Current Pharmaceutical Design

ISSN: 1381-6128

Current Pharmaceutical Design
Volume 15, Number 35, 2009

Contents


In Silico Approaches in G Protein-Coupled Receptors (GPCRs) Drug Discovery: Quo Vadis?
Executive Editor: Stefano Moro


Editorial Pp. 3992-3993 [PMID: 20028317 PubMed - indexed for MEDLINE]


Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors Pp. 3994-4002
Stefano Costanzi, Jeffrey Siegel, Irina G. Tikhonova and Kenneth A. Jacobson
[Abstract] [Purchase Article] [PMID: 20028316 PubMed - indexed for MEDLINE]


Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy Pp. 4003-4016
Irina G. Tikhonova and Stefano Costanzi
[Abstract] [Purchase Article] [PMID: 20028318 PubMed - indexed for MEDLINE]


Progress in Elucidating the Structural and Dynamic Character of G Protein Coupled Receptor Oligomers for Use in Drug Discovery Pp. 4017-4025
A. Bortolato, J.C. Mobarec, D. Provasi and M. Filizola
[Abstract] [Purchase Article] [PMID: 20028319 PubMed - indexed for MEDLINE]


Customizing G Protein-Coupled Receptor Models for Structure-Based Virtual Screening Pp. 4026-4048
Chris de Graaf and Didier Rognan
[Abstract] [Purchase Article] [PMID: 20028320 PubMed - indexed for MEDLINE]


G Protein-Coupled Receptors: Target-Based In Silico Screening Pp. 4049-4068
Hanoch Senderowitz and Yael Marantz
[Abstract] [Purchase Article] [PMID: 20028321 PubMed - indexed for MEDLINE]


Human A₃ Adenosine Receptor as Versatile G Protein-Coupled Receptor Example to Validate the Receptor Homology Modeling Technology Pp. 4069-4084
Erika Morizzo, Stephanie Federico, Giampiero Spalluto and Stefano Moro
[Abstract] [Purchase Article] [PMID: 20028322 PubMed - indexed for MEDLINE]




Abstracts


[Back to top] [PMID: 20028317 PubMed - indexed for MEDLINE]
Editorial: In Silico Approaches in G Protein-Coupled Receptors (GPCRs) Drug Discovery: Quo Vadis?

How do we communicate with the outside world? How are our senses of vision, smell, taste and pain controlled at the cellular and molecular levels? What causes medical conditions like allergies, hypertension, depression, obesity and various central nervous system disorders? G protein-coupled receptors (GPCRs) provide a major part of the answer to all of these questions. GPCRs constitute the largest family of cell-surface receptors and in humans are encoded by more than 900 genes. GPCRs convert extracellular messages into intracellular responses and are involved in essentially all physiological processes. GPCR dysfunction results in numerous human disorders, and over 50% of all prescription drugs on the market today directly or indirectly target GPCRs.

While knowledge of a protein's structure furnishes important information for understanding its function and for drug design, progress in solving GPCR structures has been slow. Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two major techniques used to determine protein structures. NMR spectroscopy has the advantages that the protein does not need to be crystallized and dynamical information can be extracted. However, high concentrations of dissolved proteins are needed; and as yet no complete GPCR structure has been solved by the method. Crystal structures are spectacular central organizing models for just about everything you can determine about a protein. Mutants, homologs, interactors, ligands -- if you have a structure to hang them on, understanding them becomes much easier. For drug development a 3D structure can be powerful advice for chemistry efforts, suggesting directions to build out a molecule or to avoid changing. Because they are large, membrane-bound proteins with lots of floppy loops, GPCRs are particularly challenging structure targets, and they are extremely difficult to crystallize. In fact, only a few GPCRs, bovine rhodopsin, human β₂- and β₁-adrenergic receptors and, very recently, the human A_2A adenosine receptor have been solved.

Homology models of GPCRs, especially those supported by experimental data, and molecular docking experiments have been widely used in computational medicinal chemistry to guide site-directed mutagenesis and for drug discovery purposes, pursued also through virtual screenings and through the generation of docking-based quantitative structure-activity relationship (QSAR) models. Encouraging results led to a general acceptance of these models, which, however, although corroborated by indirect experimental evidence could not be ultimately validated. The recent publication of the crystal structure of the human A_2A adenosine receptor conclusively that GPCRs indeed share a pretty structurally conserved 7TM core, strongly supporting the body of literature and the hypotheses that were built on the basis of homology modeling and molecular docking.

Nowadays, GPCR computational medicinal chemistry brings together the most powerful concepts in modern chemistry, biology and pharmacology, linking medicinal chemistry with genomics and proteomics. Following our experience, both ligand-based and structure-based approaches to drug discovery in the absence, but probably also in the presence, of the real 3D-structures require a multidisciplinary approach, where molecular models represent a structural context to efficiently integrate experimental data and inferences derived from molecular biological, biophysical, bioinformatic, pharmacological and organic chemical methods. Although not always achievable, the success of a synergistic effect among these disciplines is highly dependent on the experimental design. Synergy is best achieved when mutations are structurally interpretable, structural hypotheses are experimentally testable, ligands are well characterized pharmacologically, and the necessary chemical modifications of the ligands are feasible.

Will these new approaches herald a flurry of GPCR structures? Perhaps, but they hint at what a hard slog it may be. A host of additional challenges were faced, such as the crystals being so transparent it was hard to position them in the beam. Will each GPCR present its own challenges? Only time will tell.

The present special issue opens with the interesting historical retrospective by Stefano Costanzi and collaborators [1] on structural studies of G protein-coupled receptors. This chapter represents a very nice overview about the evolution of the structural biology of GPCRs, starting from the information collecting by the first bovine rhodopsin structure until to the newest human A_2A adenosine one.

In his second contribution to this issue Stefano Costanzi and collaborators [2] provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures. They also discuss the possibility of integrating homology modeling and NMR spectroscopy toward the generation of hybrid experimental and computational structure.

Marta Filiziola and collaborators [3] introduce one of the hottest topic in GPCR pharmacology regarding the elucidating the structural and dynamic character of GPCRs oligomers for use in drug discovery.

Two important contributions concerning the application of different receptor-based drug design approaches coming from Hanoch Senderowitz and Didier Rognan and their collaborators. In particular, Didier Rognan [4] focus his contribution on the construction, refinement, and validation of GPCR models for the purpose of structure-based virtual screening. On the other hand, Hanoch Senderowitz [5] presents EPIX in silico screening platform to discover novel GPCR ligands.

Finally, Stefano Moro and collaborations [6] describe the evolution of the topology of the human A3 adenosine receptor models and the implication of using different crystallographic templates in the quality of the final homology models.


REFERENCES


[1] Costanzi S, Siegel J, Tikhonova IG, Jacobson KA. Rhodopsin and the Others: a Historical Perspective on Structural Studies of G Protein-Coupled Receptors. Curr Pharm Des 2009; 15(35): 3994-4002.
[2] Tikhonova IG, Costanzi S. Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy. Curr Pharm Des 2009; 15(35): 4003-4016.
[3] Bortolato A, Mobarec JC, Provasi D, Filizola M. Progress in Elucidating the Structural and Dynamic Character of G Protein Coupled Receptor Oligomers for Use in Drug Discovery. Curr Pharm Des 2009; 15(35): 4017-4025.
[4] de Graaf C, Rognan D. Customizing G Protein-Coupled Receptor Models for Structure-Based Virtual Screening. Curr Pharm Des 2009; 15(35): 4026-4048.
[5] Senderowitz H, Marantz Y. G Protein-Coupled Receptors: Target-Based In Silico Screening. Curr Pharm Des 2009; 15(35): 4049-4068.
[6] Morizzo E, Federico S, Spalluto G, Moro S. Human A3 Adenosine Receptor as Versatile G Protein-Coupled Receptor Example to Validate the Receptor Homology Modeling Technology. Curr Pharm Des 2009; 15(35): 4069-4084.


Stefano Moro, Ph.D.
Molecular Modeling Section (MMS)
Department of Pharmaceutical Sciences
University of Padova
Via Marzolo 5, 35131 Padova
Italy
E-mail: stefano.moro@unipd.it


[Back to top] [Purchase Article] [PMID: 20028316 PubMed - indexed for MEDLINE]
Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors
Stefano Costanzi, Jeffrey Siegel, Irina G. Tikhonova and Kenneth A. Jacobson

The role of rhodopsin as a structural prototype for the study of the whole superfamily of G protein-coupled receptors (GPCRs) is reviewed in an historical perspective. Discovered at the end of the nineteenth century, fully sequenced since the early 1980s, and with direct three-dimensional information available since the 1990s, rhodopsin has served as a platform to gather indirect information on the structure of the other superfamily members. Recent breakthroughs have elicited the solution of the structures of additional receptors, namely the β₁- and β₂-adrenergic receptors and the A_2A adenosine receptor, now providing an opportunity to gauge the accuracy of homology modeling and molecular docking techniques and to perfect the computational protocol. Notably, in coordination with the solution of the structure of the A_2A adenosine receptor, the first “critical assessment of GPCR structural modeling and docking” has been organized, the results of which highlighted that the construction of accurate models, although challenging, is certainly achievable. The docking of the ligands and the scoring of the poses clearly emerged as the most difficult components. A further goal in the field is certainly to derive the structure of receptors in their signaling state, possibly in complex with agonists. These advances, coupled with the introduction of more sophisticated modeling algorithms and the increase in computer power, raise the expectation for a substantial boost of the robustness and accuracy of computer-aided drug discovery techniques in the coming years.


[Back to top] [Purchase Article] [PMID: 20028318 PubMed - indexed for MEDLINE]
Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy
Irina G. Tikhonova and Stefano Costanzi

G protein-coupled receptors (GPCRs) are a large superfamily of signaling proteins expressed on the plasma membrane. They are involved in a wide range of physiological processes and, therefore, are exploited as drug targets in a multitude of therapeutic areas. In this extent, knowledge of structural and functional properties of GPCRs may greatly facilitate rational design of modulator compounds. Solution and solid-state nuclear magnetic resonance (NMR) spectroscopy represents a powerful method to gather atomistic insights into protein structure and dynamics. In spite of the difficulties inherent the solution of the structure of membrane proteins through NMR, these methods have been successfully applied, sometimes in combination with molecular modeling, to the determination of the structure of GPCR fragments, the mapping of receptor-ligand interactions, and the study of the conformational changes associated with the activation of the receptors. In this review, we provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures.


[Back to top] [Purchase Article] [PMID: 20028319 PubMed - indexed for MEDLINE]
Progress in Elucidating the Structural and Dynamic Character of G Protein Coupled Receptor Oligomers for Use in Drug Discovery
A. Bortolato, J.C. Mobarec, D. Provasi and M. Filizola

G Protein-Coupled Receptors (GPCRs) are the most targeted group of proteins for the development of therapeutic drugs. Until the last decade, structural information about this family of membrane proteins was relatively scarce, and their mechanisms of ligand binding and signal transduction were modeled on the assumption that GPCRs existed and functioned as monomeric entities. New crystal structures of native and engineered GPCRs, together with important biochemical and biophysical data that reveal structural details of the activation mechanism(s) of this receptor family, provide a valuable framework to improve dynamic molecular models of GPCRs with the ultimate goal of elucidating their allostery and functional selectivity. Since the dynamic movements of single GPCR protomers are likely to be affected by the presence of neighboring interacting subunits, oligomeric arrangements should be taken into account to improve the predictive ability of computer-assisted structural models of GPCRs for effective use in drug design.


[Back to top] [Purchase Article] [PMID: 20028320 PubMed - indexed for MEDLINE]
Customizing G Protein-Coupled Receptor Models for Structure-Based Virtual Screening
Chris de Graaf and Didier Rognan

This review will focus on the construction, refinement, and validation of G Protein-coupled receptor models for the purpose of structure-based virtual screening. Practical tips and tricks derived from concrete modeling and virtual screening exercises to overcome the problems and pitfalls associated with the different steps of the receptor modeling workflow will be presented. These examples will not only include rhodopsin-like (class A), but also secretine-like (class B), and glutamate-like (class C) receptors. In addition, the review will present a careful comparative analysis of current crystal structures and their implication on homology modeling. The following themes will be discussed: i) the use of experimental anchors in guiding the modeling procedure; ii) amino acid sequence alignments; iii) ligand binding mode accommodation and binding cavity expansion; iv) proline-induced kinks in transmembrane helices; v) binding mode prediction and virtual screening by receptor-ligand interaction fingerprint scoring; vi) extracellular loop modeling; vii) virtual filtering schemes. Finally, an overview of several successful structure-based screening shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands.


[Back to top] [Purchase Article] [PMID: 20028321 PubMed - indexed for MEDLINE]
G Protein-Coupled Receptors: Target-Based In Silico Screening
Hanoch Senderowitz and Yael Marantz

In silico (or virtual) screening has become a common practice in current computer-aided drug design efforts. However, application to hit discovery in the G Protein-Coupled Receptors (GPCRs) arena was until recently hampered by the paucity of crystal structures available for this important class of pharmaceutical targets, forcing practitioners in the field to rely on GPCR models derived either ab initio or through homology modeling approaches. In this work we describe the EPIX in silico screening workflow which consists of the following stages: (1) Target modeling; (2) Preparation of screening library; (3) Docking; (4) Binding mode selection; (5) Scoring; (6) Consensus scoring and (7) Selection of virtual hits. This workflow was applied to the virtual screening of 13 GPCRs (5 biogenic amine receptors, 5 peptide receptors, 1 lipid receptor, 1 purinergic receptor and 1 cannabinoid receptor). Hit rates vary between 4% and 21% with higher hit rates usually obtained for biogenic amines and lower hits rates for peptide receptors. These data are analyzed in the context of the available experimental information (i.e., mutational data), the model (i.e., binding site location, and type of interactions) and the screening library. Specific examples are discussed in more detail as well as the future directions and challenges of this approach to in silico screening.


[Back to top] [Purchase Article] [PMID: 20028322 PubMed - indexed for MEDLINE]
Human A₃ Adenosine Receptor as Versatile G Protein-Coupled Receptor Example to Validate the Receptor Homology Modeling Technology
Erika Morizzo, Stephanie Federico, Giampiero Spalluto and Stefano Moro

The development of ligands for the A₃ adenosine receptor (AR) has been directed mainly by traditional medicinal chemistry, but the influence of structure-based approaches is increasing. Rhodopsin-based homology modeling had been used for many years to obtain three-dimensional models of the A₃AR, and different A₃AR models have been published describing the hypothetical interactions with known A₃AR ligands having different chemical scaffolds. The recently published structure of the human A_2AAR provides a new template for GPCR modeling, however even use of the A_2AAR as a template for modeling other AR subtypes is still imprecise. The models compared here are based on bovine rhodopsin, the human β₂-adrenergic receptor, and the A_2AAR as templates. The sequence of the human A₃AR contains only one cysteine residue (Cys166) in the second extracellular loop (EL2), which putatively forms a conserved disulfide bridge with the respective cysteine residues of TM3 (Cys83). Homology models of the A₃AR have been helpful in providing structural hypotheses for the design of new ligands. Site-directed mutagenesis of the A₃AR shows an important role in ligand recognition for specific residues in TM3, TM6 and TM7.