Biosensor Analysis of Drug–Target Interactions: Direct and Competitive Binding Assays for Investigation of Interactions between Thrombin and Thrombin Inhibitors

Karlsson, Robert; Kullman-Magnusson, Mari; Hämäläinen, M.; Remaeus, Annika; Andersson, Karl; Borg, Peter; Gyzander, Erika; Deinum, Johanna

doi:10.1006/abio.2001.5026

Cited by 33 publications

(48 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Zero-concentration blank buffer cycles were included as negative control samples. Solvent-correction procedures were included to compensate for any DMSO-related bulk refractive index variations and performed as described (34). Nonspecific odorant binding to antipolyhistidine-derivatized sensor surfaces was absent for all analyses reported.…”

Section: Methodsmentioning

confidence: 99%

“…Data were prepared by subtraction of reference surface data and blank buffer sample data, a procedure commonly referred to as ''double referencing'' (35). Solvent correction was applied as described (34). A plot of the corrected equilibrium odorant binding responses versus odorant concentration was fitted by using the following equation to yield the K D value: Req ϭ KA C Rmax/KA C ϩ 1, where Req is the odorant binding response at equilibrium, C is the odorant concentration, Rmax is the maximum binding capacity of the captured receptor, and KA is the association equilibrium constant.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

Kaiser

Graveland-Bikker

Steuerwald

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

132

108

View full text Add to dashboard Cite

High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a ␣-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.detergent screen ͉ G protein-coupled receptor purification ͉ membrane protein ͉ odorant interaction ͉ surface plasmon resonance

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

Kaiser

Graveland-Bikker

Steuerwald

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

132

108

View full text Add to dashboard Cite

show abstract

“…The major advantages are low target consumption (a few mg protein/microplate compounds), the possibility to simultaneously use reference proteins (to discriminate between selective and promiscuous binders) and that it resolves affinity into on/off-rate constants, improving structure-activity relationship studies. The study of direct binding between low molecular weight compounds and target molecules was developed by Helena Danielson at Uppsala University, Sweden in collaboration with scientists at Biacore, AB (Markgren et al, 1998(Markgren et al, , 2002Alterman et al, 2001;Hämäläinen et al, 2000;Karlsson et al, 2000). The use of optical biosensors in drug discovery has recently been reviewed (Cooper, 2002(Cooper, , 2003aMyszka and Rich, 2000;Rich and Myszka, 2004;Karlsson, 2004;Löfås, 2004).…”

Section: Introductionmentioning

confidence: 96%

A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions

Huber

2005

J. Mol. Recognit.

View full text Add to dashboard Cite

Biophysical label-free assays such as those based on SPR are essential tools in generating high-quality data on affinity, kinetic, mechanistic and thermodynamic aspects of interactions between target proteins and potential drug candidates. Here we show examples of the integration of SPR with bioinformatic approaches and mutation studies in the early drug discovery process. We call this combination 'structure-based biophysical analysis'. Binding sites are identified on target proteins using information that is either extracted from three-dimensional structural analysis (X-ray crystallography or NMR), or derived from a pharmacore model based on known binders. The binding site information is used for in silico screening of a large substance library (e.g. available chemical directory), providing virtual hits. The three-dimensional structure is also used for the design of mutants where the binding site has been impaired. The wild-type target and the impaired mutant are then immobilized on different spots of the sensor chip and the interactions of compounds with the wild-type and mutant are compared in order to identify selective binders for the binding site of the target protein. This method can be used as a cost-effective alternative to high-throughput screening methods in cases when detailed binding site information is available. Here, we present three examples of how this technique can be applied to provide invaluable data during different phases of the drug discovery process.

show abstract

“…After a period of silence, several SPR papers appeared on the topic of conformation-dependent sensing despite many considering the reports to be simply anomalous behavior (43)(44)(45)(46)(47), attributed to the following: (i) buffer mismatch, (ii) volume exclusion due to ligand density differences (43)(44)(45), (iii) nonspecific matrix interaction (46), and (iv) nonspecific reference interactions (47). Regardless, a recent paper reported that the RI sensing figures of merit were dependent on shape and the size of the Au nanoparticles (48) with sensitivities generally increasing as the nanoparticles became elongated and their apexes become sharper.…”

Section: Introductionmentioning

confidence: 99%

Origin and prediction of free-solution interaction studies performed label-free

Bornhop

Kammer

Kussrow

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for freesolution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R 2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.backscattering interferometry | assay methodology | molecular interactions | conformation change | hydration change C ontemporary assays enabling single-molecule detection (1,2) have accelerated the sequencing of the human genome (3) and facilitated imaging with extraordinary resolution without labels (4). To most closely approximate the natural state, an interaction assay methodology would interrogate the processes (reaction, molecular interaction, protein folding event, etc.) without perturbation. Label-free chemical and biochemical investigations (5, 6) transduce the desired signal without an exogenous label (fluorescent, radioactive, or otherwise) representing an essential step toward this goal. Many label-free methods require one of the interacting species to be either tethered or immobilized to the sensor surface, introducing a potential perturbation to the natural state of the species (7,8). However, back-scattering interferometry (BSI) is a free-solution label-free technique with the added benefit of sensitivity that rivals fluorescence (9). There are other techniques performed in free solution, such as MS (10, 11) and NMR (12,13) and the widely used isothermal titration calorimetry (ITC) (14, 15). As with NMR, ITC has many advantages, but exhibits modest sensitivity and often requires large sample quantities. Another increasingly popular free-solution approach is microscale thermophoresis (...

show abstract

Biosensor Analysis of Drug–Target Interactions: Direct and Competitive Binding Assays for Investigation of Interactions between Thrombin and Thrombin Inhibitors

Cited by 33 publications

References 0 publications

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

Efficient cell-free production of olfactory receptors: Detergent optimization, structure, and ligand binding analyses

A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions

Origin and prediction of free-solution interaction studies performed label-free

Contact Info

Product

Resources

About