Label-free optical biosensors can shed new light on cell biology.

High throughput and high information screening plays a critical role in drug discovery. While the approaches, tools, and strategies vary, researchers are increasing investment in cell-based assay technologies to generate higher quality leads and determine more about the pharmacology of compounds earlier in the discovery process. Recent studies indicate drug discovery laboratories seek more robust assays that are biologically relevant, sensitive to inhibitors, and enable the study of difficult targets.¹

Most cell-based assays employ radioactive or fluorescent dyes to tag one or more molecules within the cellular pathway. These labels track activity in the cell to determine particular signaling pathways. Labels, however, can potentially disrupt the intricate moments and interactions of intracellular proteins that determine signaling pathways. Furthermore, the necessity to over-express receptors of interest adds to the complexity of the screening process and drastically alters a cell’s natural condition. Moreover, label-based approaches are based on a linear assumption of one signaling pathway per ligand-receptor complex, which fails to account for the true complexity of intracellular dynamics.^2,3,4  Receptor-receptor interactions, ligand-directed functional selectivity, promiscuous binding, and receptor oligimerization represent some of the more complex relationships overlooked by conventional approaches.⁵

click to enlarge Figure 1. The principle of label-free optical biosensor detection for cell-based assays. (Source: Corning Incorporated)

Label-free optical biosensors were developed to overcome the inherent limitations of labels. Optical biosensor-based technologies demonstrate promise for cell-based assays because they provide a means of non-invasively obtaining pharmacological information in physiologically-relevant systems. Label-free, biosensor-based technologies promise to improve the drug discovery process by enabling researchers to study receptor biology and ligand pharmacology in living cells.

A phenomenon termed dynamic mass redistribution (DMR) by scientists working with optical biosensors at Corning Incorporated, serves as the basis for new insight into cell biology. Through DMR, the cellular signaling profiles of different classes of endogenously-expressed G-protein coupled receptors (GPCRs) can be detected. DMR can also be used to identify G-protein coupling of orphan GPCRs. Using specific chemical tools, DMR can be used to reveal complex signal transduction pathways and distinguish among full agonists, partial agonists, neutral antagonists, and inverse agonists. 

The greater sensitivity of label-free optical biosensors enables the use of non-engineered cells, including primary cells. While this provides the benefit of physiological relevance, it also yields more information on intracellular dynamics such as the complex signal transduction pathways that would have been otherwise overlooked by conventional approaches. Because optical biosensors show a real-time, global cellular response to a given compound, they can be used as a universal platform for a broad range of targets, including all the major classes of GPCRs (G_q, G_s G_i).

How optical biosensors work
On the Corning Epic System, optical biosensors are integrated into the individual wells of a 384-well microplate. The resonant waveguide grating (RWG) optical biosensors consists of a glass substrate with a periodic optical grating that is formed on top of it. A high index-of-refraction, dielectric waveguide coating is applied on the grating in each well, forming the integrated biosensor. When illuminated with broadband light, the biosensor in each well reflects a specific wavelength of light that is a sensitive function of the index of refraction close to the sensor surface. A cellular response (dynamic mass redistribution) causes a shift in the resonant wavelength that provides researchers with response profiles indicating specific cellular events (Figure 1).

click to enlarge Figure 2. The carbachol-induced DMR responses in both CHO-M1 and its parental CHO-K1 control cells. (Source: Corning Incorporated)

The RWG biosensors are sensitive to cellular changes within the first ~150nm from the sensor surface. In a cell-based assay, a stimulus-induced dynamic mass redistribution of cellular material within this detection zone produces real-time, global cellular responses specific to a receptor-ligand complex.

Determining GPCR signaling pathways

Label-free optical biosensors shed new light on cell-based assays for the most druggable class of drug discovery targets—GPCRs. These seven, transmembrane-spanning, cell surface receptors are involved in transmitting extracellular stimuli to a variety of intracellular responses. More than 40 percent of marketed drugs modulate GPCR function directly or indirectly.⁶ Conventional cell-based GPCR assays often require multiple technologies in order to measure all the signaling pathways.

Most cell-based assays today measure a single cellular event in a specific signaling pathway. These assays rely upon the detection of labeled secondary messengers like IP₃ and cyclic AMP (cAMP), or other intracellular targets, or the expression of a reporter gene such as luciferase, beta-lactamase, or beta-galactosidase. While capable of providing robust results, these assays are limited in their ability to detect complex cellular events and may potentially miss valuable information that could speed up the drug discovery process.

Systems cell biology studies
Biosensors enable systems cell biology studies of GPCR signaling. Label-free cell assay technologies typically measure an integrated cellular response, such as DMR signals obtained with optical biosensors. The DMR signal reflects the native signaling of a known receptor, an unknown receptor, a receptor in different cellular contexts, or even the same receptor by different ligands. These responses, coupled with knowledge of chemical biology, enable researchers to identify coupling along all three major GPCR classes: G_s, G_i, and G_q.⁷

click to enlarge Figure 3. The bradykinin-induced DMR signal in A431 cells can be inhibited by bradykinin B2 receptor selective antagonist HOE140. Left panel indicates bradykinin-induced responses in the presence and absence of HOE140. Right panel depicts the dose-dependent inhibition by HOE140 of bradykinin-induced responses in A431 cells. (Source: Corning Incorporated)

Receptor-ligand coupling
The specificity of receptor-ligand coupling has been extensively demonstrated in both recombinant and endogenously-expressing cell lines. Figure 2a depicts results from an experiment on an Epic system in which CHO-M1 cells (recombinant CHO cells over-expressing muscarinic receptor-1) were stimulated by increasing concentrations of carbachol (a non-selective agonist of muscarinic and nicotinic receptors). Carbachol failed to elicit any DMR response, even at very high concentration (3240 nM), in the parental CHO-K1 control cells (Figure 2b), indicating the specificity of carbachol-M1 receptor coupling in the recombinant cells.

The specificity of DMR response to receptor-ligand coupling has also been demonstrated in non-engineered human epidermoid carcinoma (A431) cells, which endogenously express bradykinin B2 receptors.⁷ Figure 3 illustrates the specific and dose-dependent inhibition of bradykinin-induced DMR response by HOE140 (a bradykinin B2 receptor-selective antagonist), again indicating the specificity of the DMR signal.

Chemical modulators that specifically control the activities of many different types of cellular targets can be used to intervene with a specific cell signaling component. This approach can be used to map the signaling and network interactions mediated through a receptor, including the endogenous ₂ARs and the bradykinin B2 receptors in A431 cells.^8,9

Distinguishing among antagonists

Current cell-based assays require complex protocols and appropriate reagents to distinguish between neutral antagonists and inverse agonists. Inverse agonists often are mistaken for neutral antagonists. A recent study suggests that as many as 85% of GPCR compounds thought to be antagonists really behave as inverse agonists.¹⁰

click to enlarge Figure 4. Dose-response profiles of dopamine (an agonist; 4a) and three inverse agonists: spiperone, amisulpride, and haloperidol (4b, 4c, 4d respectively) at dopamine D3 receptors over-expressed in CHO background. (Source: Corning Incorporated)

Recently, Lee et al used Epic label-free optical biosensors to identify inverse agonists.¹¹ In a double-blinded evaluation, a panel of 12 unknown compounds were screened against two recombinant GPCR cell lines—CHO-D3R (dopamine D₃ receptor) and CHO-M1R (muscarinic M₁ receptor). DMR responses from cells were used to identify compounds that functioned as agonists, neutral antagonists, or inverse agonists. DMR results were shown to be in good agreement with data obtained from cAMP and calcium flux assays for the compounds.¹¹

As expected, dopamine, a known potent agonist, induced a positive, dose-dependent DMR shift in CHO-D3R (Figure 4a). Three compounds, spiperone, amisulpride, and haloperidol, induced dose-dependent, negative DMR (N-DMR) shifts in CHO-D3R cells (Figure 4b, 4c, and 4d, respectively), indicating their inverse agonist pharmacology at D3 receptor. The positive DMR responses produced by subsequent additions of dopamine were significantly attenuated by spiperone, amisulpride, and haloperidol in a dose-dependent manner, indicating their specificity at D3 receptor (data not shown).¹¹

click to enlarge Figure 5. Dose-response profiles of a M1 receptor agonist carbachol (5a) and three known neutral antagonists, telenzepine, scopolamine, and atropine (5b, 5c, and 5d, respectively) in CHO-M1 cells. (Source: Corning Incorporated)

The negative DMR shifts seen in these three compounds against the D3R receptor suggest that they are acting as inverse agonists at the receptor and not as purely neutral antagonists as previously thought. If these compounds were purely neutral antagonists, they would most likely respond similarly to the known neutral antagonists: telenzepine, scopolamine, and atropine in the CHO-M1 cell line (Figures 5b, 5c, and 5d, respectively).  As expected, M1 receptor agonist carbachol induced a positive, dose-dependent positive DMR shift in CHO-M1 cells (Figure 5a).  

Based on these data, Lee et al concluded that using label-free optical biosensors, the observed negative DMR shifts indicate the compounds are inverse agonists, something that could have been easily missed using conventional assays and technologies.¹¹ 

Conclusions
The ability to measure DMR, made possible through the use of label-free optical biosensors, can positively impact modern-day drug discovery efforts by making more physiologically-relevant information available earlier in the process. The sensitivity and universality of label-free assays opens the door for a wide range of applications including the identification of the GPCR signaling pathways in real time. Label-free cell assays offer a universal platform for pharmaceutical evaluation including hit confirmation, potency ranking, efficacy analysis, and selectivity across a wide spectrum of receptors.

About the Authors

Ravi Marala is Business Development Manager for the Corning Epic System. He holds a BS in Pharmacy from Delhi University and a PhD in Biochemistry from University of Tennessee. He has nearly 10 years experience in drug discovery at Pfizer and has published extensively in scientific peer review journals.

Michael Hallstrom is Assistant Product Line Manager for the Corning Epic System. He holds a BS in Biotech Business from Brigham Young University.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 7, June, 2008, pp. 36-39.

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