Studies of g protein-coupled receptor structure and function: Receptor modeling and synthesis of bivalent ligands

G protein-coupled receptors (GPCRs) are therapeutically significant proteins and are targeted by over 25% of FDA approved drugs. GPCRs are highly druggable and are involved in a diverse range of disease states and, as such, are of immense interest to the pharmaceutical industry. GPCRs play an important role in cell signaling, mediating signals across the cell membrane. Recent advances in the high resolution X-ray crystallography of GPCRs make structure-based drug design significantly more feasible. Additionally, increased understanding of the arrangement of GPCRs in the cell membrane indicates that many GPCRs are likely to form dimers or higher order oligomers. In fact, dimerization is believed to be a common feature to GPCRs and may represent a novel therapeutic target for numerous disease states. In this thesis, recent high resolution crystal structures of several class A GPCRs have been used in the development and evaluation of new homology models of pharmaceutically relevant GPCRs. Additionally, a series of homobivalent ligands have been developed as pharmacological tools to investigate GPCR dimerization. Homology models for several therapeutically significant GPCRs were developed using the high resolution X-ray crystal structure of the β2-adrenergic receptor as a template (Chapter 2; McRobb, F. M. et al. J. Chem. Inf. Model. 2010, 50, 626-637). Techniques to optimize the orthosteric binding site, such as flexible receptor docking and loop refinement, were investigated. Small scale virtual screening was undertaken to evaluate the developed homology models for use in a structure-based drug design campaign. Of the nine homology models developed, six showed moderate to good enrichment in virtual screening experiments (5-HT1BR, 5-HT2AR, 5-HT2CR, D2R, D3R and M1 mAChR). These newly developed aminergic GPCR homology models supplement the limited number of freely available GPCR homology models. It is hoped that these models will provide a better starting point for structure-based drug design. As a continuation of our research, we have evaluated our GPCR modeling method using the GPCR Dock 2010 assessment (Chapter 3). GPCR Dock 2010 involved the prediction of the complex of the dopamine D3 receptor with the small molecule eticlopride, prior to the release of the high resolution X-ray crystal structure. The five top ranked models from this prediction were submitted to the GPCR Dock 2010 analysis (Kufareva, I. et al. Structure 2011, 19, 1108-1126) and are also compared to eticlopride in the dopamine D3 receptor crystal structure. Three series of homobivalent ligands of the atypical antipsychotic clozapine, were designed, synthesized and pharmacologically evaluated (Chapter 4). Clozapine exerts its therapeutic effect by antagonism of dopaminergic and serotonergic GPCRs, however, clozapine only displays moderate (sub-micromolar) affinity for the dopamine D2 receptor. Attachment of the spacer at the N4′ position of clozapine yielded a series of homobivalent ligands that displayed the most promising affinity and activity for the dopamine D2 receptor. A spacer length-dependent relationship with affinity or inhibitory potency was observed, with the 16 and 18 atom spacer bivalent ligands displaying low nanomolar affinity (1.41 nM and 1.35 nM) and a significant gain in affinity (75- and 79-fold, respectively) relative to the original pharmacophore, clozapine. Additionally, expanding on the modeling methods described in Chapters 2 and 3, four models of the dopamine D2 homodimer were built and optimized using molecular dynamics simulations, to determine the approximate distance between the adjacent orthosteric sites of the dimer. This project has successfully achieved the aims outlined, developing and evaluating homology models of aminergic GPCRs that are useful for structure-based drug design, as well as discovering a lead clozapine homobivalent ligand, with an appropriate attachment point and spacer length determined. Chapter 5 provides a brief summary of this thesis, with an evaluation of outcomes, as well as directions for future work.