Novel Approaches to 7-transmembrane Receptor Therapeutics

Organizers: Conrad L. Cowan, formerly with Cara Therapeutics, Frederick Monsma, Schering-Plough Research Institute and Jose R. Perez, Pfizer Global Research & Development

G protein-coupled receptors (GPCRs) are high impact therapeutic targets, constituting 50% of drug targets and generating annual drug sales above $50 billion (Lundstrom K, 2006). Historically, most drug discovery campaigns have sought to identify compounds that specifically activate the receptor (agonists), or block activation by an endogenous compound (antagonist), both via a G-protein-dependent signaling pathway.

In this full-day symposium, we will focus on finding novel strategies offering new opportunities for drug discovery. We will begin with a discussion of the opportunities for allosteric regulation of receptor activity and the potential for ligand mediated modulation of 7-TM receptor interacting proteins. We will then switch to a discussion of modulation by receptor heterodimerization. We will conclude with a discussion of receptor chaperones.

The BPDG at the New York Academy of Sciences represents a diverse group of scientists and others with an interest in biochemistry, molecular biology, biomedical research, and related areas. Members are from pharmaceutical and biotechnology companies, and university and medical center research facilities across the Eastern United States. The group also serves as the Biochemical Topical Group for the American Chemical Society's New York Section. The purpose of the BPDG is to bring together diverse institutions and communities, industrial and academic, to share new and relevant information at the frontiers of research and development.

ABSTRACTS

An Allosteric Approach for the Modulation of Class A GPCRs: Potential New Therapeutics for Schizophrenia and Alzheimer's Disease
Craig W Lindsley
Vanderbilt University Medical Center

M1 and M4 mAChR subtypes of the muscarinic receptor represent attractive targets for many CNS disorders including Alzheimer's disease (AD) and Schizophrenia. Traditional muscarinic agonists lack efficacy and carry severe side effects due to non-selective activation of all mAChR subtypes, and absence of selective agents has hindered basic research into the respective roles of the M1 and M4 receptors in the CNS. We report the discovery and preliminary characterization of novel muscarinic ligands that modulate either the M1 or M4 receptor in an allosteric manner with high subtype-selectivity. We also report the discovery of a highly potent M1 antagonist with unprecedented selectivity. Functional cell-based HTS and technology-enabled synthetic methods were used to identify and optimize a number of compounds. An identified allosteric agonist, TBPB, demonstrated disease modification potential for AD by decreasing Aβ secretion in an APP processing assay. TBPB also displayed in vivo antipsychotic activity in rodent studies. A novel series of centrally active M4 positive allosteric modulators have demonstrated antipsychotic efficacy in preclinical models.

Molecular Determinants of GPCR Ligand-Biaised Signaling
Geneviève Oligny-Longpré
University of Montreal

Drug efficacy is generally determined by the drug's ability to promote a quantifiable biological response. In the context of the classical receptor-occupancy theory, the efficacy is considered an intrinsic property of the ligand/receptor pair, and it is often assumed to be the same for all the responses evoked by this pair. However, recent observations suggest that efficacy may also be influenced by the signaling cascade considered, raising the possibility that drugs may have complex efficacy profiles toward the signaling activity of different effectors engaged by a unique receptor. To directly and systematically test this possibility, we assessed the ability of a panel of beta-adrenergic ligands to modulate the activity of two effector systems, the adenylyl cyclase (AC) and the mitogen-activated protein kinase (MAPK), via beta-adrenergic receptors (βARs). Although some compounds displayed similar efficacies toward the two pathways, others showed complex efficacy profiles. For example, compounds that are inverse agonists for the AC activity were found to be either agonists, neutral antagonists, or inverse agonists for the MAPK pathway. Reciprocally, agonist for the AC could be either agonist or neutral antagonists for MAPK. Given this complexity, we propose a Cartesian representation of the efficacies that takes into account the activities of the different effectors that can be engaged by a given receptor. Taken together, the results obtained are consistent with the notion that binding of different ligands can promote distinct conformational changes leading to specific signaling outcomes, often referred to as "ligand-biased signaling". To investigate the molecular correlates of such ligand-biased signaling, the ability of different β-adrenergic ligands to selectively engage distinct proximal signaling partners was evaluated and correlated with ligand-specific conformational rearrangements of receptor/G protein complexes measured by Bioluminescence Resonance Energy Transfer. The results clearly indicate that ligands with distinct efficacy profiles promote different conformational rearrangements of the signaling complexes. Recently, the structure of the β2AR has been obtained, and had allowed the formulation of testable hypotheses about the structural determinants linking drug binding to specific signaling responses. The three-dimensional structure derived from the coordinates of the β2AR crystal was used to perform in silico docking of ligands with distinct activity profiles toward AC and MAPK. The different binding modes of these ligands, which correlated with their reported efficacy profiles, suggest that it should be possible to predict the structural determinants of drug signaling efficacies.

Modulation of Function by Opioid Receptor Dimerization
Lakshmi A. Devi
Mount Sinai School of Medicine

Opioid receptors belong to the super family of G-protein coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by narcotic analgesics or by endogenous opioid peptides leads to the activation of inhibitory G-proteins followed by the activation of multiple signal transduction pathways. A number of investigations have suggested that opioid receptor types interact with each other. Previous studies using receptor selective antagonists, antisense oligonucleotides or animals lacking opioid receptors have suggested that these interactions modulate receptor activity. We examined opioid receptor interactions (homotypic and heterotypic) using biochemical, biophysical and pharmacological techniques. We show that mu and delta opioid receptors physically associate with each other to form heterodimers that exhibit altered agonist affinity, efficacy and/or potency. Using receptor type-selective antibodies, we immunoisolated interacting complexes from heterologous cells as well as endogenous tissue. Finally, we show that chronic morphine treatment upregulates the levels of mu-delta heterodimers in vivo and leads to the formation of new signaling complexes, which in turn lead to a switch in signaling by mu-delta heterodimers. Taken together, these results suggest that mu-delta heterodimers can be used as unique targets for the development of novel drugs and therapies for the treatment of chronic pain and other pathologies.

Distinct β-Arrestin and G-protein Mediated Signaling Pathways
Jonathan Violin
Trevena, Inc

Since it was first cloned 22 years ago, the molecular pharmacology of the beta-2 adrenergic receptor (B2AR) has proven to be archetypal for the family of G protein-coupled receptors. Despite extensive investigation of the molecular regulation of B2AR function, recent work has uncovered a series of surprising findings. In particular, the function of G protein-coupled receptor kinases (GRKs) and beta-arrestins as modulators of receptor signaling has proven to be an unexpected source of complexity in B2AR biology. Originally characterized for their ability to desensitize G protein signaling, beta-arrestins are now known also to promote receptor internalization and activate G protein-independent signaling at the B2AR. Each of these functions requires receptor phosphorylation by GRKs. Recent evidence demonstrating specificity of the B2AR for GRK isoforms, and differential regulation of these isoforms, has important implications for the physiological regulation of B2AR signaling. Strikingly, the beta blocker carvedilol is a "biased ligand" that selectively engages beta-arrestin and GRKs, and in this regard is unique among commonly used beta blockers. This raises the possibility that beta-arrestin and GRK function may be an important aspect of therapeutic intervention via the B2AR.

Dopamine D2 Receptors Form Higher Order Oligomers at Physiological Expression Levels
Jonathan A. Javitch
Columbia University

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.
Co-authors: Guo W, Urizar E, Kralikova M, Mobarec JC, Shi L, Filizola M

Regulation of Kappa Opioid Receptor Trafficking by GEC1
Lee-Yuan Liu-Chen
Temple University School of Medicine

The number of cell surface 7TMRs reflects a delicate balance between biosynthesis pathway and endocytosis pathway. The post-activation endocytic events such as internalization, recycling and degradation have been well-documented; however, the regulatory events occurring along the protein biosynthesis pathway are less-studied. We have demonstrated that the protein glandular epithelial cell 1 (GEC1) bound to the C-terminal domain of the human kappa opioid receptor (hKOPR) and promoted cell surface expression of the receptor by facilitating its trafficking along the secretory pathway. In addition, we showed that three hKOPR residues (F345, P346 and M350) and seven GEC1 residues (Y49, V51, L55, T56, V57, F60 and I64) are indispensable for the interaction. Modeling studies revealed that the interaction was mediated via direct contacts between the kinked hydrophobic fragment in hKOPR C-tail and the curved hydrophobic surface in GEC1 around the S2 β-strand. Intramolecular L44-Y109 interaction in GEC1 was important, likely by maintaining its structural integrity. Microtubule binding mediated by GEC1 N-terminal domain was essential for the GEC1 effect. Expression of GEC1 also increased cell surface levels of the GluR1 subunit and the prostaglandin EP3.f receptor, which have FPXXM and FPXM sequences, respectively. With its widespread distribution in the nervous system and its predominantly hydrophobic interactions, GEC1 may have chaperone-like effects for many cell surface proteins along the biosynthesis pathway.

Pharmacological Chaperones: Potential for the Treatment of Hereditary Diseases Caused by Mutant G Protein-coupled Receptors
Kenneth J. Valenzano
Amicus Therapeutics

Many human diseases result from mutations in specific genes. Once translated, the resulting proteins are often functionally competent and produced at normal levels. However, because of the mutations, these proteins may be recognized as misfolded or less stable by the quality control system of the endoplasmic reticulum (ER). As such, they are not processed and trafficked correctly, ultimately leading to cellular dysfunction and disease. Small molecule pharmacological chaperones represent a promising new therapeutic approach to the treatment of these genetic disorders. Pharmacological chaperones are designed to selectively bind to the mutated proteins and are believed to stabilize a near-native conformation. This stabilization promotes normal processing and trafficking of the mutant protein and allows passage through the ER quality control system, ultimately increasing protein levels and activity in relevant cellular locations and reducing ER accumulation, aggregation, and associated cell stress. Partial or complete restoration of normal protein target function in the presence of pharmacological chaperones has been shown for numerous types of mutant proteins, including enzymes, secreted proteins, transcription factors, ion channels, and G protein-coupled receptors. This talk will highlight in vitro and in vivo data that provide proof-of-concept for the use of pharmacological chaperones in two common lysosomal storage disorders, Gaucher and Fabry diseases, which result from mutations in the enzymes β-glucocerebrosidase and α-galactosidase A, respectively. In addition, preclinical and clinical data that support the use of pharmacological chaperones to treat X-linked nephrogenic diabetes insipidus, via restoration of mutant V2 vasopressin receptor function, will also be reviewed. Lastly, recent data will be presented that demonstrate the potential application of pharmacological chaperones for the treatment of mutant melanocortin 4 receptor-mediated obesity in humans, via restoration of cell surface localization and function.