Multi-well plate immunoassays for measuring signaling protein activations/deactivations and membrane vs. intracellular receptor levels.

We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.

Peptide inhibitors disrupt the serotonin 5-HT2C receptor interaction with phosphatase and tensin homolog to allosterically modulate cellular signaling and behavior.

Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT(2C) receptor (5-HT(2C)R) is essential in normal physiology, whereas aberrant 5-HT(2C)R function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT(2C)R interacts with specific protein partners, but the impact of such interactions on 5-HT(2C)R function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT(2C)R and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT(2C)R-mediated biology but not that of the closely homologous 5-HT(2A)R.

Exploration of synthetic approaches and pharmacological evaluation of PNU-69176E and its stereoisomer as 5-HT2C receptor allosteric modulators.

Allosteric modulators of the serotonin (5-HT) 5-HT(2C) receptor (5-HT(2C)R) present a unique drug design strategy to augment the response to endogenous 5-HT in a site- and event-specific manner with great potential as novel central nervous system probes and therapeutics. To date, PNU-69176E is the only reported selective positive allosteric modulator for the 5-HT(2C)R. For the first time, an optimized synthetic route to readily access PNU-69176E (1) and its diastereomer 2 has been established in moderate to good overall yields over 10 steps starting from commercially available picolinic acid.