Peter B. Simpson

α4β3δ GABAAReceptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential

Selective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

High Throughput Fluorescence Assays for the Measurement of Mitochondrial Activity in Intact Human Neuroblastoma Cells

The mitochondrial permeability transition event is implicated in the activation phase of apoptosis and necrosis, and is therefore postulated to play a role in many disease states. Mitochondrial permeability transition is therefore of increasing pharmaceutical interest. Drug discovery requires the rapid screening of compound libraries to identify functionally active ligands. We report the development of two fluorescence-based approaches for screening compound libraries for effects on mitochondrial function. These assays use the fluorometric imaging plate reader in 96-well format, and two commercially available dyes: JC-1 and calcein-AM. We show here that a JC-1 assay proved highly amenable to HTS implementation. By combining this with a calcein-based assay, these approaches gave complementary information: JC-1 facilitates the discovery of modulators of mitochondrial polarization from a library of -100,000 compounds screened at 8 1μM, and the calcein assay identifies permeability transition pore-specific inhibitors.

Functional characterization of bradykinin analogues on recombinant human bradykinin B1 and B2 receptors

We have examined the activity of a range of kinins on recombinant human bradykinin receptors, using a high throughput functional assay which measures intracellular Ca(2+) responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B(1) receptors were Des-Arg(9)-bradykinin (EC(50)=7.9 nM) and Des-Arg(10)-kallidin (EC(50)=8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B(2) receptors was bradykinin (EC(50)=2.0 nM). These findings confirm the validity of the recombinant system and the microtitre plate imaging-based characterization system when compared to known agonist properties of the native receptors. The concentration-response relationship for bradykinin at bradykinin B(2) receptors was potently inhibited by [D-Arg(0),Hyp(3), beta-(2-thienyl)-Ala(5),D-Tic(7),Oic(8)]-bradykinin (Hoe140) (IC(50)=71 nM), which was 500-fold more potent against the B(2)-expressing cells than the B(1) cells. Bradykinin B(1) receptor-mediated responses activated by Des-Arg(10)-kallidin were fully antagonized by Des-Arg(9)-[Leu(8)]bradykinin (IC(50)=59 nM), Des-Arg(10)-Hoe140 (IC(50)=211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic (B9858) (IC(50)=14 nM), none of which displayed any activity against the bradykinin B(2) receptor cell line up to 3 microM. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B(1) and B(2) receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used.

The Local Control of Cytosolic Ca2+ as a Propagator of CNS Communication—Integration of Mitochondrial Transport Mechanisms and Cellular Responses

Ca2+ signals propagate in wave form along individual cells of the central nervous system(CNS) and through networks of connected cells of neuronal and multiple glial cell types. Inorder for wave fronts to convey information, signaling mechanisms are required that allowwaves to propagate reproducibly and without decrement in signal strength over long distances.CNS Ca2+ waves are under specific integrated local control, made possible by interactions atlocal subcellular microdomains between endoplasmic reticulum and mitochondria. Activemitochondria located near the mouth of inositol trisphosphate receptor (InsP3R) channel clustersin glia take up Ca2+, which may prevent a buildup of Ca2+ around the InsP3R channel, therebydecreasing the rate of Ca2+-induced receptor inactivation, and prolonging channel open time.Mitochondria may amplify InsP;i3-dependent Ca2;pl signals by a transient permeability transitionin response to Ca2+ uptake into the mitochondrion. Other evidence suggests privileged accessinto mitochondria for Ca2+ entering neurons by glutamatergic receptor channels. This enablesspecific signal modulation as the Ca2+ wave is propagated into neurons, such that mitochondrialocated close to glutamate channels can prolong the neuronal cytosolic response time bysuccessive uptake and release of Ca2+. Disruption of mitochondrial function deregulates theability of CNS-derived cells to undergo normal Ca2+ signaling and wave propagation.

Implementation of a High Specification Dual-Arm Robotic Platform to Meet Flexible Screening Needs

The need for a high degree of flexibility within compound screening is a requirement throughout the drug discovery industry. The demands made of automation technology are becoming increasingly sophisticated as assay formats diversify and confidence in screening automation grows. To meet these demands for high-performance and flexibility, MSD with RTS Life Science have developed and installed a dual-robot arm screening system at our Terlings Park site to support early stage drug discovery. Plate transport on the system is via two articulated robotic Staubli arms. The system has two operating modes, combined, with the entire system operating in unison; and independent with each robot arm acting independently to perform two different screens in parallel. Additional flexibility is provided by the SPRINT software ‘dynamic batch scheduling’ feature. This allows for the scheduling of multiple screens to run automatically and for extra plates to be added and new screens to be added once the system is running without the need to stop the system and reschedule the work plan. The utility of the systems is exemplified in a kinase counter screening mode in which four different kinase assays are run against the same set of compounds in one robotic run.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signals that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signals involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of release from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Open innovation in early drug discovery: roadmaps and roadblocks

Open innovation in pharmaceutical R&D evolved from a triple helix of convergent paradigm shifts in academic, industrial and government research sectors. The birth of the biotechnology sector catalyzed shifts in location dynamics that led to the first wave of open innovation in pharmaceutical R&D between big pharma and startup companies. The National Institutes of Health (NIH) Roadmap was a crucial inflection point that set the stage for a new wave of open innovation models between pharmaceutical companies and universities that have the potential to transform the pharmaceutical R&D landscape. We highlight the attributes of leading protected open innovation models that foster the sharing of proprietary small molecule collections by lowering the risk of premature escape of intellectual property, particularly structure-activity data.

The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells.

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells, in particular in response to platelet‐derived growth factor (PDGF). PDGF‐AA, ‐AB and ‐BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF‐induced outgrowth of Tuj1‐positive precursors was abolished by the addition of EGTA, suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF‐induced outgrowth. Clostridium Toxin B increased the amount of PDGF‐induced neurite branching but had no effect on basal levels. In contrast, WHI‐P154, an inhibitor of Janus protein tyrosine kinase (JAK3), Hck and Syk, prevented PDGF‐induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross‐talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors, and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed.

Measurement and analysis of calcium signaling in heterogeneous cell cultures.

High-content imaging platforms capable of studying kinetic responses at a single-cell level have elevated kinetic recording techniques from labor-intensive low-throughput experiments to potential high-throughput screening assays. We have applied this technology to the investigation of heterogeneous cell cultures derived from primary neural tissue. The neuronal cultures mature into a coupled network and display spontaneous oscillations in intracellular calcium, which can be modified by the addition of pharmacological agents. We have developed algorithms to perform Fourier analysis and quantify both the degree of synchronization and the effects of modulators on the oscillations. Functional and phenotypic experiments can be combined using this approach. We have used post-hoc immunolabeling to identify subpopulations of cells in cocultures and to dissect the calcium responses of these cells from the population response. The combination of these techniques represents a powerful tool for drug discovery.

Estrogen receptor ligands affect mitochondrial activity in SH-SY5Y human neuroblastoma cells.

&NA; We have studied the pharmacological regulation of mitochondrial activity in a human neuroblastoma cell line. Cyclosporin A was found to directly alter mitochondrial membrane potential and to decrease mitochondrial permeability as measured using calcein. The estrogen receptor ligands tamoxifen, nafoxidine and clomiphene were identified as agents which affect mitochondrial membrane potential in a cyclosporin A‐like manner. Also when mitochondrial permeability was measured using calcein, tamoxifen, nafoxidine and clomiphene were effective in inhibiting dye loss from mitochondria. Nafoxidine and cyclosporin A inhibit effects of mastoparan on SH‐SY5Y mitochondria. These studies indicate that estrogen receptor ligands appear to affect mitochondria in a cyclosporin A‐like manner in human neuroblastoma cells.