Sujatha M. Gopalakrishnan

Validation of FLIPR Membrane Potential Dye for High Throughput Screening of Potassium Channel Modulators

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive Ki channel; KATP) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference KATP channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.

Homogeneous Time-Resolved Fluorescence Quenching Assay (LANCE) for Caspase-3

In addition to kinases and G protein—coupled receptors, proteases are one of the main targets in modern drug discovery. Caspases and viral proteases, for instance, are potential targets for new drugs. To satisfy the current need for fast and sensitive high-throughput screening for inhibitors, new homogeneous protease assays are needed. We used a caspase-3 assay as a model to develop a homogeneous time-resolved fluorescence quenching assay technology. The assay utilizes a peptide labeled with both a luminescent europium chelate and a quencher. Cleavage of the peptide by caspase-3 separates the quencher from the chelate and thus recovers europium fluorescence. The sensitivity of the assay was 1 pg/μl for active caspase-3 and 200 pM for the substrate. We evaluated the assay for high-throughput usage by screening 9600 small-molecule compounds. We also evaluated this format for absorption/distribution/metabolism/excretion assays with cell lysates. Additionally, the assay was compared to a commercial fluorescence caspase-3 assay.

Structure−Activity Studies on a Series of a 2-Aminopyrimidine-Containing Histamine H4 Receptor Ligands

A series of 2-aminopyrimidines was synthesized as ligands of the histamine H4 receptor (H4R). Working in part from a pyrimidine hit that was identified in an HTS campaign, SAR studies were carried out to optimize the potency, which led to compound 3, 4- tert-butyl-6-(4-methylpiperazin-1-yl)pyrimidin-2-ylamine. We further studied this compound by systematically modifying the core pyrimidine moiety, the methylpiperazine at position 4, the NH2 at position 2, and positions 5 and 6 of the pyrimidine ring. The pyrimidine 6 position benefited the most from this optimization, especially in analogs in which the 6- tert-butyl was replaced with aromatic and secondary amine moieties. The highlight of the optimization campaign was compound 4, 4-[2-amino-6-(4-methylpiperazin-1-yl)pyrimidin-4-yl]benzonitrile, which was potent in vitro and was active as an anti-inflammatory agent in an animal model and had antinociceptive activity in a pain model, which supports the potential of H 4R antagonists in pain.

Discovery of 4-(5-(4-Chlorophenyl)-2-methyl-3-propionyl-1H-pyrrol-1-yl)benzenesulfonamide (A-867744) as a Novel Positive Allosteric Modulator of the α7 Nicotinic Acetylcholine Receptor

The discovery of a series of pyrrole-sulfonamides as positive allosteric modulators (PAM) of alpha7 nAChRs is described. Optimization of this series led to the identification of 19 (A-867744), a novel type II PAM with good potency and selectivity. Compound 19 showed acceptable pharmacokinetic profile across species and brain levels sufficient to modulate alpha7 nAChRs. In a rodent model of sensory gating, 19 normalized gating deficits. These results suggest that 19 represents a novel class of molecules capable of allosteric modulation of the alpha7 nAChRs.

Structure-activity studies for a novel series of tricyclic dihydropyrimidines as KATP channel openers (KCOs)

A novel series of tricyclic dihydropyrimidines was synthesized and evaluated for activity as K(ATP) channel openers. The functional activity of several compounds, for example 6A (EC(50)=30nM) and its enantiomers exceeded cromakalim.

In Vitro Pharmacological Characterization of a Novel Selective α7 Neuronal Nicotinic Acetylcholine Receptor Agonist ABT-107

Enhancement of α7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimers disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective α7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to α7 nAChRs [rat or human cortex, [3H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), Ki = 0.2–0.6 nM or [3H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-α7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman α3β4, chimeric (α6/α3)β4, or 5-HT3A receptors, and weak or negligible Ca2+ responses in human neuroblastoma IMR-32 cells (α3* function) and human α4β2 and α4β4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat α7 nAChR current responses in oocytes (EC50, 50–90 nM total charge, ∼80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked α7-like currents, which were inhibited by the α7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an α7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive α7 nAChR-mediated Ca2+ signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity α7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) Induces Phosphorylation of Eukaryotic Elongation Factor-2 (eEF2) A CAUTIONARY NOTE ON THE ANTICANCER MECHANISM OF AN eEF2 KINASE INHIBITOR

Background: NH125 inhibits cancer cell growth through inhibition of eukaryotic elongation factor-2 kinase (eEF2K). Results: NH125 induces eEF2 phosphorylation (peEF2) through multiple pathways in cancer cells. Conclusion: NH125 is not an eEF2K inhibitor in cancer cells. Inhibition of cell growth correlates with induction of peEF2. Significance: NH125-induced peEF2 corrects a misconception and provides an opportunity for a new multipathway approach to anticancer therapies. Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

Role of Rho kinase pathway in chondroitin sulfate proteoglycan‐mediated inhibition of neurite outgrowth in PC12 cells

Activation of the Rho kinase (ROCK) pathway has been associated with inhibition of neurite regeneration and outgrowth in spinal cord injury. Growth‐inhibitory substances present in the glial scar such as chondroitin sulfate proteoglycans (CSPGs) have been shown to create a nonpermissive environment for axon regeneration that results in growth cone collapse. In this study, an in vitro model was developed in nerve growth factor‐differentiated PC12 cells where the Rho/ROCK pathway was modulated by CSPG. CSPG elicited concentration‐dependent inhibition of neurite outgrowth in PC12 cells, which was reversed by ROCK inhibitors such as fasudil, dimethylfasudil, and Y27632. Further studies on the interactions of CSPG with ROCK inhibitors revealed that the modulation of ROCK by CSPG is noncompetitive in nature. It was also observed that ROCK inhibitors increased neurite outgrowth in undifferentiated PC12 cells, indicating constitutive ROCK activity in the cells. Analysis of signaling pathways demonstrated that the effect of CSPG increases the phosphorylation of myosin phosphatase, a substrate immediately downstream of ROCK activation. Fasudil, dimethylfasudil, and Y27632 inhibited the phosphorylation of myosin phosphatase induced by CSPG with rank order potencies comparable to those observed in the neurite outgrowth assay. In addition, ROCK inhibitors reversed cofilin phosphorylation induced by CSPG with similar rank order potencies. Taken together, our data demonstrate that the interaction of CSPG with the ROCK pathway involves downstream effectors of ROCK such as myosin phosphatase and cofilin.

Application of Micro Arrayed Compound Screening (pcARCS) to Identify Inhibitors of Caspase-3

Micro Arrayed Compound Screening (pARCS) is a miniaturized ultra-high-throughput screening platform developed at Abbott Laboratories. In this format, 8640 discrete compounds are spotted and dried onto a polystyrene sheet, which has the same footprint as a 96-well plate. A homogeneous time-resolved fluorescence assay format (LANCE) was applied to identify the inhibitors of caspase-3 using a peptide substrate labeled with a fluorescent europium chelate and a dabcyl quencher. The caspase-3 enzyme was cast into a thin agarose gel, which was placed on a sheet containing test compounds. A second gel containing caspase substrate was then laid above the enzyme gel to initiate the reaction. Caspase-3 cleaves the substrate and separates the europium from the quencher, giving rise to a time-resolved fluorescent signal, which was detected using a ViewLux charge-coupled device imaging system. Potential inhibitors of caspase-3 appeared as dark spots on a bright fluorescent background. Results from the pARCS assay format were compared to those from a conventional 96-well plate-screening format.

Utility of Large-Scale Transiently Transfected Cells for Cell-Based High-Throughput Screens to Identify Transient Receptor Potential Channel A1 (TRPA1) Antagonists

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.