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.1999.4406

Cited by 121 publications

(45 citation statements)

References 14 publications

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“…Following DNase treatment, the RNA was cleaned up using Qiagen RNeasy columns according to the manufacturer's protocol and performing additional washes with buffer RPE and 80% ethanol to remove contaminants. The n-fold change of gene expression for yeast stimulation vs. non-stimulation was calculated using the comparative C T method (41). …”

Section: Methodsmentioning

confidence: 99%

C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic Mycosis of North America

Wang

LeBert

Hung

et al. 2014

The Journal of Immunology

120

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Vaccine immunity to the endemic mycoses of North America requires Th17 cells, but the pattern recognition receptors and signaling pathways that drive these protective responses have not been defined. We show that C-type lectin receptors (CLRs) exert divergent contributions to the development of anti-fungal Th17 cells and vaccine resistance against Blastomyces dermatitidis, Histoplasma capsulatum and Coccidioides posadasii. Acquired immunity to B. dermatitidis requires Dectin-2, whereas vaccination against H. capsulatum and C. posadasii infection depends on innate sensing by Dectin-1 and Dectin-2, but not Mincle. Tracking antigen-specific T cells in vivo established that the Card9 signaling pathway acts indispensably and exclusively on differentiation of Th17 cells, while leaving intact their activation, proliferation, survival and migration. Thus, whereas Card9 signaling is essential, CLRs offer distinct and divergent contributions to vaccine immunity against these endemic fungal pathogens. Our work provides new insight into innate immune mechanisms that drive vaccine immunity and Th17 cells.

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Section: Methodsmentioning

confidence: 99%

C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic Mycosis of North America

Wang

LeBert

Hung

et al. 2014

The Journal of Immunology

120

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“…A potential difficulty is that the method is ideally suited for monitoring large protein binding interactions because the sensitivity of the method is directly proportional to the mass of the binding ligand. However various investigators have successfully employed SPR to measure small molecule binding to proteins by increasing the surface density of the bound target [18,28]. …”

Section: Resultsmentioning

confidence: 99%

Direct-reversible binding of small molecules to G protein βγ subunits

Seneviratne¹,

Burroughs²,

Giralt

et al. 2011

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits α, β, γ mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to Gβγ and interfere with or enhance binding of select effectors with Gβγ. To understand the molecular mechanisms of selectivity and assess binding of compounds to Gβγ, we used biophysical and biochemical approaches to directly monitor small molecule binding to Gβγ. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to Gβγ with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the koff was slow for most of the compounds in physiological buffers, they could be removed from Gβγ with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-Gβγ interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of Gβγ-effector interactions is by specific direct non-covalent and reversible binding of small molecules to Gβγ. This is highly relevant to development of Gβγ targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.

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“…For example, the Biacore ™ 4000 allows up to 16 targets to be screened at once, allowing 4800 compounds to be screened over a 24 hour period without user input after starting the protocol. Primary SPR screening has been used to screen a variety of small molecule compounds for many therapeutic applications including thrombin [24], HIV protease [25–26], and DNA gyrase [27] inhibitors.…”

Section: Spr For Design and Characterization Of Non-particulate Tmentioning

confidence: 99%

Surface plasmon resonance as a high throughput method to evaluate specific and non-specific binding of nanotherapeutics

Schneider

Bhargav

Perez

et al. 2015

Journal of Controlled Release

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Surface plasmon resonance (SPR) is a powerful analytical technique used to quantitatively examine the interactions between various biomolecules, such as proteins and nucleic acids. The technique has been particularly useful in screening and evaluating binding affinity of novel small molecule and biomolecule-derived therapeutics for various diseases and applications including lupus medications, thrombin inhibitors, HIV protease inhibitors, DNA gyrase inhibitors and many others. Recently, there has been increasing interest in nanotherapeutics (nanoRx), due to their unique properties and potential for controlled release of encapsulated drugs and structure-specific targeting to diseased tissues. Targeted nanoRx offer the potential to solve many drug delivery challenges by enabling, specific interactions between molecules on the surface of the nanoparticle and molecules in the diseased tissue, while minimizing off-target interactions toward non-diseased tissues. These properties are largely dependent upon careful control and balance of nanoRx interactions and binding properties with tissues in vivo. Given the great promise of nanoRx with regard to engineering specific molecular interactions, SPR can rapidly quantify small aliquots of nanoRx formulations for desired and undesired molecular interactions. Moving forward, we believe that utilization of SPR in the screening and design of nanoRx has the potential to greatly improve the development of targeted nanoRx formulations and eventually lead to improved therapeutic efficacy. In this review, we discuss (1) the fundamental principles of SPR and basic quantitative analysis of SPR data, (2) previous applications of SPR in the study of non-particulate therapeutics and nanoRx, and (3) future opportunities for the use of SPR in the evaluation of nanoRx.

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

Cited by 121 publications

References 14 publications

C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic Mycosis of North America

C-Type Lectin Receptors Differentially Induce Th17 Cells and Vaccine Immunity to the Endemic Mycosis of North America

Direct-reversible binding of small molecules to G protein βγ subunits

Surface plasmon resonance as a high throughput method to evaluate specific and non-specific binding of nanotherapeutics

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