Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT2C receptor (5-HT2CR) is essential in normal physiology, whereas aberrant 5-HT2CR function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT2CR interacts with specific protein partners, but the impact of such interactions on 5-HT2CR function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT2CR and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT2CR-mediated biology but not that of the closely homologous 5-HT2AR. A peptide derived from the third intracellular loop of the human 5-HT2CR [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT2CR-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT2CR signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT2CR allostery and therapeutics for 5-HT2CR-mediated disorders.

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. This synthetic approach not only enables a feasible preparation of a sufficient amount of 1 for use as a reference compound for secondary pharmacological studies, but also provides an efficient synthesis of key intermediates to develop novel and simplified 5-HT 2C R allosteric modulators. Compound 1 and its diastereomer 2 were functionally characterized in Chinese hamster ovary (CHO) cells stably transfected with the 5-HT 2C R using an intracellular calcium (Ca i 2+ ) release assay. Compound 1 demonstrated efficacy and potency as an allosteric modulator for the 5-HT 2C R with no intrinsic agonist activity. Compound 1 did not alter 5-HT-evoked Ca i 2+ in CHO cells stably transfected with the highly homologous 5-HT 2A R. In contrast, the diastereomer 2 did not alter 5-HT-evoked Ca i 2+ release in 5-HT 2A R-CHO or 5-HT 2C R-CHO cells or exhibit intrinsic agonist activity.KEYWORDS: PNU-69176E, diastereomer, synthesis, allosteric modulator, 5-HT 2C receptor T he serotonin (5-HT) 2C receptor (5-HT 2C R) is implicated in a diversity of physiological functions, such as nociception, motor behavior, endocrine secretion, thermoregulation, appetite modulation, and the control of exchanges between the central nervous system (CNS) and the cerebrospinal fluid.1 This receptor has also been implicated in numerous psychiatric pathologies, and the modulation of 5-HT 2C R function holds a tremendous amount of therapeutic promise for the treatment of diseases of significant unmet medical need, including addiction, anxiety, depression, obesity/ eating disorders, Parkinson's disease, and schizophrenia.Successful development of 5-HT 2C R ligands requires selectivity versus the highly homologous 5-HT 2A R and 5-HT 2B R, as 5-HT 2A/2B R agonists can result in significant CNS (5-HT 2A R) and cardiovascular (5-HT 2B R) adverse effects.3 Allosteric modulators of 5-HT 2C R present a novel and attractive drug design strategy to augment the response to endogenous 5-HT and to achieve high receptor subtype selectivity and specificity with ligand binding to an allosteric site rather than to the orthosteric binding site that binds the endogenous agonist.

BackgroundThe serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Cai++) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.ResultsWe optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.ConclusionsThese data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.

Caveolin-1 (Cav-1) is a membrane scaffolding protein which functions to regulate intracellular compartmentalization of various signaling molecules. In the present studies, transgenic mice with a targeted disruption of the Cav-1 gene (Cav-1 −/− ) were used to assess the role of Cav-1 in acetaminophen-induced hepatotoxicity. Treatment of wild type mice with acetaminophen (300 mg/ kg) resulted in centrilobular hepatic necrosis and increases in serum transaminases. This was correlated with decreased expression of Cav-1 in the liver. Acetaminophen-induced hepatotoxicity was significantly attenuated in Cav-1 −/− mice, an effect that was independent of acetaminophen metabolism. Acetaminophen administration resulted in increased hepatic expression of the oxidative stress marker, lipocalin 24p3, as well as hemeoxygenase-1, but decreased glutathione and superoxide dismutase-1; no differences were noted between the genotypes suggesting that reduced toxicity in Cav-1 −/− mice is not due to alterations in anti-oxidant defense. In wild type mice, acetaminophen increased mRNA expression of the pro-inflammatory cytokines, interleukin-1β and monocyte chemoattractant protein-1 (MCP-1), as well as cyclooxygenase-2, while 15-lipoxygenase (15-LOX), which generates anti-inflammatory lipoxins, decreased. Acetaminophen-induced changes in MCP-1 and 15-LOX expression were greater in Cav-1 −/− mice. Although expression of tumor necrosis factor-α, a potent hepatocyte mitogen, was up-regulated in the liver of Cav-1 −/− mice after acetaminophen, expression of proliferating cell nuclear antigen and survivin, markers of cellular proliferation, were delayed which may reflect the reduced need for tissue repair. Taken together, these data demonstrate that Cav-1 plays a role in promoting inflammation and toxicity during the pathogenesis of acetaminophen-induced injury.

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