Identification of substances that change alternative pre-mRNA splicing of the serotonin 2C receptor the serotonin receptor 2C (5-HT2CR) protein is a validated anti-obesity drug target that binds to the anorectic substances fen-phen (fenfluramine and phentermin). Due to their cross-reactivity with other receptors, these drugs are no longer in use. The activity of the 5-HT2CR protein is regulated by alternative processing pathways of its pre- mRNA. Only when the alternative exon Vb is included in the mRNA, a functional receptor can be formed. This inclusion can be achieved by two mechanisms: (i) RNA editing, which generates less active receptors and (ii) promotion by specific small nuclear RNAs which generates the most active receptors. Patients with Prader-Willi syndrome do not express the small nuclear RNAs, which most likely contributes to their obesity. We propose to identify compounds that promote inclusion of the alternative exon Vb, without a change on RNA editing. This change in pre-mRNA processing will generate the most active 5-HT2CR protein that is likely to exert an anti-appetite effect. We generated cell lines containing a fluorescent-based splicing reporter construct. These cell lines were successfully validated in a pilot screen using a library of 1692 compounds. The screen was robust (Z factor 6.1) and resulted in one compound, pyrvinium pamoate that showed an IC50 of 65M in established secondary and tertiary PCR-based screens. Our preliminary analysis of pyrvinium pamoate and small nuclear RNAs acting on serotonin pre-mRNA processing suggests that exon Vb functions similar to a bacterial riboswitch. A conformational change triggered by low molecular weight compounds causes a change in its structure that allows its splice site to be recognized, leading to its inclusion. To gain insight into structure activity relationships, we therefore further propose to test the influence of pyrvinium pamoate and other validated hits on serotonin receptor 2C pre-mRNA structure. In addition to conventional structure probing experiments, we will use RNA oligonucleotides with modified bases that reflect an alteration in structure by a change in fluorescence the selective modification of the processing pathways of a structured pre-mRNA has not been attempted yet and is the major innovation of the proposal. The identification of compounds that promote the activity of the serotonin receptor 2C by changing its pre-mRNA processing would be highly significant, as they are expected to have anti-appetite properties. Identifying a compound that combats obesity would have a major therapeutic impact and the possible identification of the first mammalian riboswitch will have a strong scientific impact.
Obesity is a major health problem affecting more than 26% of adult Americans. This project will identify compounds that change the way the body makes the serotonin receptor protein. Since this protein is a major control point of hunger, the compounds are expected to decrease appetite, which could help fighting the obesity epidemic.
