ASSAY and Drug Development Technologies

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides a possible mechanism for “off-target” drug effects. We have previously shown that channels formed by the linear gramicidins are suitable probes for changes in lipid bilayer properties, as experienced by bilayer-spanning proteins. We now report a gramicidin-based fluorescence assay for changes in bilayer properties. The assay is based on measuring the time course of fluorescence quenching in fluorophore-loaded large unilamellar vesicles, due to entry of a gramicidin channel-permeable quencher. The method is scalable and suitable for both mechanistic studies and high-throughput screening for bilayer-perturbing, potential off-target effects, which we illustrate using capsaicin (Cap) and other compounds.

We present here the characterization and optimization of a novel imaging-based positional biosensor high-content screening (HCS) assay to identify disruptors of p53-hDM2 protein–protein interactions (PPIs). The chimeric proteins of the biosensor incorporated the N-terminal PPI domains of p53 and hDM2, protein targeting sequences (nuclear localization and nuclear export sequence), and fluorescent reporters, which when expressed in cells could be used to monitor p53-hDM2 PPIs through changes in the subcellular localization of the hDM2 component of the biosensor. Coinfection with the recombinant adenovirus biosensors was used to express the NH-terminal domains of p53 and hDM2, fused to green fluorescent protein and red fluorescent protein, respectively, in U-2 OS cells. We validated the p53-hDM2 PPI biosensor (PPIB) HCS assay with Nutlin-3, a compound that occupies the hydrophobic pocket on the surface of the N-terminus of hDM2 and blocks the binding interactions with the N-terminus of p53. Nutlin-3 disrupted the p53-hDM2 PPIB in a concentration-dependent manner and provided a robust, reproducible, and stable assay signal window that was compatible with HCS. The p53-hDM2 PPIB assay was readily implemented in HCS and we identified four (4) compounds in the 1,280-compound Library of Pharmacologically Active Compounds that activated the p53 signaling pathway and elicited biosensor signals that were clearly distinct from the responses of inactive compounds. Anthracycline (topoisomerase II inhibitors such as mitoxantrone and ellipticine) and camptothecin (topoisomerase I inhibitor) derivatives including topotecan induce DNA double strand breaks, which activate the p53 pathway through the ataxia telangiectasia mutated-checkpoint kinase 2 (ATM-CHK2) DNA damage response pathway. Although mitoxantrone, ellipticine, camptothecin, and topotecan all exhibited concentration-dependent disruption of the p53-hDM2 PPIB, they were much less potent than Nutlin-3. Further, their corresponding cellular images and quantitative HCS data did not completely match the Nutlin-3 phenotypic profile.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that signals through G-protein-coupled receptors to produce a range of biological responses. A recently reported LPA receptor GPR23 (LPA₄ receptor) has a low homology to the LPA_1–3 receptors identified previously. In Chinese hamster ovary cells expressing the human GPR23, LPA induced an increase in cellular cyclic adenosine monophosphate (cAMP) and calcium levels. GPR23-selective agonists or antagonists have not been reported previously. Such ligands, if available, would be valuable tools for studying the functions of this receptor. Here we report the identification of novel GPR23 agonists, inverse agonists, and a negative modulator from 2 high-throughput screens, a β-lactamase reporter screen, and a [³H]LPA-binding screen. Several screening hits were selected for mechanism of action studies using the β-lactamase reporter assay and a cAMP assay. An evaluation of their selectivity at the other LPA receptors was also conducted. This study demonstrates a strategy for the identification of GPR23 agonists and inverse agonists. We believe the strategy employed here is applicable to other constitutively active GPCRs.

The ubiquitin proteasome pathway (UPP) has been implicated in a number of pathogenic diseases: cancer, inflammation, metabolic disorders, and viral infection. The human genome contains well over 500 genes encoding proteins involved in the UPP. Ubiquitin ligases (E3s) comprise the largest subset of these genes, and together with an E2 partner, provide the substrate selectivity required for regulating cellular proteins through the covalent attachment of ubiquitin. Many ligases that have been identified in critical cellular pathways have no known substrates. Even those E3s with known substrates may have a yet unidentified role in the pathways on which they lie and as such may have additional substrates. It is critical to identify these substrates for discovery of selective small molecule inhibitors aimed at therapeutic intervention. Other methods, such as mass spectrometry, have been utilized for identifying ligase substrates, but these are labor-intensive and require a significant investment. In this study, we utilized protein microarrays for the identification of substrates of the HECT domain E3, Smurf1.

There is renewed interest in the Src family of protein tyrosine kinases (SFKs) as a result of their potential utility as molecular targets for cancer therapy. This protein family consists of 9 nonreceptor tyrosine kinases that, although implicated in a diverse array of cellular functions, possess a similar modular structure. Here we describe a simple and efficient multiplex microbead immunoassay (MMIA), based on Luminex^® xMAP technology, which allows for the simultaneous detection of 8 phosphorylated SFKs in a single assay. Microbead sets identifiable by unique fluorescence were individually coated with antibodies specific for an individual SFK member. Detection of phosphorylated SFKs was accomplished using a secondary antibody directed against phosphotyrosine. The assay requires ≤ 10 μg of cell lysate or nanogram amounts of purified SFK. The use of a generic secondary antibody allows for the expansion of the assay to include any other tyrosine kinase for which a specific antibody exists. Using either mammalian cell lines or purified, recombinant kinases as the SFK source, we demonstrate the utility of the assay by evaluating the phosphorylation status of SFK members following several in vitro manipulations designed to modulate the phosphotyrosine content of the kinases. These results show that the SFK multiplex assay is a robust tool to investigate the function of SFKs in basic and potentially in clinical research.

The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.

Ion channels have provided a diverse set of therapeutic targets across all areas of the pharmaceutical industry. Many companies are pursuing this unique class of targets for areas of unmet medical need such as neuropathic and inflammatory pains. In the past, focused library screening sets had been designed for CNS and kinase targets. Our investigations were aimed at creating a similar dynamic screening set enriched for compounds targeting ion channels to aid screening efforts of this important class of targets. The key advantages of this approach for ion channel targets would be: (1) to identify tool compounds for novel targets and assist in assay validation, (2) to serve as a focused screen for non-384-well adaptable targets, and (3) to jump start a particular program, that is, catch-up to competition for validated, well-known targets.

The oxidative stress response is an important pathway involved in maintaining redox homeostasis in cells, preventing damage induced by free radicals and reactive oxygen species. The central regulator of this response is the transcription factor Nrf2. Nrf2 modulates expression of the oxidative stress genes via the antioxidant response element (ARE). Oxidative stress in cells may be both a cause of toxicity and a result of adaptation or cell death. To investigate whether the oxidative stress genes function as a group in response to toxic insult, we have designed and validated a rapid semiquantitative PCR assay for each selected gene. We demonstrate that the oxidative stress genes are not coordinately regulated in the mouse liver upon toxic insult. Instead their combined liver expression profiles present a gene expression signature that differs depending on the toxic stress.