, peripheral and integral membrane proteins, peptides, and oligonucleotides upon binding of ligands over a wide range of affinities. In this chapter, we will provide a technology overview, detailed protocols for optimizing assays and screening, practical considerations, and an example case study to guide the reader in developing robust and informative measurements using the Biodesy Delta SHG platform...
2018: Methods in Enzymology
Christian Bergsdorf, S Kirk Wright
Over the past 30 years, drug discovery has evolved from a pure phenotypic approach to an integrated target-based strategy. The implementation of high-throughput biochemical and cellular assays has enabled the screening of large compound libraries which has become an important and often times the main source of new chemical matter that serve as starting point for medicinal chemistry efforts. In addition, biophysical methods measuring the physical interaction (affinity) between a low molecular weight ligand and a target protein became an integral part of hit validation/optimization to rule out false positives due to assay artifacts...
2018: Methods in Enzymology
Jacob Valentine, Ali Tavassoli
With the increasing utilization of high-throughput screening for lead identification in drug discovery, the need for easily constructed and diverse libraries which cover significant chemical space is greater than ever. Cyclic peptides address this need; they combine the advantageous properties of peptides (ease of production, high diversity, high potential specificity) with increased resistance to proteolysis and often increased biological activity (due to conformational locking). There are a number of techniques for the generation and screening of cyclic peptide libraries...
2018: Methods in Enzymology
Gurpreet Singh, Jayme L Dahlin, Michael A Walters
Drug discovery is inherently very risky. The management of these risks can enable the effective use of limited human and monetary resources. A careful attention to risk management in early discovery is especially important given that what happens in the early phases of a project may dictate the course of a research program for months or years. Risk management in early discovery starts with high-level managerial concerns: careful project selection, sufficient staffing and funding, and access to the appropriate instrumentation and tools...
2018: Methods in Enzymology
Challa Vijaya Kumar
No abstract text is available yet for this article.
2018: Methods in Enzymology
Maryam Khan, Qayyum Husain, Shamoon Asmat
The unexpected discovery of graphene and especially the follow-up explosion of interest in its properties and applications marked the beginning of a new carbon era. Graphene-based nanostructured materials are highly useful because they show great promise in the field of biotechnology and biomedicine. Owing to their unique structural features, exceptional chemical, electrical, and mechanical properties, and their ability to affect the microenvironment of biomolecules, graphene-armored nanomaterials are suitable for use in various applications, such as immobilization of enzymes, field-effect transistors, photovoltaic devices, and biosensors, which in turn is extremely vital to the development of biomedical instruments, clinical diagnosis, and disease treatment...
2018: Methods in Enzymology
Alexandra V Chatzikonstantinou, Elena Gkantzou, Dimitrios Gournis, Michaela Patila, Haralambos Stamatis
This chapter deals with the use of functionalized derivatives of graphene oxide as nanoscaffolds for the immobilization and stabilization of laccase from Trametes versicolor. Covalent and noncovalent immobilization approaches are described, while a novel method for the development of laccase-based multilayer nanoassemblies is also presented. Various biochemical, spectroscopic, and microscopic techniques were applied to characterize the nanobiocatalytic systems in respect to their microstructure and catalytic performance...
2018: Methods in Enzymology
Asieh Soozanipour, Asghar Taheri-Kafrani
Enzymes are used as biocatalysts for analytical purposes in diagnostics and preparative purposes in large-scale industrial processes. Despite perfect catalytic properties of enzymes, their industrial applications are limited due to the drawbacks regarding the lack of long-term stability under process conditions. The difficulties associated with recycling have to be resolved before enzyme implementation at industrial scale. Enzyme immobilization, as a novel approach, can improve the half-life, stability, catalytic activity, and reusability of enzymes...
2018: Methods in Enzymology
Yiying Sun, Jingjing Zhao, Jiafu Shi, Shaohua Zhang, Zhongyi Jiang
Immobilization of enzyme on the surface of graphene-based composite cellular foams (GCCFs) is commonly prone to acquire stable and ultrahigh loading of enzymes and fast transport of substrates during the catalytic process. In this chapter, we reported a method of preparing GCCFs through combination of redox assembly and biomimetic mineralization with in situ enzyme immobilization. Briefly, GCCFs were first prepared through redox assembly of graphene oxide (GO) nanosheets enabled by polyethyleneimine (PEI). The cationic PEI in the resultant reduced GO/PEI (rGO/PEI) cellular foams acted as the mineralization-inducing agent could catalyze the condensation of silicate to form silica (biomimetic silicification) on the reduced graphene oxide (rGO) surface, where enzyme (with penicillin G acylase as model enzyme) is in situ entrapped and shielded within the silica network...
2018: Methods in Enzymology
Ankarao Kalluri, Debika Debnath, Bhushan Dharmadhikari, Prabir Patra
Graphene and its derivatives having at least one dimension in nanoscale range have attracted tremendous attention in recent years due to their unique electronic, optical, chemical, and mechanical properties. This chapter is about graphene quantum dots (GQDs) that are zero-dimensional graphene derivatives with one to few layers of graphene sheet having size range less than 20nm. This chapter is an overview of synthesis of GQDs by top-down and bottom-up approaches, as well as detailed methods of synthesis of GQDs by acidic oxidation of carbon fibers...
2018: Methods in Enzymology
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