BSI Technology Literature
Journal references, abstracts, presentations, and company collateral are excellent resources for exploring the many ways in which BSI technology is being put to work in life science and medical research. We encourage you to return often to this page to keep abreast of new developments in Back-Scattering Interferometry.
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Fjellström O1, Larsson N2, Yasuda S3, Tsuchida T3, Oguma T3, Marley A4, Wennberg-Huldt C5, Hovdal D6, Fukuda H7, Yoneyama Y7, Sasaki K3, Johansson A1, Lundqvist S2, Brengdahl J2, Isaacs RJ8, Brown D8, Geschwindner S2, Benthem L5, Priest C4, Turnbull A9.
1Medicinal Chemistry CVMD iMed, AstraZeneca R&D Gothenburg, Mölndal, Sweden.
2Discovery Sciences, AstraZeneca R&D Gothenburg, Mölndal, Sweden.
3Pharmacology Research Laboratories II, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
4Discovery Sciences, AstraZeneca R&D, Mereside, United Kingdom.
5Bioscience CVMD iMed, AstraZeneca R&D Gothenburg, Mölndal, Sweden.
6DMPK CVMD iMed, AstraZeneca R&D Gothenburg, Mölndal, Sweden.
7DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Kawagishi, Toda-shi, Saitama, Japan.
8Molecular Sensing, Inc., Nashville, Tennessee, United States of America.
9CVMD iMed, AstraZeneca R&D Gothenburg, Mölndal, Sweden.
Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.
“BSI can determine whether a putative allosteric compound is effective in modulating the binding of an orthosteric agonist or antagonist or whether it is competing with them for binding,” adds Dr. Isaac. “BSI can also differentiate different modes of action for different types of compounds, and whether the compounds are competing for the same binding site or they are binding elsewhere.”
Phoonthawee Saetear1, Abigail J Perrin2, S Josefin Bartholdson2, Madushi Wanaguru2, Amanda Kussrow3, Darryl J Bornhop3* and Gavin J Wright2
1Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
2Cell Surface Signalling Laboratory and Malaria Programme, Wellcome Trust Sanger Institute, Hinxton, Cambridge.
3Department of Chemistry and the Vanderbilt Institute for Chemical Biology, Vanderbilt University Nashville, Tennessee.
These results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.
Guyon A, Kussrow A, Olmsted IR, Sandoz G, Bornhop DJ, Nahon JL.
Université de Nice Sophia Antipolis, Nice, France. email@example.com
CXCR4, a receptor for the chemokine CXCL12 (stromal-cell derived factor-1α), is a G-protein-coupled receptor (GPCR), expressed in the immune and CNS and integrally involved in various neurological disorders. The GABAB receptor is also a GPCR that mediates metabotropic action of the inhibitory neurotransmitter GABA and is located on neurons and immune cells as well. Using diverse approaches, we report novel interaction between GABAB receptor agents and CXCR4 and demonstrate allosteric binding of these agents to CXCR4. First, both GABAB antagonists and agonists block CXCL12-elicited chemotaxis in human breast cancer cells. Second, a GABAB antagonist blocks the potentiation by CXCL12 of high-threshold Ca(2+) channels in rat neurons. Third, electrophysiology in Xenopus oocytes and human embryonic kidney cell line 293 cells in which we coexpressed rat CXCR4 and the G-protein inward rectifier K(+) (GIRK) channel showed that GABAB antagonist and agonist modified CXCL12-evoked activation of GIRK channels. To investigate whether GABAB ligands bind to CXCR4, we expressed this receptor in heterologous systems lacking GABAB receptors and performed competition binding experiments. Our fluorescent resonance energy transfer experiments suggest that GABAB ligands do not bind CXCR4 at the CXCL12 binding pocket suggesting allosteric modulation, in accordance with our electrophysiology experiments. Finally, using backscattering interferometry and lipoparticles containing only the CXCR4 receptor, we quantified the binding affinity for the GABAB ligands, confirming a direct interaction with the CXCR4 receptor. The effect of GABAergic agents on CXCR4 suggests new therapeutic potentials for neurological and immune diseases.
Adams NM, Olmsted IR, Haselton FR, Bornhop DJ, Wright DW.
Department of Chemistry, Vanderbilt University, Nashville, TN 37235, USA.
Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.
Haddad GL, Young SC, Heindel ND, Bornhop DJ, Flowers RA 2nd.
Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA.
A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22,000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer’s disease, by detecting dual-binding interactions. E = enzyme, I = inhibitor, S = substrate.
Lau JL, Baksh MM, Fiedler JD, Brown SD, Kussrow A, Bornhop DJ, Ordoukhanian P, Finn MG.
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, United States.
A high-affinity RNA aptamer (K(d) = 50 nM) was efficiently identified by SELEX against a heteroaryldihydropyrimidine structure, chosen as a representative drug-like molecule with no cross reactivity with mammalian or bacterial cells. This aptamer, its weaker-binding variants, and a known aptamer against theophylline were each embedded in a longer RNA sequence that was encapsidated inside a virus-like particle by a convenient expression technique. These nucleoprotein particles were shown by backscattering interferometry to bind to the small-molecule ligands with affinities similar to those of the free (nonencapsidated) aptamers. The system therefore comprises a general approach to the production and sequestration of functional RNA molecules, characterized by a convenient label-free analytical technique.
Luka Z, Moss F, Loukachevitch LV, Bornhop DJ, Wagner C.
Department of Biochemistry, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Methylation of lysine residues in histones has been known to serve a regulatory role in gene expression. Although enzymatic removal of the methyl groups was discovered as early as 1973, the enzymes responsible for their removal were isolated and their mechanism of action was described only recently. The first enzyme to show such activity was LSD1, a flavin-containing enzyme that removes the methyl groups from lysines 4 and 9 of histone 3 with the generation of formaldehyde from the methyl group. This reaction is similar to the previously described demethylation reactions conducted by the enzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate serves as an accepter of the formaldehyde that is generated. We now show that nuclear extracts of HeLa cells contain LSD1 that is associated with folate. Using the method of back-scattering interferometry, we have measured the binding of various forms of folate to both full-length LSD1 and a truncated form of LSD1 in free solution. The 6R,S form of the natural pentaglutamate form of tetrahydrofolate bound with the highest affinity (K(d) = 2.8 μM) to full-length LSD1. The fact that folate participates in the enzymatic demethylation of histones provides an opportunity for this micronutrient to play a role in the epigenetic control of gene expression.
Baksh MM, Kussrow AK, Mileni M, Finn MG, Bornhop DJ.
Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.
Although membrane proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show that backscattering interferometry (BSI) can accurately quantify ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest.
Kussrow A, Baksh MM, Bornhop DJ, Finn MG.
Department of Chemistry and Vanderbilt Institute for Chemical Biology, Vanderbilt University
All binding events induce a change in refractive index, and antibody–antigen interactions are no exception. We describe the use of backscattering interferometry to quantify antibody binding without labels and with high sensitivity. As a broad range of antibodies are available, this method represents a general way to selectively detect a wide variety of trace molecules in simple or complex mixtures.
Morcos EF, Kussrow A, Enders C, Bornhop D.
Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University
Back-scattering interferometry (BSI) is a label-free, free-solution, small-volume technique used for characterizing binding interactions, which is also relevant to a growing number of biosensing applications including drug discovery. Here, we use BSI to characterize the interaction of carbonic anhydrase enzyme II with five well-known carbonic anhydrase enzyme II inhibitors (± sulpiride, sulfanilamide, benzene sulfonamide, dansylamide, and acetazolamide) in the presence of DMSO. Dissociation constants calculated for each interaction were consistent with literature values previously obtained using surface plasmon resonance and fluorescence-based competition assays. Results demonstrate the potential of BSI as a drug-screening tool which is fully compatible with DMSO and does not require immobilization or labeling, therefore allowing binding interactions to be characterized in the native state. BSI has the potential for reducing labor costs, sample consumption, and assay time while providing enhanced reliability over existing techniques.
Sétif P, Harris N, Lagoutte B, Dotson S, Weinberger SR.
iBiTec-S, URA CNRS 2096, CEA Saclay, 91191 Gif sur Yvette, France. firstname.lastname@example.org
The dissociation constant K(d) of the photosystem I (PSI):ferredoxin complex has been measured by backscattering interferometry (BSI) with cyanobacterial PSI (350 kDa) and ferredoxin (10.5 kDa). The BSI signal, consisting of shifts for interference fringes resulting from a change in refractive index due to complex formation, was monitored as ferredoxin concentration was titrated. K(d) values of 0.14-0.38 microM were obtained with wild-type PSI whereas no complex was detectable with a PSI mutant containing a single mutation (R39Q) in the PsaE extrinsic subunit. These results are in quantitative agreement with previous functional determinations consisting in the detection of fast electron transfer within the complex. They provide evidence that the main contribution for the high affinity binding of ferredoxin to PSI is due to a single region of PsaE comprising arginine 39. They do not support the existence of a secondary binding site that could have escaped functional detection.
A. Kussrow, C.S. Enders, E.F. Morcos, D.J. Bornhop
Department of Chemistry and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN
Backscattering interferometry (BSI), which uses a simple optical train comprising a He—Ne laser, a microfluidic channel, and a position sensor, has now enabled the measurement of both tethered and free-solution, label-free, molecular interactions within just nanoliters of sample. The simple macro-to-micro interface allows for a highly efficient assay work flow, which has been used to interrogate molecular binding interactions between proteins, ions and protein, and small molecules and proteins, with a high dynamic range of dissociation constants (KD) and unmatched sensitivity. With this technique, the equilibrium KD for several different binding partners was determined, typically using just picomole—micromole quantities of the binding pair at physiologically relevant concentrations.
Latham JC, Stein RA, Bornhop DJ, Mchaourab HS.
Department of Chemistry and The Vanderbilt Institute for Chemical Biology, Vanderbilt University,
We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.
A. Kussrow, D.J. Bornhop, N.H. Harris, S. Dotson, S. Weinberger, W.E. Rich
Molecular Sensing, Inc.
BSI is a novel interferometric technology for measuring mass-independent molecular interactions allowing quantification of binding affinity over a large dynamicrange in KD, in a homogeneous or heterogeneous assay format, and requiring just picograms to nanograms of the protein target. Unique to BSI, small molecule inhibitors and even ions interacting with high molecular weight proteins can be studied with little a priori information about the interaction system. It is our belief that BSI shows great promise in the study of molecular interactions in drug discovery, particularly for small molecule and fragment-based
Bornhop DJ, Latham JC, Kussrow A, Markov DA, Jones RD, Sørensen HS.
Department of Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University
Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules and protein, were determined with high dynamic range dissociation constants (Kd spanning six decades) and unmatched sensitivity (picomolar Kd’s and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G, interleukin-2 with its monoclonal antibody, and calmodulin with calcium ion Ca2+, a small molecule inhibitor, the protein calcineurin, and the M13 peptide. The high sensitivity of back-scattering interferometry and small volumes of microfluidics allowed the entire calmodulin assay to be performed with 200 picomoles of solute.
1. Introduction to Label-free Methods
2. Drug Discovery Applications
3. Case Studies
The real value of label-free:
• Highly sensitive, information rich methods that have a wide application across the early drug discovery value chain
• Ability to use in combination with other label-free, biochemical or cell-based methods for improved success rates
• Increased application and understanding of the power of combinations of label-free and traditional assays will help to improve in vivo activity predictions
• Most sensitive (pM), molecular interaction platform available; homogeneous and heterogeneous assays; mass and complex matrix independent detection
• Breakthrough in membrane bound protein – ligand interaction characterization
• Overcomes the limitations of SPR & ITC & labeled methods
• Quantitative assay performance with pM LOD sensitivity
Scot R. Weinberger, Fei Shen, Richard J. Isaacs; Molecular Sensing, Inc., Nashville, Tennessee, USA
Presented during SLAS 2015 on February 2015 in Washington, DC.
1Molecular Sensing, Inc., Nashville, Tennessee, USA,
2AstraZeneca Discovery Sciences, Mölndal, Sweden,
3AstraZeneca CVMD iMED, Mölndal, Sweden
Presented during Discovery on Target 2014
1Molecular Sensing, Inc.
2Vanderbilt University Medical Center, Dept. of Pharmacology
Presented during Discovery on Target on September 2013 at Boston, MA
Presented during NovAliX Conference 2013: Biophysics in Drug Discovery on October 2013 in Strasbourg, France