Models For Understanding The Effects Of Allosteric Modulators

The recognition that GPCRs can possess binding sites in addition to the orthosteric site has necessitated the development of mathematical models to facilitate descriptions and understanding of allosteric drug-receptor interactions. The binding of an allosteric ligand to its site will change the conformation of the receptor, which means that the geography of the orthosteric binding pocket and any other potential receptor-ligand protein interfaces, may also change. As a consequence, the binding...

Receptor Binding Assays

The biological identification and pharmacological characterization of receptors have been aided immensely by the advent of radiolabeled agents that retained their biological activity after iodination or tritiation, allowing the development of a method known as the radioligand binding or receptor binding technique. Due to the relative easy way with which radioligand binding assays can be performed, they are still widely used in both academic and industrial laboratories. The quality of data...

The Search For Other Gpcr Structures 1441 Expression of Recombinant Receptors

In the absence of tissues expressing high quantities of homogenous native receptors, much of the work leading up to the crystallization of other GPCRs has focused on their expression. The expression of membrane proteins is notoriously difficult and has been one of the major bottlenecks in the structural biology of GPCRs, which typically require milligram quantities of active protein. A number of expression systems have been developed for the expression of GPCRs, each with their own particular...

Pharmacological Models of Allosteric Interactions

The classical form of the TCM suggests that the probe dependence of the G protein defines the ability of the orthosteric ligand to promote the formation of the ternary complex and as such, the efficacy of an orthosteric ligand (Fig. 2.1b, middle). Within this model, the G protein has positive cooperativity with an orthosteric agonist, negative cooperativity with an orthosteric inverse agonist, and neutral cooperativity with an orthosteric antagonist. The TCM can also be used as a more general...

In Cubo Crystallization

In contrast to the original human p2AR 2 and turkey p1AR 4 , which were crystallized using hanging drop vapor diffusion, both the p2AR and A2a T4L Figure 14.4 Comparison of the structures of the PAR showing the positions of the antibody and T4L insertion used to facilitate crystallization. Figure 14.4 Comparison of the structures of the PAR showing the positions of the antibody and T4L insertion used to facilitate crystallization. fusions were crystallized using the cubic phase method to grow...

Crystallization Chaperones

To obtain well-ordered crystals of membrane proteins, it is often necessary to include a protein that will act as a crystallization chaperone. Such proteins include antibodies - 164 or DARPins - 165 , or smaller more crystallizable proteins may be inserted into the target protein of interest in the form of a fusion protein. These chaperones facilitate crystallization by using a number of different mechanisms. First, the bound chaperone increases the overall hydrophilicity of the complex. This...

The Ionic Lock

In the inactive state of rhodopsin, located on the intracellular side, the ionic lock, which constrains H6 inside the helical bundle, is comprised of an extended hydrogen-bonded network involving Arg135 (H3), Glu247 (H6), Glu134 (H3), and Thr251 (H6), where Glu134 and Arg135 are the first and second residues of the conserved D(or E)RY motif (see Fig. 16.2a-e). The presence of the ionic lock is understood to characterize the inactive state and is supported by biophysical data 47, 48 ....

The Operational Model Of Drug Action

While Stephenson's treatment of drug receptor-mediated response can be used to fit data to molecular models, it still utilized efficacy essentially as a fitting parameter tailored to make experimental data fit the model. This arbitrary nature of efficacy led Black and Leff to postulate a new model of drug action based on observed effects of drugs in tissues they called this approach the operational model of drug action 12 . This model is based on the premise that the efficacy term emerges from...

Introduction

The members of the G protein- coupled receptor (GPCR) superfamily are important contributors to many processes of development and disease 1, 2 . They consist of at least 300 nonsensory human family members that are known or predicted to be activated by endogenous ligands. GPCRs are an important drug target class for the pharmaceutical industry, and more than 20 of currently top selling drugs are GPCR related 3, 4 . However, only a small proportion of the GPCR family has been fully exploited,...

Controversial Issues

Deorphanization of 7TM proteins has become increasingly difficult. New screening strategies will help to deorphanize further orphans, and new concepts may highlight additional functions that may or may not depend on ligand binding to the predicted orthosteric binding site. However, deorphanization of the remaining orphan GPCRs is expected to remain a slow and tedious task. From the early days of deorphanization on, researchers have been often confronted with high levels of apparent constitutive...

Inverse Agonism Detected at the Level of the Receptor

FRET-based detection of receptor conformational changes which are directly related to activation proved to be a useful tool to determine the action of inverse agonists on GPCRs. Both inverse agonists for a2A- adrenergic receptors, as well as those acting on P1-adrenergic receptors have been studied using this approach 32, 78 . All inverse agonists tested induced a FRET change in the opposite direction compared to agonists. In the case of a2A- adrenergic receptor, the introduction of a point...

Glutamate Class C

The Class C GPCR family comprises 22 human receptors 141 , including such therapeutically important targets as the metabotropic glutamate receptors (GRM1. 8) 142-144 . GABA binding receptors (GABABRs), and the calcium-sensing receptor (CaR). For example, the GABAB agonist baclofen is used in the clinic as an anticonvulsant 122 , while the allosteric CASR modulator cinacalcet is known to normalize calcium level in patients with hyper-parathyroidism 145 . Class C receptors bind their endogenous...

Structure and Modeling of Loop Regions

The seven transmembrane GPCR helices are connected by three intracellular (ICL) and three ECL loops. The shorter loops ICL1, ICL2, and ECL1 are well resolved in the crystal structures and are amenable to homology modeling for most Class A GPCRs. However, modeling of the other loop regions (ICL3, ECL3, and ECL2) is very challenging due to their conformational flexibility, large length variation, and low sequence conservation among GPCRs. For example ICL3, which is closely involved in G protein...

References

Bouvier, M. (2001) Oligomerization of G-protein-coupled transmitter receptors. Nat Rev Neurosci. 2, 274-286. 2. Milligan, G., Ramsay, D., Pascal, G., Carrillo, J.J. (2003) GPCR dimerisation. Life Sci. 74,181-188. 3. James, J.R., Oliveira, M.I., Carmo, A.M., Iaboni, A., Davis, S.J. (2006) A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer. Nat Methods. 3,1001-1006. 4. Bouvier, M., Heveker, N., Jockers, R., Marullo, S.,...

Ntroduction

Despite a common structural scaffold of seven transmembrane helices, the G protein-coupled receptor (GPCR) superfamily enables recognition of a broad range of extracellular stimuli encompassing odorants, peptides, lipids, biogenic amines, hormones, nucleotides, and even light (Fig. 15.1). Phylogenetic analysis of the human GPCR superfamily has identified 860 distinct receptors, of which approximately 300 are nonolfactory 1, 2 . Given this remarkable diversity in sequence and function,...

Chapter

Secretin Receptor Dimerization A Possible Functionally Important Paradigm for Family B G Protein-Coupled Receptors* KALEECKAL G. HARIKUMAR, MAOQING DONG, and LAURENCE J. MILLER Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ Protein-protein interactions represent a molecular mechanism for signal propagation and for regulation that is utilized extensively in cell biology and physiology. The interaction between single transmembrane tyrosine kinase...

GPCR Heterodimerization

For certain class C GPCRs, such as the GABAb receptor and taste receptors, the evidence for heterodimerization is overwhelming. However, there is no reported heterodimerization among different metabotropic glutamate receptors, despite several closely related receptor subtypes, coexpressed in the same neurons 51, 98 . Ability to form class C dimers is clearly a tightly regulated phenomenon. What is then to be made of FRET studies which indicate numerous interacting combinations of different...

Stabilization of XRay Structures

Due to its high abundance in bovine retina 19 and the limitations in the purification and crystallization of other GPCR proteins 20 , bovine rhodopsin, until recently, was the only GPCR for which a three-dimensional (3D) structure was determined. Creating challenges to crystallization, other GPCR proteins are found in low concentrations, are conformationally flexible, and are unstable in detergent solutions. Since the bovine rhodopsin X-ray determinations, the first GPCR structures published...

Functional Assays For Deorphanization

The nature of the functional assay used to deorphanize 7TM proteins is crucial for the success rate of ligand identification. A number of expression systems TABLE 7.2 Identified Functions of Orphan 7TM Proteins Since 2006 Constitutive inhibition of cAMP production through G Constitutive inhibition of cAMP production through Goi Protective role in response to hemodynamic stress Constitutive stimulation of cAMP production Interacts with dopamine transporter and modulates its activity...

Some Examples Of Gpcr Allosteric Modulators 361 Small Molecule Allosteric Modulators

Clearly, the most obvious potential for exploiting allosteric sites on GPCRs is with respect to the discovery of novel small molecule chemotypes, which traditionally are seen as the most suitable agents amenable for drug development. In recent years, small molecule allosteric modulators have been discovered to act at all three major families of GPCRs (see Table 3.1 for some representative examples). While many of these have remained pharmacological tools, they have highlighted the applicability...

Radioligand Binding Assays

Equilibrium Binding Assays Detection of allosteric interactions using an equilibrium binding assay relies on the ability of a putative allosteric ligand to alter the binding affinity of a radiolabeled orthosteric ligand to either increase or decrease the specific binding of the orthosteric probe. The most common assay in this regard is to perform a modulator titration curve against a single fixed concentration of orthosteric radioligand. According to the simple ATCM, the maximum fractional...

Modeling Secretin Class B Family GPCRs

The secretin (Class B) family comprises at least 15 GPCRs that share significant sequence similarity and an extracellular hormone-binding amino-terminal domain. Structures of isolated amino-termini for corticotropin-releasing factor (CRF), pituitary adenylate cyclase-activating polypeptide (hPAC1), gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH1) receptors have been recently solved by NMR and crystallography -127-129 . Though highly divergent in amino acid sequence, the...

Second Messenger Assays

CAMP The concept of transmembrane signaling via GPCRs, which transduces extracellular signals into intracellular messages, was established with the discovery of cAMP by Sutherland et al. 35 . Specifically, binding of an agonist to a GPCR could either increase (via coupling through Gas) or decrease (via coupling through Gai) the rate at which cAMP is generated in cells through activation or inhibition of adenylyl cyclase, respectively 7 . There are a variety of assays designed to directly...

Importance Of Target Cells In Gpcr Hts Assays

Generally, cell-based assays involve the expression of recombinant GPCRs in modified tumor cell lines. By expressing the molecular target in such cells, large quantities of the target are generated for testing in HTS assays. A major advantage of these approaches is that the immortalized recombinant cells provide a relatively naive background for target expression and a relatively homogenous target expression system that facilitates consistency in screening. Furthermore, not only can the GPCR be...

Class A GPCRs

Melanocortin Receptors The melanocortin system consists of five GPCRs that all stimulate the adenylyl cyclase signal transduction pathway. The endogenous agonists are derived by posttranslational processing of the proopiomela-nocortin gene transcript by the prohormone convertases PC1 and PC2 to generate adrenocorticotropin (ACTH) and the a-, P-, and y-melanocyte-stimulating hormones (MSH). The melanocortin system also has the only two known endogenous GPCR antagonists, termed agouti and...

Contents

The Evolution of Receptors From On-Off Switches 1.2. The Receptor as an On-Off Switch 1 1.3. Historical Background and Classical Receptor Theory 2 1.4. The Operational Model of Drug Action 7 1.5. Receptor Antagonism 8 1.6. Specific Models of GPCRs (7TM Receptors) 11 1.7. The Receptor as Microprocessor Ternary Complex Models 13 1.8. Receptors as Basic Drug Recognition Units 17 1.9. Receptor Structure 19 1.10. Future Considerations 19 References 22 2. The Evolving Pharmacology of GPCRs 27 Lauren...

Frizzled Signaling

Signaling through FZDs is mainly subdivided into P-catenin-dependent 36 and P-catenin-independent pathways 37 . These branches are also known as canonical and noncanonical WNT pathways, respectively. However, the terminology refers to the historical chronology of the discovery of the pathways, rather than indicating that P - catenin is the WNT- default pathway. Indeed, P-catenin signaling can be activated by other GPCRs unrelated to FZD (e.g., JAK2 GSK- GSK- PLK CDK Phosphorylase ABL EGFR...

Recent Assays in GPCR Deorphanization

Recent advances clearly show that not all receptor actions rely on the activation of heterotrimeric G proteins but rather can be promoted via activation of G protein - independent and or P-arrestin-dependent pathways 45 . Two orphans that do not signal through G protein-dependent pathways are GPCR C5L2 also known as GPR77 46, 47 and the D6 chemokine receptor 48 . Whereas the first binds to C5a anaphylotoxin, the latter binds to chemokines. No evidence has been found for the activation of...

Automated Microscopes and Microbalances

Automated microscopes, such as the very recently launched Cell-IQ Chip-man Technologies, Tampere, Finland or IncuCyte instruments Essen Instruments, Ann Arbor, MI , offer unattended, label-free, and real-time kinetic analysis of cell proliferation, cell migration, or neurite outgrowth in a quantitative way. Both instruments are the result of continued development of classical phase contrast microscopy combined with sophisticated software for image analysis. Both instruments offer a technical...

Beyond Affinityfunctional Assays Efficacy And Allosteric Agonism

The drawbacks of screening for allosteric modulators using a radioligand binding assay are highlighted in Section 12.2. For these reasons, the most common method to identify such molecules is to run a functional assay to measure a signal transduction event downstream of receptor activation, for example, 35S -GTPyS binding, cAMP accumulation, inositol phosphate accumulation, intracellular Ca2 flux, or reporter gene activation. Among the earliest known examples of allosteric modulators of GPCRs...

Class C GPCRs

CaR Recently, the potential for pharmacological chaperone activity has been demonstrated in vitro for the CaR, which belongs to the Class C GPCR family and contains a large calcium-binding ECD. The CaR plays a critical role in calcium homeostasis, translating changes in plasma calcium levels into reciprocal changes in parathyroid hormone secretion from the parathyroid glands. Inactivating mutations in CaR produce a benign form of hypercalcemia when present in the heterozygous state, termed...

Allosteric Modulator Titration Curves

In a modern context of high-throughput screening, running multiple curves to screen a single compound is considered inefficient. Therefore, it has become commonplace for researchers to simplify assay design. Rather than running multiple curves to an agonist in the presence of varying concentrations of a putative modulator as described above , it is now commonplace to select a single concentration of agonist and examine the effects of a range of putative modulator concentrations on the response....

Contributors

Ruben Abagyan, Bioinformatics and Systems Biology Graduate Program, University of California, La Jolla, CA Elfrida R. Benjamin, Amicus Therapeutics, Cranbury, NJ Magdalena Birker-Robaczewska, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland Michel Bouvier, D partement de Biochimie,Universit de Montr al,Montr al, Quebec, Canada Moritz Bunemann, Department of Pharmacology and Toxicology, Philipps-Universitaet Marburg, Marburg, Germany Nathalie Clement, Institut Cochin, Universit Paris...

The Frizzled Family

The Frizzled family of receptors for recent reviews, see References 9, 10 includes 10 mammalian isoforms. Along with the related receptor Smoothened SMO 11 . Frizzleds were recently classified by the International Union of Basic and Clinical Pharmacology IUPHAR as a novel family of GPCRs 12 and taken up into the IUPHAR's database of GPCRs and list of 7TM receptors see also www.iuphar.org . The recommended nomenclature for the mammalian Frizzleds 1-10 is FZD1-10. Based on homology studies, FZDs...

Functional Assays

While the past success of the receptor binding assays cannot be understated, advances in assay methodologies that measure GPCR function have begun to circumvent limitations of the ligand binding assay, such as the inability to specifically screen for agonists versus antagonists, the inability to identify allosteric modulators and potentiators, or the requirement of a high-affinity labeled ligand. The use of functional assays for primary HTS in place of traditional receptor binding assays has...

The Adenosine A2a Receptor

The T4L fusion strategy in combination with the lipidic cubic phase crystallization method that resulted in the structure of the p2AR has also been applied successfully to the adenosine A2a receptor. This suggests that the T4L technology is transferable across different subfamilies of Class A GPCRs. In this structure, obtained to 2.6 A resolution, the receptor is crystallized in complex with the high-affinity antagonist ZM241385. Incorporation of the T4L moiety shifts the conformation to an...

Historical Background And Classical Receptor Theory

What is considered a given today, namely that drugs interact with specific binding sites on the cell membrane called receptors, is an extremely important tenet of pharmacology. As stated by Rang 1 , it indeed was Pharmacology's big idea. The main reason it is such an important cornerstone of pharmacology is that it introduces order into the apparent chaos of physiology. For example, a simple molecule such as epinephrine mediates a myriad of physiological processes. A large subset of these,...

Classical Assays of GPCR Deorphanization

Whenever high-affinity radioligands are available, these pharmacological tools are an integral part of the ligand screening process. Chemical libraries or tissue extracts are typically screened for compounds that compete with the radioli-gand for the same binding site. Binding of agonists to GPCRs induces a conformational change and promotes the activation of heterotrimeric G proteins. Their activation can be most easily and directly detected by monitoring the binding of nonhydrolyzable...

ImpedanceRWG

Two very recently introduced technologies, based on impedance and RWG, have received considerable attention from cell biologists and, in particular, from researchers interested in GPCR signaling. Both platforms carry transla-tional potential and open the stage for new insights into GPCR- induced signaling pathways using a combination of label-free detection and real-time kinetics with high accuracy and throughput. In addition, these technologies offer enhanced information content over other...

1

Orphan 7TM accessory protein Orphan 7TM allosteric modulator CRLR RAMPs GPR56 CD81 CD9 GPR37 DAT Smo Ptch GABAb2 GABAb1 GPR50 MT1 Mrg E Mrg D TIR3 TIR1 TIR2 CRLR RAMPs GPR56 CD81 CD9 GPR37 DAT Smo Ptch Figure 7.3 Novel functions of 7TM proteins. Association of 7TM proteins with deor-phanized GPCRs or other accessory membrane proteins. Activation of orphan 7TM proteins by allosteric modulators. See color insert. Three further cases illustrate the impact of orphan 7TM proteins on the signaling...

The Impact Of Functional Assay Format On Allosteric Modulator Screening

In the sections above, functional assays have been spoken about as a homogenous group of assays in which a whole range of allosteric effects can be detected. It is true that all of the functional assays described can detect allosteric modulation of agonist affinity, efficacy, and allosteric agonism. However, the specific assay format chosen can greatly impact the results that are obtained. This section will discuss the issues associated with the use of transient read systems such as...