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Kenneth Lundstrom

Publications and source records attributed to Kenneth Lundstrom.

At least 19 recordsLinked to original sources
PubMed2006

Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems.

Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.

Animals↗
PubMed2006

Structural genomics: the ultimate approach for rational drug design.

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 success rate for structure resolution of membrane proteins structural genomics networks have been established.

Animals↗
PubMed2006

Latest development in drug discovery on G protein-coupled receptors.

G protein-coupled receptors (GPCRs) represent the family of proteins with the highest impact from social, therapeutic and economic point of view. Today, more than 50% of drug targets are based on GPCRs and the annual worldwide sales exceeds 50 billion dollars. GPCRs are involved in all major disease areas such as cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases. The classical drug discovery process has relied on screening compounds, which interact favorably with the GPCR of interest followed by further chemical engineering as a mean of improving efficacy and selectivity. In this review, methods for sophisticated chemical library screening procedures will be presented. Furthermore, development of cell-based assays for functional coupling of GPCRs to G proteins will be discussed. Finally, the possibility of applying structure-based drug design will be summarized. This includes the application of bioinformatics knowledge and molecular modeling approaches in drug development programs. The major efforts established through large networks of structural genomics on GPCRs, where recombinantly expressed GPCRs are subjected to purification and crystallization attempts with the intention of obtaining high-resolution structures, are presented as a promising future approach for tailor-made drug development.

Animals↗
PubMed2005

Structural biology of G protein-coupled receptors.

More than 60% of the current drugs are based on G protein-coupled receptors. Paradoxically, high-resolution structures are not available to facilitate rational drug design. Difficulties in expression, purification, and crystallization of these transmembrane receptors are the reasons for the low success rate. Recent individual and network-based technology development has significantly improved our knowledge of structural biology and might soon bring a major breakthrough in this area.

Animals↗
PubMed2005

Semliki Forest virus vectors for overexpression of 101 G protein-coupled receptors in mammalian host cells.

Semliki Forest virus vectors were applied for the evaluation of 101 G protein-coupled receptors in three mammalian cell lines. Western blotting demonstrated that 95 of the 101 tested GPCRs showed positive signals. A large number of the GPCRs were expressed at high levels suggesting receptor yields in the range of 1 mg/L or higher, suitable for structural biology applications. Specific binding assays on a selected number of GPCRs were carried out to compare the correlation between total and functional protein expression. Ligands and additives supplemented to the cell culture medium were evaluated for expression enhancement. Selected GPCRs were also expressed from mutant SFV vectors providing enhanced protein expression and reduced host cell toxicity in attempts to further improve receptor yields.

Animals↗
PubMed2005

Structural genomics of GPCRs.

G protein-coupled receptors (GPCRs) are targets for 60-70% of drugs in development today. Traditionally, the drug discovery process has relied on screening of chemical compounds to identify novel and more-efficient drug molecules. Structure-based drug design, however, provides a targeted approach but has been severely hampered by limited knowledge of high-resolution structures of GPCRs owing to the difficulties encountered in their expression, purification and crystallization. In addition to individual laboratories studying specific GPCRs, structural genomics initiatives have been established as large networks with a wide range of expertise in protein expression, purification and crystallography. Several of these national and international consortia have included GPCRs in their programs. Milligram quantities of GPCRs can now be expressed in several expression systems and purified to high homogeneity. However, success in crystallization still requires major technological improvement.

Crystallization↗
PubMed2005

The future of G protein-coupled receptors as targets in drug discovery.

G protein-coupled receptors (GPCRs) represent the most abundant drug targets today. A large number of GPCR-based drugs have already been developed for a variety of indications in human disease. However, orphan receptors with unidentified ligands serve as potential targets still to be explored. Moreover, research on the interaction of GPCRs with different molecules in the signal transduction pathways, and further studies on receptor dimerization may also lead to the discovery of new drugs. Structure-based drug design will eventually play a key role in generating better and more selective drugs more rapidly when high-resolution structures of GPCRs can be provided by expression, purification and crystallography technologies.

Animals↗
PubMed2004

Gene therapy applications of viral vectors.

Viral vectors have frequently been applied in gene therapy with the final goal of treating various diseases in the areas of neurology, neurodegeneration, metabolic disease, and cancer. Vectors have been engineered based on AAV, adenoviruses, alphaviruses, herpes simplex viruses, lentiviruses, and retroviruses. Some vectors are suitable for short-term episomal transgene expression, whereas others are integrated into the host cell genome to provide long-term expression. Additionally, hybrid vectors with favorable features from different viruses have been developed. Therapeutic genes of choice have typically been toxic genes such as thymidine kinase, pro-apoptotic genes like Bax, and immunostimulatory genes (for instance, interleukin-12). A large number of animal studies have demonstrated proof of concept of viral gene therapy. Many types of viral vectors have been employed in more than 700 clinical trials that have been carried out or are currently in progress.

Adenoviridae↗
PubMed2004

Structural genomics on membrane proteins: the MePNet approach.

Of the 25,000 crystal structure entries deposited in public databases less than 1% are for membrane proteins; this gives a clear indication of the difficulties relating to structural biology and determination of this type of protein. To date, structural genomics networks have mainly focused on soluble proteins, despite the fact that membrane proteins constitute 60 to 70% of current drug targets. Technological improvement on a broad basis is the key to higher success rates, but this is generally not possible unless large functional networks are established. Several structural genomics initiatives, concentrating partly or fully on membrane proteins, have been established during the last few years and the Membrane Protein Network (MePNet; BioXtal, Switzerland) focuses specifically on the pharmaceutically attractive G protein-coupled receptors (GPCRs). Within MePNet, over 100 full-length GPCRs have been subjected to overexpression in bacterial, yeast and mammalian cells, followed by purification and crystallization studies.

Animals↗
PubMed2003

Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins.

Semliki Forest virus (SFV) vectors have been applied for the expression of recombinant integral membrane proteins in a wide range of mammalian host cells. More than 50 G protein-coupled receptors (GPCRs), several ion channels and other types of transmembrane or membrane-associated proteins have been expressed at high levels. The establishment of large-scale SFV technology has facilitated the production of large quantities of recombinant receptors, which have then been subjected to drug screening programs and structure-function studies on purified receptors. The recent Membrane Protein Network (MePNet) structural genomics initiative, where 100 GPCRs are overexpressed from SFV vectors, will further provide new methods and technologies for expression, solubilization, purification and crystallization of GPCRs.

Animals↗
PubMed2003

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors.

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed2003

Latest development in viral vectors for gene therapy.

Gene therapy includes the application of various viral vectors, which represent most types and families of viruses, suitable for infection of mammalian host cells. Both hereditary diseases and acquired illnesses, such as cancer, can be targeted. Because of the various properties of each viral vector, the definition of their application range depends on factors such as packaging capacity, host range, cell- or tissue-specific targeting, replication competency, genome integration and duration of transgene expression. Recent engineering of modified viral vectors has contributed to improved gene delivery efficacy.

Adenoviridae↗
PubMed2003

Novel Semliki Forest virus vectors with reduced cytotoxicity and temperature sensitivity for long-term enhancement of transgene expression.

Alphaviral vectors inhibit host cell protein synthesis and are cytotoxic. To overcome these limitations, we modified the nonstructural protein-2 (nsP2) gene in the Semliki Forest virus (SFV) vector, pSFV1. Packaging of SFV replicons with two point mutations in nsP2 resulted in high-titer recombinant SFV(PD) particles. SFV(PD) led to more efficient host cell protein synthesis, exhibited reduced cytotoxicity and improved cell survival, and allowed greater and prolonged transgene expression than the original vector, SFV. In dissociated hippocampal neurons and organotypic rat hippocampal slices, SFV(PD) infection preserved neuronal morphology and synaptic function more efficiently than SFV. Combination of the two point mutations with a replication-persistent mutation in nsP2 resulted in a highly temperature-sensitive vector, SFV(PD713P), which efficiently transduced neurons in hippocampal slice cultures. At 31 degrees C, SFV(PD713P) allowed continuous transgene expression in BHK cells, at amounts comparable to SFV(PD). These new SFV mutants are expected to substantially broaden the application of alphaviral vectors in neurons and other mammalian cells.

Animals↗
PubMed2003

Homer-1a/Vesl-1S enhances hippocampal synaptic transmission.

Homer/Vesl proteins are involved in regulating metabotropic glutamate receptors, synaptogenesis, dendritic spine development and axonal pathfinding. We investigated the potential modulation of glutamatergic synaptic transmission by the immediate early gene product Homer-1a/Vesl-1S and by the constitutively expressed long-form Homer-1c/Vesl-1L in CA1 pyramidal cells from cultured rat hippocampal slices. Semliki Forest virus vector-mediated overexpression of Homer-1a enhanced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function, but did not detectably affect N-methyl-d-aspartate (NMDA) receptor function and presynaptic glutamate release. Overexpression of Homer-1c, by contrast, did not alter synaptic transmission. To corroborate our electrophysiological results obtained in slice cultures, we performed quantitative immunocytochemistry in cultures of dissociated hippocampal neurons. Homer-1a also increased synaptic clustering of AMPA but not NMDA receptors, whereas Homer-1c had no detectable effect. Our results show that Homer-1a potentiates synaptic AMPA receptor function, supporting a critical role for Homer-1a in hippocampal synaptic plasticity.

Animals↗
PubMed2003

Semliki Forest virus A7(74) transduces hippocampal neurons and glial cells in a temperature-dependent dual manner.

In central nervous system (CNS) tissue preparations, wild-type Semliki Forest virus (SFV) mainly infects neurons, and in vivo it causes lethal encephalitis in neonatal and adult rodents. The SFV strain A7(74), by contrast, is avirulent in adult rodents, triggering only limited CNS infection. To examine A7(74) infection in hippocampal tissue, the authors constructed a replicon, termed SFV(A774nsP)-GFP, expressing green fluorescent protein. The results were compared to replication-proficient recombinant A7(74) encoding GFP, named VA7-EGFP. As nonstructural gene mutations can confer temperature sensitivity, the authors also tested whether infection was temperature-dependent. Indeed, at 31 degrees C both viral recombinants transduced significantly more baby hamster kidney cells than at 37 degrees C. When rat hippocampal slices and dissociated cells were incubated at 37 degrees C, SFV(A774nsP)-GFP transduced glial cells but virtually no neurons-the opposite of conventional SFV. For VA7-EGFP at 37 degrees C, the preferred GFP-positive cells in hippocampal slices were also non-neuronal cells. At 31 degrees C, however, a more wild-type phenotype was found, with 33% and 94% of the GFP-positive cells being neurons for SFV(A774nsP)-GFP in slices and dissociated cells, respectively, and 94% neurons for VA7-EGFP in slices. Immunochemical and electrophysiological analyses confirmed that at 37 degrees C virtually all cells transduced by SFV(A774nsP)-GFP in slices were astrocytes, while at 31 degrees C they also contained neurons. These results show that in addition to the developmental age, the temperature determines which cell type becomes infected by A7(74). Our data suggest that A7(74) is avirulent in adult animals because it does not readily replicate in mature neurons at body temperature, whereas it still does so at lower temperatures.

Animals↗
PubMed2003

Viral and non-viral vectors in gene therapy: technology development and clinical trials.

Gene therapy as part of modern molecular medicine holds great promise for the treatment of both acute and chronic diseases and has the potential to bring a revolutionary era to cancer treatment. Gene therapy has been named the medicine of the future. For the past 10 years various viral and non-viral vectors have been engineered for improved gene and drug delivery. Although various diseases have been targeted, cancer therapy has been addressed to a large extent because of the straight forward approach. Delivery of toxic or immunostimulatory genes by viral and non-viral vectors has been investigated and encouraging results have been obtained in animal models. A large number of clinical trials have been conducted with some highly promising outcome. We propose that combinations of viruses with liposomes or polymers will solve the problem of systemic viral delivery and tumor targeting, bringing a revolution in molecular medicine and in applications of gene therapy in humans.

Animals↗