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1.
Chimia (Aarau) ; 75(11): 936-942, 2021 Nov 24.
Article in English | MEDLINE | ID: mdl-34798915

ABSTRACT

We present a short overview of the way Novartis chemists interact and collaborate with the academic chemistry community in Switzerland. This article exemplifies a number of collaborations, and illustrates opportunities to foster research synergies between academic and industrial researchers. It also describes established programs available to academic groups, providing them access to Novartis resources and expertise.


Subject(s)
Industry , Research Personnel , Humans , Switzerland
2.
Protein Expr Purif ; 167: 105545, 2020 03.
Article in English | MEDLINE | ID: mdl-31778786

ABSTRACT

The generation of integral membrane proteins (IMPs) in heterologous systems and their characterization remains a major challenge in biomedical research. Significant efforts have been invested both in academia and in the pharmaceutical industry to establish technologies for the expression, isolation and characterization of IMPs. Here we summarize some of the key aspects, which are important to support structure-based drug design (SBDD) in drug discovery projects. We furthermore include timeline estimates and an overview of the target selection and biophysical screening approaches.


Subject(s)
Membrane Proteins , Animals , Antibodies , Baculoviridae/genetics , Biophysics , Cell Line , Drug Design , Drug Industry , Gene Expression , Humans , Insecta/genetics , Mammals/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/chemistry , Membrane Proteins/immunology , Membrane Proteins/isolation & purification , Receptors, G-Protein-Coupled/biosynthesis , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/immunology , Receptors, G-Protein-Coupled/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification
3.
Nature ; 574(7779): 581-585, 2019 10.
Article in English | MEDLINE | ID: mdl-31645725

ABSTRACT

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3-5. Because SUCNR1 senses succinate as an immunological danger signal6-which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8-it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.


Subject(s)
Biphenyl Compounds/chemistry , Biphenyl Compounds/pharmacology , Piperazines/chemistry , Piperazines/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/chemistry , Animals , Apoproteins/antagonists & inhibitors , Apoproteins/chemistry , Apoproteins/metabolism , Crystallography, X-Ray , Humans , Models, Molecular , Rats , Receptors, G-Protein-Coupled/metabolism , Receptors, Purinergic P2Y1/chemistry , Signal Transduction , Single-Domain Antibodies/chemistry , Species Specificity , Succinic Acid/metabolism
4.
J Biomol Screen ; 21(3): 243-51, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26644402

ABSTRACT

Integral membrane proteins (IMPs) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand-target interactions, robust, reliable, high-throughput, and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimetry (DSF) screening method using the thiol-reactive BODIPY FL-cystine dye to monitor thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2. To validate this method, the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners.


Subject(s)
Coloring Agents , Drug Discovery/methods , Fluorometry/methods , Ligands , Receptors, Interleukin-8B/metabolism , Sulfhydryl Compounds , Calorimetry, Differential Scanning/methods , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Membrane Proteins/metabolism , Protein Binding , Protein Stability , Protein Unfolding , Receptors, Interleukin-8B/chemistry , Receptors, Interleukin-8B/genetics , Receptors, Interleukin-8B/isolation & purification , Recombinant Fusion Proteins , Small Molecule Libraries
5.
Biophys Chem ; 165-166: 56-61, 2012 May.
Article in English | MEDLINE | ID: mdl-22542136

ABSTRACT

To broaden the use of the recombinant high-density lipoprotein (rHDL) approach to the characterization of lead compounds, we investigated the pharmacology of the human beta-2-adrenoceptor in nanolipid bilayers (rHDL) with a broad set of beta-adrenoceptor antagonists. To that end, we developed a homogeneous copper-chelate scintillation proximity binding assay (SPA) in order to compare receptor-ligand binding affinities before and after reconstitution into rHDLs. Our results clearly show that the beta-2-adrenoceptor reconstituted in rHDLs display the same pharmacology as that in cell membranes and that rHDLs can be used not only to measure affinities for a range of ligands but also to study binding kinetics.


Subject(s)
Drug Discovery , Lipid Bilayers/metabolism , Lipoproteins, HDL/metabolism , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-2 Receptor Antagonists/metabolism , Animals , Apolipoprotein A-I/metabolism , HEK293 Cells , Humans , Kinetics , Lipid Bilayers/chemistry , Models, Molecular , Protein Binding , Protein Conformation , Receptors, Adrenergic, beta-2/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
6.
PLoS Biol ; 6(2): e44, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18303950

ABSTRACT

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein-VirE2-enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated-using single-molecule techniques-that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.


Subject(s)
Bacterial Proteins/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Ion Channels/metabolism , Rhizobium/metabolism , Base Sequence , Biological Transport , Biopolymers/metabolism , DNA Primers , Kinetics , Protein Binding
7.
J Struct Biol ; 159(2): 179-93, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17368047

ABSTRACT

In native tissues, the majority of medically important membrane proteins is only present at low concentrations, making their overexpression in recombinant systems a prerequisite for structural studies. Here, we explore the commonly used eukaryotic expression systems-yeast, baculovirus/insect cells (Sf9) and Semliki Forest Virus (SFV)/mammalian cells-for the expression of seven different eukaryotic membrane proteins from a variety of protein families. The expression levels, quality, biological activity, localization and solubility of all expressed proteins are compared in order to identify the advantages of one system over the other. SFV-transfected mammalian cell lines provide the closest to native environment for the expression of mammalian membrane proteins, and they exhibited the best overall performance. But depending on the protein, baculovirus-infected Sf9 cells performed almost as well as mammalian cells. The lowest expression levels for the proteins tested here were obtained in yeast.


Subject(s)
Gene Expression , Membrane Proteins/metabolism , Animals , Cell Line , Genetic Vectors , Humans , Membrane Proteins/genetics , Patch-Clamp Techniques , Plasmids/genetics , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solubility
8.
Mol Microbiol ; 58(4): 1130-42, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16262795

ABSTRACT

The VirE2 protein is crucial for the transfer of single-stranded DNA (ssDNA) from Agrobacterium tumefaciens to the nucleus of the plant host cell because of its ssDNA binding activity, assistance in nuclear import and putative ssDNA channel activity. The native form of VirE2 in Agrobacterium's cytoplasm is in complex with its specific chaperone, VirE1. Here, we describe the ability of the VirE1VirE2 complex to both bind ssDNA and form channels. The affinity of the VirE1VirE2 complex for ssDNA is slightly reduced compared with VirE2, but the kinetics of binding to ssDNA are unaffected by the presence of VirE1. Upon binding of VirE1VirE2 to ssDNA, similar helical structures to those reported for the VirE2-ssDNA complex were observed by electron microscopy. The VirE1VirE2 complex can release VirE1 once the VirE2-ssDNA complexes assembled. VirE2 exhibits a low affinity for small unilamellar vesicles composed of bacterial lipids and a high affinity for lipid vesicles containing sterols and sphingolipids, typical components of animal and plant membranes. In contrast, the VirE1VirE2 complex associated similarly with all kind of lipids. Finally, black lipid membrane experiments revealed the ability of the VirE1VirE2 complex to form channels. However, the majority of the channels displayed a conductance that was a third of the conductance of VirE2 channels. Our results demonstrate that the binding of VirE1 to VirE2 does not inhibit VirE2 functions and that the effector-chaperone complex is multifunctional.


Subject(s)
Agrobacterium tumefaciens/metabolism , Bacterial Proteins/metabolism , DNA, Single-Stranded/metabolism , DNA-Binding Proteins/metabolism , Ion Channels/metabolism , Molecular Chaperones/metabolism , Electrophoretic Mobility Shift Assay , Lipid Metabolism , Macromolecular Substances/metabolism , Membranes/metabolism , Microscopy, Electron
9.
FEMS Microbiol Lett ; 223(1): 1-6, 2003 Jun 06.
Article in English | MEDLINE | ID: mdl-12798992

ABSTRACT

Agrobacterium tumefaciens has evolved a unique mechanism to solve the problem of transferring DNA across five bilayers; the inner and outer membranes of the bacterium, the plasma membrane of the plant cell and the double membrane formed by the nuclear envelope. The two first and two last seem to be mediated by, respectively, the type IV secretion system in Agrobacterium and the nuclear pore complex in the plant cell, but the mechanism by which the transferred DNA (T-DNA) crosses the plant membrane still remains a mystery. New biophysical experiments suggest that, in addition to its established role as a single-stranded DNA (ssDNA)-binding protein, the VirE2 protein forms a channel in the plant membrane allowing the passage of the T-DNA into the cell. Such a role would be reminiscent of translocator molecules secreted by the type III secretion system of pathogenic bacteria and inserting into the host eukaryotic plasma membrane. The implications for the structure of the protein, its regulation and role in vivo are discussed.


Subject(s)
Agrobacterium tumefaciens/genetics , Bacterial Proteins , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Ion Channels/genetics , Ion Channels/metabolism , DNA-Binding Proteins/chemistry , Ion Channels/chemistry , Protein Structure, Tertiary
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