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Many cell-based assays interrogating cell pathway activation employ protocols that require microscopic imaging techniques. However, such assays are not in general widely adopted for primary screening. Protein complementation, particularly of enzymes, provides an alternative approach for cell pathway analysis, with a principal advantage that is amenable to high throughput screening using microtiter plate protocols. Notably, α complementation of the enzyme β-galactosidase has been exploited as a technology in this regard, using substrates that generates luminescent signals. This review describes the various uses of this flexible technology to cell-based assay development.

Immortalized cells, generated from two-dimensional cell culture techniques, are widely used in compound screening, lead optimization, and drug candidate selection. However, such cells lack many characteristics of cells in vivo. This could account for the high failure rates of lead candidates in clinical evaluation. New approaches from cell biology, materials science, and bioengineering are increasing the utility of three-dimensional (3D) culture. These approaches have become more compatible with automation and, thus, provide more physiologically relevant cells for high-throughput/high-content screening, notably in oncology drug discovery. Techniques range from simple 3D spheroids, comprising one or more cell types, to complex multitissue organoids cultured in extracellular matrix gels or microfabricated chips. Furthermore, each approach can be applied to stem cells, such as induced pluripotent stem cells, thereby providing additional phenotypic relevance and the exciting potential to enable screening in disease-specific cell types.

Activation of cells by the tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) cytokines results in activation of the nuclear factor-kappaB (NF-kappaB) via proteasomal degradation of an associated IkappaB molecule. To monitor cellular IkappaB, the protein was recombinantly expressed as a fusion protein with a novel enzymatic tag, ProLabel (PL). ProLabel is a small 5.5-kDa sequence from the amino-terminal amino acids of beta-galactosidase, possesses a simple ribbon structure, and can be fused to many proteins via the amino or carboxyl terminus. Expression of this construct allows quantitative detection of the recombinant protein in crude lysates by using a method based on beta-galactosidase enzyme fragment complementation (EFC). Transient transfection of IkappaB-PL in HeLa cells generated an EFC signal that was highly correlated with a western analysis of the protein construct. ProLabel expressed alone in the cells did not show any EFC activity, due to rapid proteolytic degradation, indicating a very low background signal from the protein tag. TNF-alpha and IL-1 treatment induced a concentration-dependent degradation of IkappaB-PL, with potency values similar to those reported using other methods. IkappaBM-PL (mutant of IkappaB-PL), in contrast, did not undergo degradation for concentrations up to and including 10 ng/ml TNF-alpha or IL-1, demonstrating that degradation of IkappaB-PL was specific to the NF-kappaB pathway activation. TNF-alpha and IL-1 induced maximal IkappaB-PL degradation within 30 min of induction. This was reversed by several agents that ablate this pathway, including anti-TNF-alpha antibodies and the proteasome inhibitor, MG-132. The assay was amenable to HTS systems, with good precision and reproducibility. Z' values and coefficients of variance for IkappaB-PL degradation were 0.6 and9%, respectively.

Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.

Protein kinases are important targets in drug discovery programs aimed at treating many devastating diseases, including cancer, autoimmune disorders, diabetes, and neurological disorders. Most "classical" drug discovery efforts employ rational drug design methods based upon structural information to identify compounds targeting the enzyme catalytic domain. Novel information on kinase biology is opening up other approaches in the design of selective inhibitors that may provide more subtle modulation of these drug discovery targets. The identification of such modulators requires adoption of a new generation of high-throughput screening techniques. These approaches will allow measurement of conformational changes in kinases, as well as protein-protein interactions via assessment of functional responses such as cellular translocation. Therefore a range of novel techniques, together with the understanding that numerous "orphan" kinases will provide targets for therapeutics, suggests that a new era of kinase therapies is rapidly emerging.

The G protein-coupled receptor (GPCR) family is a major target for drug discovery, and most, if not all, GPCRs can couple to Ca2+ signaling. Consequently, there are a number of cellbased, primary, high-throughput screening (HTS) assays used for drug discovery that assess changes in intracellular Ca2+ as a functional readout of GPCR activation. Historically, changes in intracellular Ca2+ levels have been readily detected using fluorescent dyes that emit light in proportion to changes in intracellular Ca2+ concentration. An alternative approach to indirectly measure changes in Ca2+ concentrations involves the use of recombinantly expressed biosensor photoproteins, of which aequorin is a prototypic example. These biosensors have the advantage that they provide an intense luminescent signal in response to elevations in intracellular Ca2+. This exquisite sensitivity, the high signal-to-noise ratios, and the ability to target expression to discrete subcellular sites (in order to detect Ca2+ microdomains) have made photoproteins a principal choice in a wide range of GPCR drug discovery programs. Photoproteins are also finding increasing use in detecting activation of other molecular target classes such as ligand-gated ion channels and transporters. This review focuses upon the use of calcium photoproteins principally for use in GPCR drug discovery and HTS.

Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses. Such "designer" drugs currently include allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. Moreover, the large family of orphan GPCRs provides a rich and novel field of targets to discover drugs with unique therapeutic properties. The numerous technologies to discover GPCR drugs have also greatly advanced over the years, facilitating compound screening against known and orphan GPCRs, as well as in the identification of unique designer GPCR drugs. Indeed, high throughput screening (HTS) technologies employing functional cell-based approaches are now widely used. These include measurement of second messenger accumulation such as cyclic AMP, calcium ions, and inositol phosphates, as well as mitogen-activated protein kinase activation, protein-protein interactions, and GPCR oligomerization. This review focuses on how the improved understanding of the molecular pharmacology of GPCRs, coupled with a plethora of novel HTS technologies, is leading to the discovery and development of an entirely new generation of GPCR-based therapeutics.

Positional complementation describes the use of homogeneous assays using beta- galactosidase (beta gal) enzyme fragment complementation to detect cellular protein translocation. This phenomenon occurs when the protein of interest, recombinantly expressed as a fusion protein with a modified alpha fragment of beta gal, translocates to a cellular compartment expressing an enzyme acceptor fragment of the enzyme. When these fragments interact, high-affinity complementation occurs, and a signal is generated that is then detected upon cell lysis. In the present paper the use of positional complementation is exemplified by measuring nuclear translocation of the glucocorticoid receptor in Chinese hamster ovary-K1 cells. The approach thus provides for homogeneous protocols, in an endpoint microtiter plate assay format, without the use of either imaging or reporter gene techniques. Consequently, these characteristics suggest that the technique is suitable for automated instrumentation protocols used in high throughput screening campaigns designed to identify activators or inhibitors of nuclear translocation.

High-affinity complementation of a small fragment of beta-galactosidase to an inactive deletion mutant of the enzyme forms a stable heteromeric enzyme complex capable of hydrolyzing substrates to produce either chemiluminescent or fluorescent signals. This review describes a series of screening assays in which the small beta-galactosidase fragment, Enzyme Donor or ProLabel, is either chemically conjugated or recombinantly fused to small molecules or proteins, respectively. Chemical conjugation forms the basis of several HitHunter HTS assays in which competitive displacement of the ProLabel conjugate from either a binding protein (receptor or antibody) is induced by the analyte in question. In this manner, a calibration curve is generated, to measure cellular analytes including 3',5'-cyclic AMP. Changes in this second messenger, occurring due to G protein-coupled receptor (GPCR) activation, can thus be easily measured in a homogeneous assay. Similar assays have been developed for tyrosine kinases, serine threonine kinases, nuclear hormone receptors, and proteases. A second form of assay technology involves measurement of cellular protein expression, in which the protein is fused to ProLabel. Analysis can be undertaken in crude cell lysates, or with intact cells, using beta-galactosidase complementation in a microtiter plate. This homogeneous technology is highly sensitive and has been developed to measure protein expression changes occurring in response to pathway activation by targets such as GPCRs, tyrosine kinase receptors, and proteases. In summary, the DiscoveRx technology using beta-galactosidase complementation provides a robust and flexible assay technology for use in cell-free and cell-based HTS.