ZoBio. BV

Leiden, Netherlands

ZoBio. BV

Leiden, Netherlands
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Grant
Agency: European Commission | Branch: FP7 | Program: MC-ITN | Phase: PEOPLE-2007-1-1-ITN | Award Amount: 5.80M | Year: 2008

Membrane proteins (MPs) are known to be key molecules in cellular communications, from signal transduction to transport of ions, metabolites and other molecules. They also participate in the synthesis of ATP, the import of soluble or MPs from the cytosol, and they protect living organisms from toxic factors. The proposal consists in a joint training effort involving the major biophysical methods that are -or soon will be- the major techniques used in the field of structural biology of MPs. A collaborative effort is essential for the training of the future generation of biologists dedicated to membrane proteins. It will pave the way to an integrative approach for the study of structure-function relationships of membranes. It will therefore open new strategies for structure-based drug design, in particular toward G-protein coupled receptors (GPCR), which are major drug targets (GPCRs represent 30% of current drug targets). The training proposed in this program will not only form high-level academic researchers but will also largely contribute in forming the main actors of the future developments in biotechnology and personalized medicine. This network combines 12 academic research groups and 3 industrial companies interested in collaborating with these groups and involved in drug discovery or scientific equipment for SBMP. These groups are internationally recognized for analysing the structure and dynamics of membrane proteins by a combination of experimental and theoretical approaches: in vivo and in vitro expressions systems, functional/biochemical/biophysical characterisation, X-Ray diffraction, electron microscopy (EM), atomic force microscopy (AFM), single-molecule force spectroscopy (SMFS), liquid and solid state NMR, numerical simulations. Seven partners from 6 different countries are involved: France, Poland, Portugal, Switzerland, Germany and the Netherlands.


Pritisanac I.,University of Oxford | Degiacomi M.T.,University of Oxford | Alderson T.R.,University of Oxford | Carneiro M.G.,ZoBio BV | And 3 more authors.
Journal of the American Chemical Society | Year: 2017

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy. © 2017 American Chemical Society.


Chen D.,ZoBio BV | Errey J.C.,Heptares Therapeutics | Heitman L.H.,Leiden University | Marshall F.H.,Heptares Therapeutics | And 3 more authors.
ACS Chemical Biology | Year: 2012

Fragment-based drug discovery (FBDD) has proven a powerful method to develop novel drugs with excellent oral bioavailability against challenging pharmaceutical targets such as protein-protein interaction targets. Very recently the underlying biophysical techniques have begun to be successfully applied to membrane proteins. Here we show that novel, ligand efficient small molecules with a variety of biological activities can be found by screening a small fragment library using thermostabilized (StaR) G protein-coupled receptors (GPCRs) and target immobilized NMR screening (TINS). Detergent-solubilized StaR adenosine A2A receptor was immobilized with retention of functionality, and a screen of 531 fragments was performed. Hits from the screen were thoroughly characterized for biochemical activity using the wild-type receptor. Both orthosteric and allosteric modulatory activity has been demonstrated in biochemical validation assays. Allosteric activity was confirmed in cell-based functional assays. The validated fragment hits make excellent starting points for a subsequent hit-to-lead elaboration program. © 2012 American Chemical Society.


Shah D.M.,Leiden University | Shah D.M.,ZoBio BV | Ab E.,ZoBio BV | Diercks T.,Center for Co operative Research in Biosciences | And 4 more authors.
Journal of Medicinal Chemistry | Year: 2012

An efficient way to rapidly generate protein-ligand costructures based on solution-NMR using sparse NOE data combined with selective isotope labeling is presented. A docked model of the 27 kDa N-terminal ATPase domain of Hsp90 bound to a small molecule ligand was generated using only 21 intermolecular NOEs, which uniquely defined both the binding site and the orientation of the ligand. The approach can prove valuable for the early stages of fragment-based drug discovery. © 2012 American Chemical Society.


Van Melckebeke H.,ETH Zurich | Wasmer C.,ETH Zurich | Lange A.,ETH Zurich | Lange A.,Max Planck Institute for Biophysical Chemistry | And 5 more authors.
Journal of the American Chemical Society | Year: 2010

We present a strategy to solve the high-resolution structure of amyloid fibrils by solid-state NMR and use it to determine the atomic-resolution structure of the prion domain of the fungal prion HET-s in its amyloid form. On the basis of 134 unambiguous distance restraints, we recently showed that HET-s(218-289) in its fibrillar state forms a left-handed β-solenoid, and an atomic-resolution NMR structure of the triangular core was determined from unambiguous restraints only. In this paper, we go considerably further and present a comprehensive protocol using six differently labeled samples, a collection of optimized solid-state NMR experiments, and adapted structure calculation protocols. The high-resolution structure obtained includes the less ordered but biologically important C-terminal part and improves the overall accuracy by including a large number of ambiguous distance restraints. © 2010 American Chemical Society.


Kobayashi M.,ZoBio B.V | Retra K.,VU University Amsterdam | Figaroa F.,ZoBio B.V | Hollander J.G.,ZoBio B.V | And 5 more authors.
Journal of Biomolecular Screening | Year: 2010

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM KD is limited. Similarly, they find that SPR detection is most suited to ligands that bind with KD better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies. © 2010 Society for Laboratory Automation and Screening.


Fruh V.,Leiden University | Zhou Y.,University of Virginia | Chen D.,ZoBio BV | Loch C.,ZoBio BV | And 6 more authors.
Chemistry and Biology | Year: 2010

Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here, we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The Escherichia coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1071 drug fragments for binding using target immobilized NMR Screening. Biochemical and biophysical validation of the eight most potent hits revealed an IC50 range of 7-200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins. © 2010 Elsevier Ltd.


Chen D.,ZoBio BV | Ranganathan A.,Science for Life Laboratory | Ranganathan A.,University of Stockholm | Ijzerman A.P.,Leiden University | And 4 more authors.
Journal of Chemical Information and Modeling | Year: 2013

Fragment-based lead discovery (FBLD) is becoming an increasingly important method in drug development. We have explored the potential to complement NMR-based biophysical screening of chemical libraries with molecular docking in FBLD against the A2A adenosine receptor (A2AAR), a drug target for inflammation and Parkinson's disease. Prior to an NMR-based screen of a fragment library against the A2AAR, molecular docking against a crystal structure was used to rank the same set of molecules by their predicted affinities. Molecular docking was able to predict four out of the five orthosteric ligands discovered by NMR among the top 5% of the ranked library, suggesting that structure-based methods could be used to prioritize among primary hits from biophysical screens. In addition, three fragments that were top-ranked by molecular docking, but had not been picked up by the NMR-based method, were demonstrated to be A2AAR ligands. While biophysical approaches for fragment screening are typically limited to a few thousand compounds, the docking screen was extended to include 328,000 commercially available fragments. Twenty-two top-ranked compounds were tested in radioligand binding assays, and 14 of these were A2AAR ligands with Ki values ranging from 2 to 240 μM. Optimization of fragments was guided by molecular dynamics simulations and free energy calculations. The results illuminate strengths and weaknesses of molecular docking and demonstrate that this method can serve as a valuable complementary tool to biophysical screening in FBLD. © 2013 American Chemical Society.


Grant
Agency: European Commission | Branch: H2020 | Program: MSCA-ITN-ETN | Phase: MSCA-ITN-2015-ETN | Award Amount: 3.84M | Year: 2016

The promise of more efficient lead discovery is fuelling the enthusiasm for fragment-based lead discovery (FBLD). In this approach, highly sensitive biochemical and biophysical screening technologies are being used to detect the low affinity binding of low molecular weight compounds (the so-called fragments) to protein targets that are involved in pathophysiological processes. By investigating the molecular interactions between fragment hit(s) and the target protein, a detailed understanding of the binding event is obtained. This enables the rational and efficient optimisation of the hit fragment. The optimised compounds represent high quality leads for drug development. The necessary FBLD technologies and approaches have emerged mainly from small and specialised biotech companies. At present, FBLD is being adopted throughout pharmaceutical sciences, including by pharmaceutical companies, SMEs and academic research groups. So far, the necessary training that is needed to obtain an holistic view of the possibilities and opportunities that FBLD provides is missing, most likely because the highly multidisciplinary nature of the FBLD work is difficult to capture within one (academic) institute. Therefore, we have established FragNet as a dedicated FBLD training network. The consortium consists of the most prominent pharmaceutical companies, biotech companies and academic groups that have jointly shaped the FBLD research area. FragNet is committed to train 15 ESRs in all facets of FBLD using the combined technologies, skills and knowledge. This will include both research (e.g., technologies on the interface of chemistry and biology) and transferable skill sets (e.g., writing, media training, entrepreneurship and thorough understanding of scientific knowledge transfer). This will enable the ESRs to excel in todays drug discovery and chemical biology programmes that are performed in public and private organisations in the pharmaceutical sciences.


Kobayashi M.,Leiden University | Kobayashi M.,ZoBio BV | Ab E.,University Utrecht | Ab E.,ZoBio BV | And 2 more authors.
Journal of Biological Chemistry | Year: 2010

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5′-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra α-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5′-phosphate recognition by the BRCT domains of bacterial NAD +-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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