共查询到20条相似文献,搜索用时 15 毫秒
1.
Lundstrom K 《Journal of cellular and molecular medicine》2007,11(2):224-238
Structure determination has already proven useful for lead optimization and direct drug design. The number of high-resolution structures available in public databases today exceeds 30,000 and will definitely aid in structure-based drug design. Structural genomics approaches covering whole genomes, topologically similar proteins or gene families are great assets for further progress in the development of new drugs. However, membrane proteins representing 70% of current drug targets are poorly characterized structurally. The problems have been related to difficulties in obtaining large amount of recombinant membrane proteins as well as their purification and structure determination. Structural genomics has proven successful in developing new methods in areas from expression to structure determination by studying a large number of target proteins in parallel. 相似文献
2.
High-throughput, automated or semiautomated methodologies implemented by companies and structural genomics initiatives have accelerated the process of acquiring structural information for proteins via x-ray crystallography. This has enabled the application of structure-based drug design technologies to a variety of new structures that have potential pharmacologic relevance. Although there remain major challenges to applying these approaches more broadly to all classes of drug discovery targets, clearly the continued development and implementation of these structure-based drug design methodologies by the scientific community at large will help to address and provide solutions to these hurdles. The result will be a growing number of protein structures of important pharmacologic targets that will help to streamline the process of identification and optimization of lead compounds for drug development. These lead agonist and antagonist pharmacophores should, in turn, help to alleviate one of the current critical bottlenecks in the drug discovery process; that is, defining the functional relevance of potential novel targets to disease modification. The prospect of generating an increasing number of potential drug candidates will serve to highlight perhaps the most significant future bottleneck for drug development, the cost and complexity of the drug approval process. 相似文献
3.
Cost and time reduction are two of the driving forces in the development of new strategies for protein crystallization and subsequent structure determination. Here, we report the analysis of the Thermotoga maritima proteome, in which we compare the proteins that were successfully expressed, purified and crystallized versus the rest of the proteome. This set of almost 500 proteins represents one of the largest, internally consistent, protein expression and crystallization datasets available. The analysis shows that individual parameters, such as isoelectric point, sequence length, average hydropathy, low complexity regions (SEG), and combinations of these biophysical properties for crystallized proteins define a distinct subset of the T. maritima proteome. The distribution profiles of the various biophysical properties in the expression/crystallization set are then used to extract rules to improve target selection and improve the efficiency and output of structural genomics, as well as general structural biology efforts. 相似文献
4.
Savage DF Anderson CL Robles-Colmenares Y Newby ZE Stroud RM 《Protein science : a publication of the Protein Society》2007,16(5):966-976
Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level. 相似文献
5.
Sugar FJ Jenney FE Poole FL Brereton PS Izumi M Shah C Adams MW 《Journal of structural and functional genomics》2005,6(2-3):149-158
As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects
is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination,
with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression
clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression.
In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of
recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA)
against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE
gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them
successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher
than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening
tool for more efficient protein production. 相似文献
6.
7.
Slabinski L Jaroszewski L Rodrigues AP Rychlewski L Wilson IA Lesley SA Godzik A 《Protein science : a publication of the Protein Society》2007,16(11):2472-2482
The process of experimental determination of protein structure is marred with a high ratio of failures at many stages. With availability of large quantities of data from high-throughput structure determination in structural genomics centers, we can now learn to recognize protein features correlated with failures; thus, we can recognize proteins more likely to succeed and eventually learn how to modify those that are less likely to succeed. Here, we identify several protein features that correlate strongly with successful protein production and crystallization and combine them into a single score that assesses "crystallization feasibility." The formula derived here was tested with a jackknife procedure and validated on independent benchmark sets. The "crystallization feasibility" score described here is being applied to target selection in the Joint Center for Structural Genomics, and is now contributing to increasing the success rate, lowering the costs, and shortening the time for protein structure determination. Analyses of PDB depositions suggest that very similar features also play a role in non-high-throughput structure determination, suggesting that this crystallization feasibility score would also be of significant interest to structural biology, as well as to molecular and biochemistry laboratories. 相似文献
8.
Smyth DR Mrozkiewicz MK McGrath WJ Listwan P Kobe B 《Protein science : a publication of the Protein Society》2003,12(7):1313-1322
The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. 相似文献
9.
P. A. Boriack-Sjodin S. Zeitlin H. H. Chen L. Crenshaw S. Gross A. Dantanarayana P. Delgado J. A. May T. Dean D. W. Christianson 《Protein science : a publication of the Protein Society》1998,7(12):2483-2489
X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bicyclic thienothiazene-6-sulfonamide-1,1-dioxide inhibitor skeleton in comparison with a monocyclic 2,5-thiophenedisulfonamide skeleton; (2) optimal substituents attached to a secondary sulfonamide group targeted to interact with hydrophobic patches defined by Phe131, Leu198, and Pro202; and (3) optimal stereochemistry and configuration at the C-4 position of bicyclic thienothiazene-6-sulfonamides; the C-4 substituent can interact with His64, the catalytic proton shuttle. Structure-activity relationships rationalize affinity trends observed during the development of brinzolamide (Azopt), the newest carbonic anhydrase inhibitor approved for the treatment of glaucoma. 相似文献
10.
《Expert review of proteomics》2013,10(4):349-351
Baukje de Roos is a principal investigator at the University of Aberdeen, Rowett Institute of Nutrition and Health. She investigates mechanisms through which dietary fats and fatty acids, and also polyphenols, affect parameters involved in the development of heart disease in vivo. This is achieved not only by measuring their effect on conventional risk markers for heart disease but also by assessing their effect on new markers that are being developed through proteomic and mass spectrometry methods. She obtained her PhD in Human Nutrition at Wageningen University, The Netherlands, in January 2000, after which she was appointed as a post-doctoral research fellow at the Department of Vascular Biochemistry, Glasgow Royal Infirmary, in collaboration with GlaxoSmithKline. In June 2001 she joined the Rowett Research Institute in Aberdeen. She is currently working for the University of Aberdeen, where her research is funded by the Scottish Government Rural and Environment Research and Analysis Directorate (RERAD). She is an active member of the European Nutrigenomics Organisation (NuGO), an EU-funded Network of Excellence, which merges the nutrigenomics activities of its 23 partners across Europe. 相似文献
11.
Fleming CD Bencharit S Edwards CC Hyatt JL Tsurkan L Bai F Fraga C Morton CL Howard-Williams EL Potter PM Redinbo MR 《Journal of molecular biology》2005,352(1):165-177
Human carboxylesterase 1 (hCE1) exhibits broad substrate specificity and is involved in xenobiotic processing and endobiotic metabolism. We present and analyze crystal structures of hCE1 in complexes with the cholesterol-lowering drug mevastatin, the breast cancer drug tamoxifen, the fatty acyl ethyl ester (FAEE) analogue ethyl acetate, and the novel hCE1 inhibitor benzil. We find that mevastatin does not appear to be a substrate for hCE1, and instead acts as a partially non-competitive inhibitor of the enzyme. Similarly, we show that tamoxifen is a low micromolar, partially non-competitive inhibitor of hCE1. Further, we describe the structural basis for the inhibition of hCE1 by the nanomolar-affinity dione benzil, which acts by forming both covalent and non-covalent complexes with the enzyme. Our results provide detailed insights into the catalytic and non-catalytic processing of small molecules by hCE1, and suggest that the efficacy of clinical drugs may be modulated by targeted hCE1 inhibitors. 相似文献
12.
Page R Deacon AM Lesley SA Stevens RC 《Journal of structural and functional genomics》2005,6(2-3):209-217
Currently, 119 high resolution structures of Thermotoga maritima proteins have been determined by the Joint Center for Structural Genomics (JCSG, www.jcsg.org). Sixty-seven of these were
solved using the first implementation of the multi-tiered crystallization strategy at the JCSG for the efficient crystallization
of large numbers of protein targets. Previously, we reported the analysis of all proteins crystallized using this multi-tiered
strategy [Lesley, S.A. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 11664–11669; Page, R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037]. Here, we extend the analysis and describe the crystallization characteristics of those proteins that produced
diffraction quality crystals, ultimately resulting in high resolution structures. First, we found that over 77% (52) of the
crystals used for structure determination were produced directly from high-throughput coarse screens, indicating that less
than one quarter of the crystals (15) required fine screening. In addition, as observed for the proteome screen [Page, R.
et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037], the majority of conditions that produced crystals for natively expressed proteins, whose structures have been
determined, were distinct from those of their more extensively purified and selenomethionine-labeled counterparts. Finally,
99% of the proteins whose structures were solved crystallized in conditions contained in the JCSG Minimal Core Screen [Page,
R. et al. (2003) Acta Crystallogr. D Biol. Crystallogr. 59, 1028–1037; Page, R. and Stevens, R.C. (2004) Methods 34, 373–389], a set of 67 conditions previously identified as those most likely to produce crystals of a diverse set of proteins,
confirming its success for rapid identification of proteins with a natural propensity to crystallize. 相似文献
13.
《Expert review of proteomics》2013,10(3):281-291
Despite the rapid growth of postgenomic data and fast-paced technology advancement, drug discovery is still a lengthy and difficult process. More effective drug design requires a better understanding of the interaction between drug candidates and their targets/off-targets in various situations. The ability of chemical proteomics to integrate a multiplicity of disciplines enables the direct analysis of protein activities on a proteome-wide scale, which has enormous potential to facilitate drug target elucidation and lead drug verification. Over recent years, chemical proteomics has experienced rapid growth and provided a valuable method for drug target identification and inhibitor discovery. This review introduces basic concepts and technologies of different popular chemical proteomic approaches. It also covers the essential features and recent advances of each approach while underscoring their potentials in drug discovery and development. 相似文献
14.
Donovan RS Datti A Baek MG Wu Q Sas IJ Korczak B Berger EG Roy R Dennis JW 《Glycoconjugate journal》1999,16(10):607-615
Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37°C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well -counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewisx in -glycans. A glycopolymer acceptor for 1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5–6 fold increase in throughput compared to the corresponding solution-phase assay. 相似文献
15.
Natasja Brooijmans Yu‐Wei Chang Dominick Mobilio Rajiah A. Denny Christine Humblet 《Protein science : a publication of the Protein Society》2010,19(4):763-774
The development of a kinase structural database, the kinase knowledge base (KKB), is described. It covers all human kinase domain structures that have been deposited in the Protein Data Bank. All structures are renumbered using a common scheme, which enables efficient cross‐comparisons and multiple queries of interest to the kinase field. The common numbering scheme is also used to automatically annotate conserved residues and motifs, and conformationally classify the structures based on the DFG‐loop and Helix C. Analyses of residue conservation in the ATP binding site using the full human‐kinome–sequence alignment lead to the identification of a conserved hydrogen bond between the hinge region backbone and a glycine in the specificity surface. Furthermore, 90% of kinases are found to have at least one stabilizing interaction for the hinge region, which has not been described before. 相似文献
16.
Lundstrom K 《Molecular biotechnology》2006,34(2):205-212
Structural genomics can be defined as structural biology on a large number of target proteins in parallel. This approach plays
an important role in modern structure-based drug design. Although a number of structural genomics initiatives have been initiated,
relatively few are associated with integral membrane proteins. This indicates the difficulties in expression, purification,
and crystallization of membrane proteins, which has also been confirmed by the existence of some 100 high-resolution structures
of membrane proteins among the more than 30,000 entries in public databases. Paradoxically, membrane proteins represent 60–70%
of current drug targets and structural knowledge could both improve and speed up the drug discovery process. In order to improve
the sucess rate for structure resolution of membrane proteins structural genomics networks have been established. 相似文献
17.
High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived
from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins
is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing
partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts
than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this
purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas
are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins
that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large,
human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55
distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed
for binding specificity and soluble scFv can be readily produced and purified via the appended His6 epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex
formation and are suitable for co-crystallization trials. 相似文献
18.
Reynolds MA 《Journal of industrial microbiology & biotechnology》2002,28(3):180-185
Incyte Genomics' GEM™ Gene Expression Microarray is a proven genomics tool used by a large number of pharmaceutical companies
to speed up the drug discovery and development process. The development and integration of this technology, together with
Incyte's sequence databases and clone resources, have resulted in GEM microarrays that span approximately 60,000 human genes
as well as approximately 60,000 plant, rat, mouse, yeast, and bacterial genes. The technology underlying the use of these
arrays and their application to the drug discovery process is highlighted. Journal of Industrial Microbiology & Biotechnology (2002) 28, 180–185 DOI: 10.1038/sj/jim/7000136
Received 16 November 2000/ Accepted in revised form 01 March 2001 相似文献
19.
Bonanno JB Almo SC Bresnick A Chance MR Fiser A Swaminathan S Jiang J Studier FW Shapiro L Lima CD Gaasterland TM Sali A Bain K Feil I Gao X Lorimer D Ramos A Sauder JM Wasserman SR Emtage S D'Amico KL Burley SK 《Journal of structural and functional genomics》2005,6(2-3):225-232
Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the
New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual
core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family
classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies,
structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC
will be capable of producing 100–200 experimentally determined protein structures annually. All Consortium activities can
be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes
both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported
by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal
Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed
of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org)
and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In
addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC
aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry.
The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR
spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural
biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress
toward development of a high-throughput Gene→Structure platform. An analysis of current and projected consortium metrics reflects
progress to date and delineates opportunities for further technology development. 相似文献
20.