Ann M. Ferrie

Label-free optical biosensor for ligand-directed functional selectivity acting on β2 adrenoceptor in living cells

Recent realization of ligand‐directed functional selectivity demands high‐resolution tools for studying receptor biology and ligand pharmacology. Here we use label‐free optical biosensor to examine the dynamic mass redistribution of human epidermoid A431 cells in response to diverse β2‐adrenoceptor ligands. Multi‐parameter analysis reveals distinct patterns in activation and signaling of the receptor induced by different agonists. Sequential and co‐stimulation assays categorize various ligands for their ability to modulate signaling induced by catechol, a structural component of catecholamines. This study documents multiple ligand‐specific states of the β2‐adrenoceptor and highlights the power of the biosensor assays for screening pathway‐biased ligands.

GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis

G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both normal and cancer cells. However, little is known about the response in cytosolic metabolic pathways to GPCR-mediated signaling. Here, we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. By screening a library of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gαi-coupled receptors correlates with purinosome assembly/disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling as well as the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly/disassembly may represent one of downstream events of mitogenic GPCR signaling in human cancer cells.

Probing cytoskeleton modulation by optical biosensors

This paper reported the use of resonant waveguide grating biosensors for studying the cytoskeleton structure in cells. This was achieved by measuring the changes in mass within the bottom portion of cells upon exposure to saponin in the absence and presence of cytoskeleton modulators. Treatment of Chinese hamster ovary cells with saponin led to a dose‐dependent and dynamic mass changes. When a higher concentration of saponin (>60 μg/ml) was used, a net loss in mass was observed. This is probably resulted from the diffusion of soluble intracellular materials away from the bottom portion of cells after pore formation in the cell plasma membranes by saponin. The pretreatment of cells with actin disruption agents, cytochalasin B and latrunculin A, led to significantly increased loss in cell mass induced by either 75 or 125 μg/ml saponin. These results suggested that optical biosensors provide an attractive means to study the cytoskeleton structure and screen modulators that affect the cytoskeleton structure.

Resonant waveguide grating imager for live cell sensing

We report on a resonant waveguide grating imager for high throughput screening using live cells. This imager can generate a snapshot image of all biosensors in a 384-well microtiter plate with a time resolution of ∼3 s and a spatial resolution of 80 μm. This imager is well tolerant to variability in plate configurations and cell confluency. The resonant wavelength and its shifts induced by cell responses at each pixel correlate well with cell confluency. Data filtration protocol can be used to improve assay quality for partially confluent cells.

Label-free integrative pharmacology on-target of drugs at the β 2 -adrenergic receptor

We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the β2-adrenergic receptor. This method combines dynamic mass redistribution (DMR) assays using an array of probe molecule-hijacked cells with similarity analysis. The whole cell DMR assays track cell system-based, ligand-directed, and kinetics-dependent biased activities of the drugs, and translates their on-target pharmacology into numerical descriptors which are subject to similarity analysis. We demonstrate that the approach establishes an effective link between the label-free pharmacology and in vivo therapeutic indications of drugs.

Discovery of 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives as G protein-coupled receptor 35 (GPR35) agonists.

Screening with dynamic mass redistribution (DMR) assays in a native cell line HT-29 led to identification of two novel series of chemical compounds, 2-(4-methylfuran-2(5H)-ylidene)malononitrile and thieno[3,2-b]thiophene-2-carboxylic acid derivatives, as GPR35 agonists. Of these, 2-(3-cyano-5-(3,4-dichlorophenyl)-4,5-dimethylfuran-2(5H)-ylidene)malononitrile (YE120) and 6-bromo-3-methylthieno[3,2-b]thiophene-2-carboxylic acid (YE210) were found to be the two most potent GPR35 agonists with an EC50 of 32.5 ± 1.7 nM and 63.7 ± 4.1 nM, respectively. Both agonists exhibited better potency than that of zaprinast, a known GPR35 agonist. DMR antagonist assays, knockdown of GPR35 with interference RNA, receptor internalization assays, and Tango β-arrestin translocation assays confirmed that the agonist activity of these ligands is specific to GPR35. The present study provides novel chemical series as a starting point for further investigations of GPR35 biology and pharmacology.

Agonist-directed desensitization of the β2-adrenergic receptor.

The β2-adrenergic receptor (β2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level.

Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells

BackgroundProtease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown.ResultsHere, we show that non-invasive resonant waveguide grating (RWG) biosensor differentiates signaling of endogenous protease activated receptor subtype 1 (PAR1) and 2 (PAR2) in human epidermoid carcinoma A431 cells. The biosensor directly measures dynamic mass redistribution (DMR) resulted from ligand-induced receptor activation in adherent cells. In A431, both PAR1 and PAR2 agonists, but neither PAR3 nor PAR4 agonists, trigger dose-dependent Ca2+ mobilization as well as Gq-type DMR signals. Both Ca2+ flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR1 and PAR2 exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca2+ flux signals show almost identical dependence on cell surface cholesterol level, but their corresponding DMR signals present different sensitivities.ConclusionOptical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR1 and PAR2 in A431.

Label-Free Profiling of Ligands for Endogenous GPCRs Using a Cell-Based High-Throughput Screening Technology

This article reports the use of Corning Epic system— a label-free and noninvasive optical system that is centered on resonant waveguide grating biosensors—to profile endogenous G protein-coupled receptors (GPCRs) in living cells under physiologically relevant conditions. The endogenous GPCRs examined were bradykinin B2 receptor in A431 cells and protease-activated receptor subtype 1 (PAR1) in Chinese hamster ovary (CHO) cells. The activation of either receptor led to Gq-mediated signaling in the respective cells, as confirmed by Fluo-3 assays. Stimulation of CHO cells with thrombin, a PAR1 natural agonist, resulted in an optical response relating to dynamic mass redistribution that is similar to that induced by bradykinin in A431 cells. Based on the kinetics of agonist-mediated optical signatures, two time points, one before and another 5 min after the stimulation, were chosen to develop high-throughput (HT) screening assays. Results showed that such endpoint measurements enable not only HT screening of compounds using endogenous GPCRs, but also determining the efficacies of agonists. Those results suggested that the Corning Epic label-free system is an easily scaleable biosensor, amenable as an HTS platform for GPCR drug discovery and deorphanization. (JALA 2006;11:181–7)

High resolution resonant waveguide grating imager for cell cluster analysis under physiological condition

We report on a spatially resolved resonant waveguide grating imager for cell cluster analysis under physiological condition. Compared to results obtained under ambient condition, the activation of a receptor resulted in a similar biosensor signature but with faster kinetics and greater amplitude. The imager further detected receptor signaling in and movements of single cells within small cell clusters. This opens possibility to investigate the heterogeneity and robustness of receptor signaling from single cells to cell systems.