Intensity interrogation near cutoff resonance for label-free cellular profiling.

We report a method enabling intensity-based readout for label-free cellular assays, and realize a reader device with the same footprint as a microtiter plate. For unambiguous resonance intensity measurements in resonance waveguide grating (RWG) sensors, we propose to apply resonances near the substrate cutoff wavelength. This method was validated in bulk refractive index, surface bilayer and G protein-coupled receptor (GPCR) experiments. The significantly reduced size of the reader device opens new opportunities for easy integration into incubators or liquid handling systems.

Label-free functional selectivity assays.

G protein-coupled receptors (GPCRs) represent the largest class of drug targets. Ligand-directed functional selectivity or biased agonism opens new possibility for discovering GPCR drugs with better efficacy and safety profiles. However, quantification of ligand bias is challenging. Herein, we present five different label-free dynamic mass redistribution (DMR) approaches to assess ligand bias acting at the ß2-adrenergic receptor (ß2AR). Multiparametric analysis of the DMR agonist profiles reveals divergent pharmacology of a panel of ß2AR agonists. DMR profiling using catechol as a conformational probe detects the presence of multiple conformations of the ß2AR. DMR assays under microfluidics, together with chemical biology tools, discover ligand-directed desensitization of the receptor. DMR antagonist reverse assays manifest biased antagonism. DMR profiling using distinct probe-modulated cells detects the biased agonism in the context of self-referenced pharmacological activity map.

Label-free single cell kinetics of the invasion of spheroidal colon cancer cells through 3D Matrigel.

This article reports label-free, real-time, and single-cell quantification of the invasion of spheroidal colon cancer cells through three-dimensional (3D) Matrigel using a resonant waveguide grating (RWG) imager. This imager employs a time-resolved swept wavelength interrogation scheme to monitor cell invasion and adhesion with a temporal resolution up to 3 s and a spatial resolution of 12 µm. As the model system, spheroids of human colorectal adenocarcinoma HT-29 cells are generated by culturing the cells in 96-well round-bottom ultralow attachment plates. 3D Matrigel is formed by its gelation in 384-well RWG biosensor microplates. The invasion and adhesion of spheroidal HT29 cells is initiated by placing individual spheroids onto the Matrigel-coated biosensors. The time series RWG images are obtained and used to extract the optical signatures arising from the adhesion after the cells are dissociated from the spheroids and invade through the 3D Matrigel. Compound profiling shows that epidermal growth factor accelerates cancer cell invasion, while vandetanib, a multitarget kinase inhibitor, dose-dependently inhibits invasion. This study demonstrates that the label-free imager can monitor in real-time the invasion of spheroidal cancer cells through 3D matrices.

Divergent label-free cell phenotypic pharmacology of ligands at the overexpressed ß2-adrenergic receptors.

We present subclone sensitive cell phenotypic pharmacology of ligands at the ß2-adrenergic receptor (ß2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged ß2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled ß2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

A label-free optical biosensor with microfluidics identifies an intracellular signalling wave mediated through the ß(2)-adrenergic receptor.

The canonical model of G protein-coupled receptor (GPCR) signalling states that it is solely initiated at the cell surface. In recent years, a handful of evidence has started emerging from high-resolution molecular assays that the internalized receptors can mediate the third wave of signalling, besides G protein- and ß-arrestin-mediated signalling both initiating at the cell surface. However, little is known about the functional consequences of distinct waves of GPCR signalling, in particular, at the whole cell system level. We here report the development of label-free biosensor antagonist reverse assays and their use to differentiate the signalling waves of an endogenous ß2-adrenergic receptor (ß2-AR) in A431 cells. Results showed that the persistent agonist treatment activated the ß2-ARs, leading to a long-term sustained dynamic mass redistribution (DMR) signal, a whole cell phenotypic response. Under the persistent treatment scheme in microplates, a panel of known ß-blockers all dose-dependently and completely reversed the DMR signal of epinephrine at a relatively low dose (10 nM), except for sotalol which partially reversed the DMR. Under the perfusion conditions with microfluidics, the subsequent perfusion with sotalol only reversed the DMR induced by epinephrine or isoproterenol at 10 nM, but not at 10 µM. Furthermore, the degree of the DMR reversion by sotalol was found to be in an opposite relation with the duration of the initial agonist treatment. Together, these results suggest that the hydrophilic antagonist sotalol is constrained outside the cells throughout the assays, and the early signalling wave initiated at the cell surface dominates the DMR induced by epinephrine or isoproterenol at relatively low doses, while a secondary and late signalling wave is initiated once the receptors are internalized and contributes partially to the long-term sustainability of the DMR of epinephrine or isoproterenol at high doses.

Dynamic mass redistribution assay decodes differentiation of a neural progenitor stem cell.

Stem cells hold great potential in drug discovery and development. However, challenges remain to quantitatively measure the functions of stem cells and their differentiated products. Here, we applied fluorescent imaging, quantitative real-time PCR, and label-free dynamic mass redistribution (DMR) assays to characterize the differentiation process of the ReNcell VM human neural progenitor stem cell. Immunofluorescence imaging showed that after growth factor withdrawal, the neuroprogenitor stem cell was differentiated into dopaminergic neurons, astrocytes, and oligodendrocytes, thus creating a neuronal cell system. High-performance liquid chromatography analysis showed that the differentiated cell system released dopamine upon depolarization with KCl. In conjunction with quantitative real-time PCR, DMR assays using a G-protein-coupled receptor agonist library revealed that a subset of receptors, including dopamine D(1) and D(4) receptors, underwent marked alterations in both receptor expression and signaling pathway during the differentiation process. These findings suggest that DMR assays can decode the differentiation process of stem cells at the cell system level.

Discovery of Natural Phenols as G Protein-Coupled Receptor-35 (GPR35) Agonists.

We report the discovery and characterization of natural phenols as G protein-coupled receptor-35 (GPR35) agonists. Pharmacological characterization using label-free dynamic mass redistribution and Tango ß-arrestin translocation assays revealed that GPR35-active natural phenols are divergent in their biased agonism.

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 healthy and cancerous cells. However, little is known about the response of 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. Through a library screen 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 Gα(i)-coupled receptors correlates with purinosome assembly and disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling and of the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly and disassembly may be one of the downstream events of mitogenic GPCR signaling in human cancer cells.

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 EC(50) 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 ß-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 ß2-adrenergic receptor.

The ß(2)-adrenergic receptor (ß(2)AR) 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 ß(2)AR. 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 ß(2)AR desensitization at the whole cell level.

Label-free integrative pharmacology on-target of drugs at the ß(2)-adrenergic receptor.

We describe a label-free integrative pharmacology on-target (iPOT) method to assess the pharmacology of drugs at the ß(2)-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.

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.

Resonant waveguide grating biosensor for whole-cell GPCR assays.

Current drug discovery campaigns for G protein-coupled receptors (GPCRs) heavily rely on assay technologies that use artificial cell systems tailored to a point-of-contact readout and as a consequence are mostly pathway biased. Recently, we have developed label-free optical biosensor cellular assays that are capable of examining systems cell biology of endogenous receptors and systems cell pharmacology of GPCR ligands in both physiologically and disease relevant environments. We have shown that these biosensor assays enable high-throughput screening of pathway-biased ligands acting on endogenous beta(2)-adrenergic receptor in cells. These biosensor cellular assays hold the potential to reduce attrition rates in drug discovery and development process.

Label-free optical biosensor for ligand-directed functional selectivity acting on beta(2) 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 beta(2)-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 beta(2)-adrenoceptor and highlights the power of the biosensor assays for screening pathway-biased ligands.

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

BACKGROUND: Protease 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. RESULTS: Here, 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. CONCLUSION: Optical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR1 and PAR2 in A431.

Non-invasive optical biosensor for assaying endogenous G protein-coupled receptors in adherent cells.

INTRODUCTION: Screening drugs against G protein-coupled receptors (GPCRs) - the single largest family of drug targets in the human genome - is still a major effort in pharmaceutical and biotech industries. Conventional cell-based assays generally measure a single cellular event, such as the generation of a second messenger or the relocation of a specific protein target. However, manipulation or engineering of cells is often a prerequisite for these technologies to achieve desired sensitivities. The present study is focused on the use of non-invasive and manipulation-free optical biosensors for assaying endogenous GPCRs in adherent cells. METHODS: Resonant waveguide grating (RWG) biosensor was applied to manifest ligand-induced dynamic mass redistribution (DMR) within the bottom portion of adherent cell layer. The DMR signatures mediated through the activation of several endogenous GPCRs in cells were characterized. Endogenous receptor panning was examined at cell system level by using a panel of agonists known to activate many GPCRs, and also at family receptor level by determining the efficacies of a set of family-specific agonists. RESULTS: Three major types of optical signatures were identified; each was correlated with the activation of a class of GPCRs, depending on the G protein with which the receptor is coupled (i.e., G(q), G(s) and G(i)). The characteristics of DMR signals, mostly the amplitude and kinetics of a DMR event, were dependent on the doses of agonists and the expression levels of endogenous receptors. All three classes of endogenous receptors were found in human epidermoid carcinoma A431 cells. Interestingly, the dose-dependent switching from one type of DMR signal to another was observed for several GPCR agonists examined. A small panel of P2Y receptor agonists exhibited distinct efficacies in three cell lines examined. DISCUSSIONS: The RWG biosensors were applicable to study the activation of endogenous GPCRs. Like second messengers or gene expression, the DMR signals obtained could be considered as novel and quantifiable physiological responses of living cells mediated through GPCRs and used for studying receptor biology.

Resonant waveguide grating biosensor for living cell sensing.

This article presents theoretical analysis and experimental data for the use of resonant waveguide grating (RWG) biosensors to characterize stimulation-mediated cell responses including signaling. The biosensor is capable of detecting redistribution of cellular contents in both directions that are perpendicular and parallel to the sensor surface. This capability relies on online monitoring cell responses with multiple optical output parameters, including the changes in incident angle and the shape of the resonant peaks. Although the changes in peak shape are mainly contributed to stimulation-modulated inhomogeneous redistribution of cellular contents parallel to the sensor surface, the shift in incident angle primarily reflects the stimulation-triggered dynamic mass redistribution (DMR) perpendicular to the sensor surface. The optical signatures are obtained and used to characterize several cellular processes including cell adhesion and spreading, detachment and signaling by trypsinization, and signaling through either epidermal growth factor receptor or bradykinin B2 receptor. A mathematical model is developed to link the bradykinin-mediated DMR signals to the dynamic relocation of intracellular proteins and the receptor internalization during B2 receptor signaling cycle. This model takes the form of a set of nonlinear, ordinary differential equations that describe the changes in four different states of B2 receptors, diffusion of proteins and receptor-protein complexes, and the DMR responses. Classical analysis shows that the system converges to a unique optical signature, whose dynamics (amplitudes, transition time, and kinetics) is dependent on the bradykinin signal input, and consistent with those observed using the RWG biosensors. This study provides fundamentals for probing living cells with the RWG biosensors, in general, optical biosensors.

Cellular functions of cholesterol probed with optical biosensors.

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.

Air-stable G protein-coupled receptor microarrays and ligand binding characteristics.

This paper described novel strategies to achieve air-stable G protein-coupled receptor (GPCR) microarrays and the uses of the microarrays for ligand profiling. Specifically, GPCR cell membrane fragments were suspended in a buffered solution containing bovine serum albumin (BSA) and disaccharide sucrose or trehalose and used for fabricating GPCR microarrays. During the array fabrication and postfabrication processes, BSA molecules were found to effectively form packed layer(s) surrounding the GPCR membranes immobilized onto the predetermined printing area, thereby stabilizing the membrane microspots. The use of disaccharides was shown to protect the integrity and functionality of GPCR microarrays from the typical deterioration of the membranes when fabricated and stored under dry conditions. To utilize the ability of fluorescence technology for multichannel detection as well as to maximize the capability of GPCR microarrays for multiplexed binding assays, several fluorescently labeled ligands were synthesized and optimized for multiplexing binding assays. A schematic microarray of five GPCRs had been used as a model for characterizing the association and dissociation rate constants of labeled ligands binding to their respective receptors in the microarrays. Interestingly, distinct receptor-ligand interactions exhibited different dependence on the type of pH reagent as well as the species and concentration of cations used in a binding assay buffered solution. The potential mechanisms and implications for the uses of air-stable GPCR microarrays were discussed.

Functional GPCR microarrays.

This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.