Absence of direct effects on the dopamine D2 receptor by mGluR2/3-selective receptor agonists LY 354,740 and LY 379,268.

We previously reported the absence of high-affinity binding of the group II metabotropic glutamate receptor agonists LY 354,740 and LY 379,268 to the D2L dopamine receptor. A rebuttal to our findings has since been reported (see Introduction section); this study represents our response. Analysis by LCMS of LY 354,740 and LY 379,268 used in this study revealed the correct molecular mass for these compounds. Both LY 354,740 and LY 379,268 exhibited potent agonist activity for mGluR2 in the ³5S-GTPγS assay. Functionally, neither compound displayed antagonist activity in the GTPγS assay with recombinant D2. At concentrations up to 10 µM, both compounds failed to displace [³H]-raclopride, [³H]-PHNO, or [³H]-domperidone in filter-binding assays under isotonic (120 mM NaCl or N-methyl glucamine) or low-ionic strength (no NaCl or N-methyl glucamine) conditions. Some displacement of [³H]-domperidone (20-40%) was observed at 30 µM of LY 354,740 under low-ionic strength and under isotonic conditions in the absence of NaCl. No displacement of [³H]-domperidone was detected in a two site model at lower (<100 nM) concentrations of either compound. Moreover, no D2 activity was observed for LY 354,740 or LY 379,268 in the CellKey™ (cellular dielectric spectroscopy) assay. In this communication, we discuss the possible reasons for differences in our study and the previously published work and implications of these studies for mechanisms of antipsychotic action.

Developing dual functional allosteric modulators of GABA(A) receptors.

Positive modulators at benzodiazepine sites of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Negative allosteric modulators of α5-containing GABA(A) receptors enhance cognition. By oocyte two-electrode voltage clamp and subsequent structure-activity relationship studies, we discovered cinnoline and quinoline derivatives that were both positive modulators at α2-/α3-containing GABA(A) receptors and negative modulators at α5-containing GABA(A) receptors. In addition, these compounds showed no functional activity at α1-containing GABA(A) receptors. Such dual functional modulators of GABA(A) receptors might be useful for treating comorbidity of anxiety and cognitive impairments in neurological and psychiatric illnesses.

Evaluation of cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs.

Cellular dielectric spectroscopy (CDS) is an emerging technology capable of detecting a range of whole-cell responses in a label-free manner. A new CDS-based instrument, CellKey, has been developed that is optimized for G-protein coupled receptor (GPCR) detection and has automated liquid handling in microplate format, thereby making CDS accessible to lead generation/optimization drug discovery. In addition to having sufficient throughput, new assay technologies must pass rigorous standards for assay development, signal window, dynamic range, and reproducibility to effectively support drug discovery SAR studies. Here, the authors evaluated CellKey with 3 different G(i)-coupled GPCRs for suitability in supporting SAR studies. Optimized assay conditions compatible with the precision, reproducibility, and throughput required for routine screening were quickly achieved for each target. Across a 1000-fold range in compound potencies, CellKey results correlated with agonist and antagonist data obtained using classical methods ([(35)S]GTPgammaS binding and cAMP production). For partial agonists, relative efficacy measurements also correlated with GTPgammaS data. CellKey detection of positive allosteric modulators appeared superior to GTPgammaS methodology. Agonist and antagonist activity could be accurately quantified under conditions of low receptor expression. CellKey is a new technology platform that uses label-free detection in a homogeneous assay that is unaffected by color quenching and is easily integrated into existing microtiter-based compound testing and data analysis procedures for drug discovery.

Mechanism of gamma-secretase cleavage activation: is gamma-secretase regulated through autoinhibition involving the presenilin-1 exon 9 loop?

Maturation of gamma-secretase requires an endoproteolytic cleavage in presenilin-1 (PS1) within a peptide loop encoded by exon 9 of the corresponding gene. Deletion of the loop has been demonstrated to cause familial Alzheimer's disease. A synthetic peptide corresponding to the loop sequence was found to inhibit gamma-secretase in a cell-free enzymatic assay with an IC(50) of 2.1 microM, a value similar to the K(m) (3.5 microM) for the substrate C100. Truncation at either end, single amino acid substitutions at certain residues, sequence reversal, or randomization reduced its potency. Similar results were also observed in a cell-based assay using HEK293 cells expressing APP. In contrast to small-molecule gamma-secretase inhibitors, kinetic inhibition studies demonstrated competitive inhibition of gamma-secretase by the exon 9 peptide. Consistent with this finding, inhibitor cross-competition kinetics indicated noncompetitive binding between the exon 9 peptide and L685458, a transition-state analogue presumably binding at the catalytic site, and ligand competition binding experiments revealed no competition between L685458 and the exon 9 peptide. These data are consistent with the proposed gamma-secretase mechanism involving separate substrate-binding and catalytic sites and binding of the exon 9 peptide at the substrate-binding site, but not the catalytic site of gamma-secretase. NMR analyses demonstrated the presence of a loop structure with a beta-turn in the middle of the exon 9 peptide and a loose alpha-helical conformation for the rest of the peptide. Such a structure supports the hypothesis that this exon 9 peptide can adopt a distinct conformation, one that is compact enough to occupy the putative substrate-binding site without necessarily interfering with binding of small molecule inhibitors at other sites on gamma-secretase. We hypothesize that gamma-secretase cleavage activation may be a result of a cleavage-induced conformational change that relieves the inhibitory effect of the intact exon 9 loop occupying the substrate-binding site on the immature enzyme. It is possible that the DeltaE9 mutation causes Alzheimer's disease because cleavage activation of gamma-secretase is no longer necessary, alleviating constraints on Abeta formation.

A homogeneous in vitro functional assay for estrogen receptors: coactivator recruitment.

Estrogen receptor (ER)-mediated gene transcription occurs via the formation of a multimeric complex including ligand-activated receptors and nuclear coactivators. We have developed a homogeneous in vitro functional assay to help study the ligand-dependent interaction of ERs with various nuclear coactivators. The assay consists of FLAG-tagged ERalpha or ERbeta ligand binding domain (LBD), a biotinylated coactivator peptide, europium-labeled anti-FLAG antibody, and streptavidin-conjugated allophycocyanin. Upon agonist binding, the biotinylated coactivator peptide is recruited to FLAG-tagged ER LBD to form a complex and thus allow fluorescence resonance energy transfer (FRET) to occur between europium and allophycocyanin. Compounds with estrogen antagonism block the agonist-mediated recruitment of a coactivator and prevent FRET. The assay was used to evaluate the preference of ERs for various coactivators and ligands. Both ERalpha and ERbeta exhibited strong preferences for coactivator peptides corresponding to steroid receptor coactivator-1 and PPARgamma coactivor-1 vs. peroxisome proliferator-activated receptor-interacting protein and cAMP response element binding protein-binding protein. 17beta-Estradiol acted as a nonselective agonist for ERalpha and ERbeta. Genistein showed full agonism for ERalpha and only partial agonism for ERbeta, but with higher potency for ERbeta than ERalpha. Both raloxifene and tamoxifen behaved as full antagonists in this functional assay. The results obtained using compounds with a wide range of potency correlated well with those from a cell-based reporter gene assay. Therefore, this simple in vitro functional assay is predictive of ligand-dependent transactivation function of the receptor and, as such, is useful in nuclear receptor applications including mechanistic studies.