Title

Utilizing a cysteine substitution strategy to elucidate key residues in human CB2 and mouse CB2 binding pocket: Ligand-based structural biology

Date of Completion

January 2007

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The cannabinoid CB2 receptor is an increasingly important therapeutic target for neuropathic pain, inflammation, cancer treatment, as well as a variety of other physiological disorders. A target-based approach for the design and synthesis of novel CB2 selective compounds requires structural data on the manner with which ligands interact with this GPCR. To characterize the ligand binding motifs for CB2, we have designed and synthesized high affinity cannabinergic compounds representing the major chemical classes of CB2 ligands and carrying suitable groups capable of forming covalent attachments with different residues within the binding domain. ^ Our approach which we named "Ligand Based Structural Biology (LBSB)" involves a combination of biochemical and chemical methods aimed at identifying the ligand binding motifs at the CB2 receptor. In this dissertation, a number of high affinity covalent ligands (pharmacophores conjugated to isothiocyanate (NCS)) have been used in order to study the ligand-receptor interactions and to map the receptor's binding site(s) including: AM841, AM1336, AM4772, AM6719, AM6720, AM6721 and etc. Using a global set of cysteine substitution (cysteine to serine and/or alanine) mutant transgenic cell lines, the critical amino acid(s) in human and mouse CB2 for the irreversible attachment of each specific covalent probe were identified; ligands showing species differences to the orthologous CB2 receptors were discovered and investigated; relationships between the ligand binding motif(s) and the CB2 receptor activation has been studied. Taken together, the data provided detail as to how the compound docks into its specific binding domain and how the receptor downstream function is related to its structure. ^