Design and synthesis of allosteric modulators of CB1 cannabinoid receptor

Abstract

Cannabinoid receptors are a major class of cell-membrane receptors which belong to the super-family of G protein-coupled receptors (GPCRs) and are targeted for the treatment of several diseases including neurodegenerative diseases, cancer, obesity, inflammation and neuropathic and inflammatory pain. Two subtypes of cannabinoid receptors, namely, CB1 and CB2, have been cloned and studied more intensively. CB1 receptor is the most abundant GPCR in the brain, and a wide range of selective and potent CB1-receptor ligands for its orthosteric site have been developed. However, their therapeutic utility has been limited due to side effects associated with indiscriminate cannabinoid receptor activation and propensity for receptor desensitization. This problem is exemplified by the recent cancellation of the Phase III clinical trials of the CB1 antagonists / inverse agonists Taranabant and Otenabant and the manufacturer's (Sanofi-Aventis) voluntary withdrawal of the marketed drug Rimonabant in the European Union. Rimonabant (Acomplia), which was approved as an adjunctive weight-loss drug in Europe, suffered serious-dose related gastrointestinal and psychiatric side effects such as depression and suicidal ideation. Other approaches such as development of CB1 neutral as well as peripherally-acting antagonists have shown therapeutic promise and reduced side effects in recently published preclinical studies.A promising alternative approach is the development of CB1 allosteric modulators which by binding to a sub-type specific and topographically distinct site from the orthosteric site can either enhance or inhibit the action of endocannabinoids and thus act more selectively to tune the CB signaling in a site- and event-specific manner. Recently high-throughput screening (HTS) from two different laboratories has identified two different classes of ligand (e.g., Org27569 and PSNCBAM-1) exhibiting negative allosteric modulation at CB1 receptors. Due to the unavailability of the cannabinoid receptor's crystal structure, characterization of the binding site(s) of these allosteric modulators is lacking. Availability of such data will prove instrumental in elucidating their molecular basis for activity and in developing highly selective, potent CB1 allosteric modulators. The objective of the present study is to develop covalent probes (both photo-activatable and electrophilic) based upon the parent structure of Org27569 bearing azido and isothiocyanate functionality at the judiciously chosen positions. Using Ligand-Assisted Protein Structure approach (LAPS), which involves use of such probes for labeling the receptor covalently followed by MS analysis of the protein and validating the resulting data with site-directed mutagenesis and molecular modeling studies, the chemical nature and tertiary structure of the active allosteric sites of CB1 can be elucidated. Additionally, we propose to synthesize an iodinated analog of Org27569 to facilitate development of radiometric competitive binding assays directed at CB1 allosteric site. We also propose to synthesize two hybrid analogs of Org27569 and PSNCBAM-1 to help understand structural requirements for CB1 allosteric site and facilitate development of future structure-activity relationship studies.