Publications by authors named "Michael Sundström"

26 Publications

  • Page 1 of 1

Identifying novel B-cell targets for chronic inflammatory autoimmune disease by screening of chemical probes in a patient-derived cell assay.

Transl Res 2021 Mar 22;229:69-82. Epub 2020 Sep 22.

Division of Rheumatology, Department of Medicine Solna, Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden. Electronic address:

B-cell secretion of autoantibodies drives autoimmune diseases, including systemic lupus erythematosus and idiopathic inflammatory myositis. Few therapies are presently available for treatment of these patients, often resulting in unsatisfactory effects and helping only some of the patients. We developed a screening assay for evaluation of novel targets suspending B-cell maturation into antibody secreting cells, which could contribute to future drug development. The assay was employed for testing 43 high quality chemical probes and compounds inhibiting under-explored protein targets, using primary cells from patients with autoimmune disease. Probes inhibiting bromodomain family proteins and histone methyl transferases demonstrated abrogation of B-cell functions to a degree comparable to a positive control, the JAK inhibitor tofacitinib. Inhibition of each target rendered a specific functional cell and potential disease modifying effect, indicating specific epigenetic protein targets as potential new intervention points for future drug discovery and development efforts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.trsl.2020.09.003DOI Listing
March 2021

Anti-Inflammatory Properties of Chemical Probes in Human Whole Blood: Focus on Prostaglandin E Production.

Front Pharmacol 2020 6;11:613. Epub 2020 May 6.

Division of Rheumatology, Department of Medicine, Solna, Karolinska Institutet, and Karolinska University Hospital, Stockholm, Sweden.

We screened 57 chemical probes, high-quality tool compounds, and relevant clinically used drugs to investigate their effect on pro-inflammatory prostaglandin E (PGE) production and interleukin-8 (IL-8) secretion in human whole blood. Freshly drawn blood from healthy volunteers and patients with systemic lupus erythematosus (SLE) or dermatomyositis was incubated with compounds at 0.1 or 1 µM and treated with lipopolysaccharide (LPS, 10 µg/ml) to induce a pro-inflammatory condition. Plasma was collected after 24 h for lipid profiling using liquid chromatography tandem mass spectrometry (LC-MS/MS) and IL-8 quantification using enzyme-linked immunosorbent assay (ELISA). Each compound was tested in at least four donors at one concentration based on prior knowledge of binding affinities and activity. Our screening suggested that PD0325901 (MEK-1/2 inhibitor), trametinib (MEK-1/2 inhibitor), and selumetinib (MEK-1 inhibitor) decreased while tofacitinib (JAK inhibitor) increased PGE production. These findings were validated by concentration-response experiment in two donors. Moreover, the tested MEK inhibitors decreased thromboxane B (TXB) production and IL-8 secretion. We also investigated the lysophophatidylcholine (LPC) profile in plasma from treated whole blood as these lipids are potentially important mediators in inflammation, and we did not observe any changes in LPC profiles. Collectively, we deployed a semi-high throughput and robust methodology to investigate anti-inflammatory properties of new chemical probes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2020.00613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218097PMC
May 2020

A genetics-led approach defines the drug target landscape of 30 immune-related traits.

Nat Genet 2019 07 28;51(7):1082-1091. Epub 2019 Jun 28.

Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK.

Most candidate drugs currently fail later-stage clinical trials, largely due to poor prediction of efficacy on early target selection. Drug targets with genetic support are more likely to be therapeutically valid, but the translational use of genome-scale data such as from genome-wide association studies for drug target discovery in complex diseases remains challenging. Here, we show that integration of functional genomic and immune-related annotations, together with knowledge of network connectivity, maximizes the informativeness of genetics for target validation, defining the target prioritization landscape for 30 immune traits at the gene and pathway level. We demonstrate how our genetics-led drug target prioritization approach (the priority index) successfully identifies current therapeutics, predicts activity in high-throughput cellular screens (including L1000, CRISPR, mutagenesis and patient-derived cell assays), enables prioritization of under-explored targets and allows for determination of target-level trait relationships. The priority index is an open-access, scalable system accelerating early-stage drug target selection for immune-mediated disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41588-019-0456-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124888PMC
July 2019

Large-Scale Biophysical Evaluation of Protein PEGylation Effects: In Vitro Properties of 61 Protein Entities.

Mol Pharm 2016 05 12;13(5):1587-98. Epub 2016 Apr 12.

Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.6b00049DOI Listing
May 2016

Preclinical target validation using patient-derived cells.

Nat Rev Drug Discov 2015 Mar 20;14(3):149-50. Epub 2015 Feb 20.

Structural Genomics Consortium, Department of Medicine, Karolinska University Hospital and Karolinska Institutet, 171 76 Stockholm, Sweden.

The Structural Genomics Consortium (SGC) and its clinical, industry and disease-foundation partners are launching open-source preclinical translational medicine studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nrd4565DOI Listing
March 2015

Recently targeted kinases and their inhibitors-the path to clinical trials.

Curr Opin Pharmacol 2014 Aug 9;17:58-63. Epub 2014 Aug 9.

Department of Medicine, Karolinska University Hospital and Karolinska Institutet, 171 76 Stockholm, Sweden. Electronic address:

Protein kinases have emerged as one of the most important drug target families for the treatment of cancer. To date, 28 inhibitors with reported activity versus one or multiple kinases have been approved for clinical use. However, the majority of new clinical trials are focused on new subindications using already approved kinase inhibitors or target well validated kinase targets with novel inhibitors. In contrast, relatively few clinical trials have been initiated using specific inhibitors that inhibit novel kinase targets, despite significant validation efforts in the public domain. Analysis of the target validation history of first in class kinase inhibitors revealed a long delay between initial disease association and development of inhibitors. As part of this analysis, we have investigated which first in class inhibitor that entered phase I clinical trials over the last five years and also considered which research approaches that were used to validate them.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.coph.2014.07.015DOI Listing
August 2014

Heterologous expression and purification of an active human TRPV3 ion channel.

FEBS J 2013 Dec 30;280(23):6010-21. Epub 2013 Sep 30.

Protein Function and Interactions, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Denmark.

The transient receptor potential vanilloid 3 (TRPV3) cation channel is widely expressed in human tissues and has been shown to be activated by mild temperatures or chemical ligands. In spite of great progress in the TRP-channel characterization, very little is known about their structure and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. Here, we report on the high-level Escherichia coli expression of the human TRPV3 channel, for which no structural information has been reported to date. We selected a suitable detergent and buffer system using analytical size-exclusion chromatography and a thermal stability assay. We demonstrate that the recombinant purified protein contains high α-helical content and migrates as dimers and tetramers on native PAGE. Furthermore, the purified channel also retains its current inducing activity, as shown by electrophysiology experiments. The ability to produce the TRPV3 channel heterologously will aid future functional and structural studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.12520DOI Listing
December 2013

Functional proteomics defines the molecular switch underlying FGF receptor trafficking and cellular outputs.

Mol Cell 2013 Sep 5;51(6):707-22. Epub 2013 Sep 5.

Department of Proteomics, NNF Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.

The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4 recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching in mouse lung explants. Altogether, these results identify an intriguing ligand-dependent mechanism for the control of receptor fate and cellular outputs that may explain the pathogenic role of deregulated FGFR2b, thus offering therapeutic opportunities.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2013.08.002DOI Listing
September 2013

A secretory system for bacterial production of high-profile protein targets.

Protein Sci 2011 Mar;20(3):597-609

Facility for Protein Science and Technology, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.

Escherichia coli represents a robust, inexpensive expression host for the production of recombinant proteins. However, one major limitation is that certain protein classes do not express well in a biologically relevant form using standard expression approaches in the cytoplasm of E. coli. To improve the usefulness of the E. coli expression platform we have investigated combinations of promoters and selected N-terminal fusion tags for the extracellular expression of human target proteins. A comparative study was conducted on 24 target proteins fused to outer membrane protein A (OmpA), outer membrane protein F (OmpF) and osmotically inducible protein Y (OsmY). Based on the results of this initial study, we carried out an extended expression screen employing the OsmY fusion and multiple constructs of a more diverse set of human proteins. Using this high-throughput compatible system, we clearly demonstrate that secreted biomedically relevant human proteins can be efficiently retrieved and purified from the growth medium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pro.593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064838PMC
March 2011

Screening of genetic parameters for soluble protein expression in Escherichia coli.

Protein Expr Purif 2011 May 2;77(1):104-11. Epub 2010 Dec 2.

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.

Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts. Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth factor binding proteins. This rapid screening approach appears highly suitable for high-throughput efforts targeting either large sets of proteins or more focused investigations regarding individual high-profile targets.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pep.2010.11.016DOI Listing
May 2011

Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.

Proc Natl Acad Sci U S A 2008 Apr 23;105(17):6457-62. Epub 2008 Apr 23.

Structural Genomics Consortium, Oxford University, Oxford OX3 7DQ, United Kingdom.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0801508105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2359823PMC
April 2008

A systematic interaction map of validated kinase inhibitors with Ser/Thr kinases.

Proc Natl Acad Sci U S A 2007 Dec 11;104(51):20523-8. Epub 2007 Dec 11.

Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Oxford OX3 7LD, United Kingdom.

Protein kinases play a pivotal role in cell signaling, and dysregulation of many kinases has been linked to disease development. A large number of kinase inhibitors are therefore currently under investigation in clinical trials, and so far seven inhibitors have been approved as anti-cancer drugs. In addition, kinase inhibitors are widely used as specific probes to study cell signaling, but systematic studies describing selectivity of these reagents across a panel of diverse kinases are largely lacking. Here we evaluated the specificity of 156 validated kinase inhibitors, including inhibitors used in clinical trials, against 60 human Ser/Thr kinases using a thermal stability shift assay. Our analysis revealed many unexpected cross-reactivities for inhibitors thought to be specific for certain targets. We also found that certain combinations of active-site residues in the ATP-binding site correlated with the detected ligand promiscuity and that some kinases are highly sensitive to inhibition using diverse chemotypes, suggesting them as preferred intervention points. Our results uncovered also inhibitor cross-reactivities that may lead to alternate clinical applications. For example, LY333'531, a PKCbeta inhibitor currently in phase III clinical trials, efficiently inhibited PIM1 kinase in our screen, a suggested target for treatment of leukemia. We determined the binding mode of this inhibitor by x-ray crystallography and in addition showed that LY333'531 induced cell death and significantly suppressed growth of leukemic cells from acute myeloid leukemia patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0708800104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2154464PMC
December 2007

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins.

J Struct Funct Genomics 2007 Sep 12;8(2-3):107-19. Epub 2007 Oct 12.

Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Oxford, UK.

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein-ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10969-007-9027-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140095PMC
September 2007

Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity.

Nature 2007 Jul 24;448(7149):87-91. Epub 2007 Jun 24.

Structural Genomics Consortium, Botnar Research Center, University of Oxford, Oxford OX3 7LD, UK.

Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature05971DOI Listing
July 2007

Insights for the development of specific kinase inhibitors by targeted structural genomics.

Drug Discov Today 2007 May 29;12(9-10):365-72. Epub 2007 Mar 29.

University of Oxford, Structural Genomics Consortium, Botnar Research Centre, Nuffield Othopaedic Centre, Old Road, Oxford, OX3 7LD, UK.

Many protein kinases are validated intervention points for drug development, however active site similarities often lead to a lack of selectivity and unwanted side effects in the clinic. To address this issue, it is desirable to increase the number of high resolution crystal structures and complexes with non-adenosine ligands available for the rational design of more selective inhibitors. Recent progress in protein crystallography and biotechnology has enabled structural genomics projects to target challenging proteins successfully, including protein kinases. As we discuss here, this effort has resulted in a considerable increase in the number of available high resolution structures and inhibitor complexes and has identified novel structural motifs that are available for drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.drudis.2007.03.006DOI Listing
May 2007

Structural basis for protein-protein interactions in the 14-3-3 protein family.

Proc Natl Acad Sci U S A 2006 Nov 3;103(46):17237-42. Epub 2006 Nov 3.

Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD, United Kingdom.

The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0605779103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1859916PMC
November 2006

Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.

Proc Natl Acad Sci U S A 2006 Oct 11;103(43):15835-40. Epub 2006 Oct 11.

Structural Genomics Consortium, University of Toronto, 100 College Street, Toronto, ON, Canada M5G 1L5.

The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening platforms, based on either fluorimetry or static light scattering, to measure the increase in protein thermal stability upon binding of a ligand without the need to monitor enzyme activity. In total, 221 different proteins from humans and human parasites were screened against one or both of two sorts of small-molecule libraries. The first library comprised different salts, pH conditions, and commonly found small molecules and was applicable to all proteins. The second comprised compounds specific for protein families of particular interest (e.g., protein kinases). In 20 cases, including nine unique human protein kinases, a small molecule was identified that stabilized the proteins and promoted structure determination. The methods are cost-effective, can be implemented in any laboratory, promise to increase the success rates of purifying and crystallizing human proteins significantly, and identify new ligands for these proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0605224103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1595307PMC
October 2006

High-throughput structural characterisation of therapeutic protein targets.

Expert Opin Drug Discov 2006 Jul;1(2):123-36

The Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Oxford, OX3 7LD, UK.

The overarching scientific objective for high-throughput structural biology projects targeting human proteins or relevant proteins of human pathogens is to generate a detailed structural insight of biologically relevant structural features of soluble or membrane protein families of therapeutic relevance. These data will better enable structure-guided drug discovery in pharmaceutical and biotechechnology companies. In particular, detailed structural descriptions for entire protein families will be highly valuable for the design of drugs and other bioactive compounds that are selective for individual members or groups of proteins within protein families. In addition to determined structural information, targeted high-throughput structural biology will also provide reagents, methodologies, technologies and small molecules that bind selectively to targets that are suitable as intervention points in the treatment of disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/17460441.1.2.123DOI Listing
July 2006

Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase.

Proc Natl Acad Sci U S A 2006 May 4;103(20):7637-42. Epub 2006 May 4.

Structural Genomics Consortium, Botnar Research Centre, University of Oxford, Oxford OX3 7LD, United Kingdom.

Growth hormone (GH) signaling is tightly controlled by ubiquitination of GH receptors, phosphorylation levels, and accessibility of binding sites for downstream signaling partners. Members of the suppressors of cytokine signaling (SOCS) family function as key regulators at all levels of this pathway, and mouse knockout studies implicate SOCS2 as the primary suppressor. To elucidate the structural basis for SOCS2 function, we determined the 1.9-A crystal structure of the ternary complex of SOCS2 with elongin C and elongin B. The structure defines a prototypical SOCS box ubiquitin ligase with a Src homology 2 (SH2) domain as a substrate recognition motif. Overall, the SOCS box and SH2 domain show a conserved spatial domain arrangement with the BC box and substrate recognition domain of the von Hippel-Lindau (VHL) tumor suppressor protein, suggesting a common mechanism of ubiquitination in these cullin-dependent E3 ligases. The SOCS box binds elongin BC in a similar fashion to the VHL BC box and shows extended structural conservation with the F box of the Skp2 ubiquitin ligase. A previously unrecognized feature of the SOCS box is revealed with the burial of the C terminus, which packs together with the N-terminal extended SH2 subdomain to create a stable interface between the SOCS box and SH2 domain. This domain organization is conserved in SOCS1-3 and CIS1, which share a strictly conserved length of their C termini, but not in SOCS4, 5, and 7, which have extended C termini defining two distinct classes of inter- and intramolecular SOCS box interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0601638103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1472497PMC
May 2006

Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening.

Proteins 2006 Jul;64(1):60-7

Department of Chemistry, Nerviano Medical Sciences, Nerviano, MI, Italy.

The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prot.20955DOI Listing
July 2006

Structural proteomics: lessons learnt from the early case studies.

Farmaco 2002 Nov;57(11):947-51

Biovitrum, Department of Structural Chemistry, Nordenflychtsvägen 62:6, SE-112 76 Stockholm, Sweden.

The genomics efforts have identified a large number of novel genes and thus provided a pool of interesting but not functionally characterized target proteins. It has been suggested that structural proteomics will significantly impact the success rate of functional characterization of such identified genes and proteins by providing structure-function hypotheses by fold and feature recognition and analysis. Structural proteomics initiatives, both in academic and industrial settings, are today generating protein structures at an unprecedented rate although relatively few large-scale efforts have been displayed in the public domain. However, a number of individual studies have provided a 'road-map' for selected approaches that hold the promise to significantly impact the process of deriving function from structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0014-827x(02)01212-0DOI Listing
November 2002

Structural proteomics: developments in structure-to-function predictions.

Trends Biotechnol 2002 Feb;20(2):79-84

Biovitrum, Department of Structural Chemistry., Stockholm, Sweden.

The major challenge for post-genomic research is to functionally assign and validate a large number of novel target genes and their corresponding proteins. Functional genomics approaches have, therefore, gained considerable attention in the quest to convert this massive data set into useful information. One of the crucial components for the functional understanding of unassigned proteins is the analysis of their experimental or modeled 3D structures. Structural proteomics initiatives are generating protein structures at an unprecedented rate but our current knowledge of 3D-structural space is still limited. Estimates on the completeness of the 3D-structural coverage of proteins vary but it is generally accepted that only a minority of the structural proteome has a template structure from which reliable conclusions can be drawn. Thus, structural proteomics has set out to build a map of protein structures that will represent all protein folds included in the 'global proteome'.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0167-7799(01)01884-4DOI Listing
February 2002