Identifying nonselective hits from a homogeneous calcium assay screen.

The authors used a homogeneous calcium dye kit with a cell line transfected using a recombinant protein construct to screen a 50,000 compound library for G-protein coupled receptor (GPCR) agonists. Only 1 of the 365 primary hits activated Gq-coupled GPCRs, as shown using IP-ONE HTRF. Furthermore, an agonist screen against the entire compound library and same heterologous cell line using AequoScreen technology generated no false positives and identified the same positive hit. Next, a multiplex assay composed of both Fluo-3 and Fura-2-loaded cells identified 1 false positive and the same true-positive hit out of the 365 primary hits. Finally, rescreening the 365 primary hits against the parental cell line loaded using the homogeneous calcium dye kit confirmed the specificity of the same true-positive hit only. In summary, the results suggest that AequoScreen technology, IP-ONE HTRF, and multiplex assays are unique, orthogonal technologies to identify nonspecific hits.

Metastin (KiSS-1) mimetics identified from peptide structure-activity relationship-derived pharmacophores and directed small molecule database screening.

Metastin, also known as KiSS-1, the cognate ligand for the metastin receptor GPR54, is a peptide known to dramatically reduce metastasis in experimental models. Despite this, there is no reported structure for metastin nor any small molecule modulators of metastin function that could be used either clinically or experimentally. Here we report the NMR solution structure of a 13-residue metastin peptide in a membrane-like environment (SDS micelles) and find it to have a relatively stable helix conformation from residues 7 to 13. In assays for metastin receptor binding and calcium flux with receptor-transfected HEK-293 cells, we demonstrate through alanine scanning and amino acid substitutions that the peptide C-terminus shows helix periodicity in an NMR structural model and that Phe9, Arg12, and Phe13 are crucial to the activity of the peptide. These three residues lie on one face of the helix and define a pharmacophore site for metastin. We used these pharmacophore features in small molecule database searches to identify hits with submicromolar affinity for the metastin receptor. We also show here that molecules mimicking key elements of this pharmacophore site bind to the metastin receptor and act as full agonists, albeit with reduced potency compared to that of metastin itself. Together this structure-activity approach may yield pharmacologically useful compounds relevant in defining and modulating metastin receptor function.

3-(4-Piperidinyl)indoles and 3-(4-piperidinyl)pyrrolo-[2,3-b]pyridines as ligands for the ORL-1 receptor.

A novel series of indoles and 1H-pyrrolo[2,3-b]pyridines having a piperidine ring at the 3-position were synthesized and found to bind with high affinity to the ORL-1 receptor. Structure-activity relationships at the piperidine nitrogen were investigated in each series. Substitution on the phenyl ring and nitrogen atom of the indole and 1H-pyrrolo[2,3-b]pyridine cores generated several selective high-affinity ligands that were agonists of the ORL-1 receptor.

Medication safety--reliability of preference cards.

A CLINICAL ANALYSIS of surgeons' preference cards was initiated in one hospital as part of a comprehensive analysis to reduce medication-error risks by standardizing and simplifying the intraoperative medication-use process specific to the sterile field. THE PREFERENCE CARD ANALYSIS involved two subanalyses: a review of the information as it appeared on the cards and a failure mode and effects analysis of the process involved in using and maintaining the cards. THE ANALYSIS FOUND that the preference card system in use at this hospital is outdated. Variations and inconsistencies within the preference card system indicate that the use of preference cards as guides for medication selection for surgical procedures presents an opportunity for medication errors to occur.

Preparation of 3-spirocyclic indolin-2-ones as ligands for the ORL-1 receptor.

A novel series of indolin-2-ones having a spirocyclic piperidine ring at the 3-position was synthesized and found to bind with high affinity to the ORL-1 receptor. Structure-activity relationships at the piperidine nitrogen were investigated. Substitution on the phenyl ring and nitrogen atom of the indolin-2-one core generated several selective high-affinity ligands that were antagonists of the ORL-1 receptor.

8-(Heteroaryl)phenalkyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones as opioid receptor modulators.

A series of N-biarylalkyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones were prepared and evaluated for biological activity at opioid (mu, delta, kappa) and opioid receptor like-1 (ORL-1) G-protein coupled receptors. Substitution on the biaryl moiety produced enhanced affinity for the mu-opioid receptor.

The nociceptin pharmacophore site for opioid receptor binding derived from the NMR structure and bioactivity relationships.

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4-17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.

Synthesis and biological evaluation of the major metabolite of atomoxetine: elucidation of a partial kappa-opioid agonist effect.

The major human metabolite of atomoxetine (4-hydroxyatomoxetine) was tested against a panel of receptors and enzymes, and was found to interact with the mu, delta, and kappa-opioid receptors based upon studies involving both binding and functional assays. 4-hydroxyatomoxetine was determined to be a partial agonist of the kappa-opioid receptor.