Frank Wunder

Functional Cell-Based Assays in Microliter Volumes for Ultra-High Throughput Screening

Functional cell-based assays have gained increasing importance for microplate-based high throughput screening (HTS). The use of high-density microplates, most prominently 1536-well plates, and miniaturized assay formats allow screening of comprehensive compound collections with more than 1 million compounds at ultra-high throughput, i.e. in excess of 100,000 samples per day. uHTS operations with numerous campaigns per year should generally support this throughput at all different steps of the process, including the underlying compound logistics, the (automated) testing of the corporate compound collection in the bioassay, and the subsequent follow-up studies for hit confirmation and characterization. A growing number of reports document the general feasibility of cell-based uHTS in microliter volumes. In addition, full automation with integrated robotic systems allows the realization of also complex assay protocols with multiple liquid handling and signal detection steps. For this review, cell-based assays are categorized based on the kinetics of the cellular response to be quantified in the test and the readout method employed. Thus, assays measuring fast cellular responses with high temporal resolution, e.g., receptor mediated calcium signals or changes in membrane potential, are at one end of this spectrum, while tests quantifying cellular transcriptional responses mark the opposite end. Trends for cell-based uHTS assays developed at Bayer-Schering Pharma are, first, to incorporate assay integral reference signals allowing the experimental differentiation of target hits from non-specifically acting compounds, and second, to make use of kinetic, real-time readouts providing additional information on the mode-of-action of test compounds.

Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-mediated inhibition of cAMP production

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

Design and synthesis of the first NO- and haem-independent sGC activator BAY 58–2667 for the treatment of acute decompensated heart failure

Soluble guanylate cyclase (sGC) is a signal-transduction enzyme activated by nitric oxide (NO) and plays a key role in a variety of physiological processes such as vasodilatation, antiaggregation, antiproliferation and neuronal signaling as well as in a variety of disorders of these functions. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules and lack of response and the development of tolerance [1,2]. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages.

Pharmacological characterization of receptor guanylyl cyclase reporter cell lines.

Receptor guanylyl cyclases are implicated in a growing number of pathophysiologies and, therefore, represent an important target class for drug development. We report here the generation and pharmacological characterization of three particulate guanylyl cyclase (pGC) reporter cell lines. Plasmid constructs encoding the natriuretic peptide receptors GC-A and GC-B, and the heat-stable enterotoxin receptor GC-C, were stably transfected in a parental reporter cell line expressing a cyclic nucleotide-gated (CNG) cation channel, acting as the biosensor for intracellular cGMP. In our reporter cell lines pGC activity can be monitored in living cells in real-time . By using different natural as well as synthetic receptor ligands of the natriuretic and guanylin peptide families, we show that our reporter assay monitors pGC activity with very high sensitivity. In contrast to previous findings, we could detect significant stimulation of GC-A and GC-B by each of the natriuretic peptides ANP, BNP and CNP. In addition, the clearance receptor ligand Cys-ANF(4-18) and the ANP receptor antagonist Arg-ANF(6-18) were characterized as partial GC-A agonists. The results imply that our novel pGC reporter cell lines are well suited for the characterization of receptor pharmacology and may be used for natural ligand characterization of guanylyl cyclase orphan receptors.

Residues stabilizing the heme moiety of the NO sensor soluble guanylate cyclase

Soluble guanylate cyclase (sGC), the intracellular receptor for the ubiquitous biological messenger NO, exists as heterodimer consisting of an αand β-subunit. sGC contains a non-covalently linked heme moiety that is crucial for the NO-induced enzyme activation. This heme is bound to the β-subunit via the axial ligand His-105 and the recently identified counterparts of the heme propionic acids, Tyr135 and Arg-139.

Antifibrotic effects of an sGC activator in rat models of liver fibrosis

Background Liver fibrosis and cirrhosis are late complications common to liver diseases of different etiology such as viral hepatitis and alcoholic liver disease. Irrespective of the initial cause of liver disease, activation of hepatic stellate cells is a crucial step in the fibrotic pathomechanism. Activated hepatic stellate cells produce excess collagen as well as profibrotic cytokines and change to a contractile phenotype which reduces the diameter of the hepatic sinusoids. Activation of hepatic stellate cells is reduced by an increase in intracellular cyclic guanosine monophoshate (cGMP). Stable cGMP analogues also reduce the contractile response of hepatic stellate cells. However, cGMP production is downregulated in the cirrhotic liver due to reduced activity of the endothelial NO synthase. The recently discovered activators of soluble guanylate cyclase (sGC) increase cGMP production independently of NO. We therefore investigated the effects of an sGC activator in two classical rat models of liver fibrosis, the pig serum model and the carbon tetrachloride model.

Beyond NO and heme: biochemical and pharmacological opportunities

Address: 1Pharma Research Center, Bayer HealthCare, Aprather Weg 18a, 42096 Wuppertal, Germany., 2Department of Pharmacology, Monash University, Melbourne, Clayton, VIC 3800, Australia., 3Martin-Luther-University, School of Pharmacy, Wolfgang-Langenbeck-Strasse 4, 06120 Halle, Germany. and 4Herzzentrum Wuppertal, Helios-Klinikum, Medizinische Klinik, Arrenberger Strasse 20, 42117 Wuppertal, Germany.

A luminescence-based assay for sensitive nitric oxide detection

Nitric oxide (NO) plays an important role in the protection against the onset and progression of various cardiovascular diseases, including hypertension and atherosclerosis, which are associated with an apparently reduced NO bioavailability. Therefore, the NO/cGMP signaling pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based and highly sensitive nitric oxide reporter assay which is suitable for ultra-high-throughput screening. In our coculture system, endothelial nitric oxide synthase (eNOS) mediated NO generation is monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx through the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor [1]. Using this NO reporter assay, a fully automated highthroughput screening campaign for stimulators of NO synthesis was performed. The coculture system reflects most aspects of the natural NO/cGMP signaling pathway. Namely, Ca2+-dependent and Ca2+-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability, as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP pathway. References 1. Wunder F, Stasch JP, Hutter J, Alonso-Alija C, Huser J, Lohrmann E: A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the NO/cGMP pathway. Anal Biochem 2005, 339:104-112. from 3rd International Conference on cGMP Generators, Effectors and Therapeutic Implications Dresden, Germany. 15–17 June 2007

Characterization of the novel PDE9 inhibitor BAY 73-6691 using a cGMP reporter cell line

We report here the generation and characterization of a stably transfected PDE9 CHO cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system can be used for the characterization of the cellular activity of PDE9 inhibitors and for the search of new PDE9 inhibitors by ultra-high-throughput screening.