A Strategy Combining Differential Low‐Throughput Screening and Virtual Screening (DLS‐VS) Accelerating the Discovery of new Modulators for the Orphan GPR34 Receptor

The DLS‐VS strategy was developed as an integrated method for identifying chemical modulators for orphan GPCRs. It combines differential low‐throughput screening (DLS) and virtual screening (VS). The two cascaded techniques offer complementary advantages and allow the experimental testing of a minimal number of compounds. First, DLS identifies modulators specific for the considered receptor among a set of receptors, through the screening of a small library with diverse chemical compounds. Then, an active molecular model of the receptor is built by homology to a validated template, and it is progressively refined by rotamers modification for key side‐chains, by VS of the already screened library, and by iterative selection of the model generating the best enrichment. The refined active model is finally used for the VS of a large chemical library and the selection of a small set of compounds for experimental testing. Applied to the orphan receptor GPR34, the DLS‐VS strategy combined the experimental screening of 20 000 compounds and the virtual screening of 1 250 000 compounds. It identified one agonist and eight inverse agonists, showing a high chemical diversity. We describe the method. The strategy can be applied to other GPCRs.

[1]  Oliver P. Ernst,et al.  Crystal structure of opsin in its G-protein-interacting conformation , 2008, Nature.

[2]  H. Heng,et al.  Discovery of three novel orphan G-protein-coupled receptors. , 1999, Genomics.

[3]  R. Abagyan,et al.  Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists , 2010, Science.

[4]  C. Venkatachalam,et al.  LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. , 2003, Journal of molecular graphics & modelling.

[5]  Tsutomu Kouyama,et al.  Crystal structure of squid rhodopsin , 2008, Nature.

[6]  O. Civelli,et al.  Orphan GPCR research , 2008, British journal of pharmacology.

[7]  Ruben Abagyan,et al.  Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment. , 2011, Structure.

[8]  B. Roth,et al.  Deorphanization of Novel Peptides and Their Receptors , 2010, The AAPS Journal.

[9]  J. Baldwin,et al.  An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. , 1997, Journal of molecular biology.

[10]  Peter Kolb,et al.  Structure-based discovery of β2-adrenergic receptor ligands , 2009, Proceedings of the National Academy of Sciences.

[11]  P. Casellas,et al.  A Selective Inverse Agonist for Central Cannabinoid Receptor Inhibits Mitogen-activated Protein Kinase Activation Stimulated by Insulin or Insulin-like Growth Factor 1 , 1997, The Journal of Biological Chemistry.

[12]  T. Klabunde,et al.  Structure-based drug discovery using GPCR homology modeling: successful virtual screening for antagonists of the alpha1A adrenergic receptor. , 2005, Journal of medicinal chemistry.

[13]  J. Wess,et al.  Conformational changes involved in G-protein-coupled-receptor activation. , 2008, Trends in pharmacological sciences.

[14]  Gebhard F. X. Schertler,et al.  Structure of a β1-adrenergic G-protein-coupled receptor , 2008, Nature.

[15]  B. Kobilka.  Structural insights into adrenergic receptor function and pharmacology. , 2011, Trends in pharmacological sciences.

[16]  T. Kenakin,et al.  Inverse, protean, and ligand‐selective agonism: matters of receptor conformation , 2001, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  M. Ebisawa,et al.  Identification of a lysophosphatidylserine receptor on mast cells. , 2006, Biochemical and biophysical research communications.

[18]  Claudio N. Cavasotto,et al.  Docking-based virtual screening for ligands of G protein-coupled receptors: not only crystal structures but also in silico models. , 2011, Journal of molecular graphics & modelling.

[19]  Maria A Miteva,et al.  Structure‐based virtual ligand screening with LigandFit: Pose prediction and enrichment of compound collections , 2007, Proteins.

[20]  M. Abbracchio,et al.  Deorphanisation of G protein-coupled receptors: A tool to provide new insights in nervous system pathophysiology and new targets for psycho-active drugs , 2008, Neurochemistry International.

[21]  R. Lefkowitz.  Historical review: a brief history and personal retrospective of seven-transmembrane receptors. , 2004, Trends in pharmacological sciences.

[22]  Ruben Abagyan,et al.  Structure of the human histamine H1 receptor complex with doxepin , 2011, Nature.

[23]  J. Reagan,et al.  High throughput screening for orphan and liganded GPCRs. , 2008, Combinatorial chemistry & high throughput screening.

[24]  Vadim Cherezov,et al.  A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. , 2008, Structure.

[25]  K. Sangkuhl,et al.  Altered Immune Response in Mice Deficient for the G Protein-coupled Receptor GPR34* , 2010, The Journal of Biological Chemistry.

[26]  T. Schöneberg,et al.  The Structural Evolution of a P2Y-like G-protein-coupled Receptor* , 2003, Journal of Biological Chemistry.

[27]  Tetsuya Hori,et al.  Crystal Structure of Squid Rhodopsin with Intracellularly Extended Cytoplasmic Region , 2008, Journal of Biological Chemistry.

[28]  P. Ferrara,et al.  High‐level synthesis of human prolactin in Chinese‐hamster ovary cells , 2000, Biotechnology and applied biochemistry.

[29]  Xueliang Fang,et al.  Molecular modeling of the three-dimensional structure of dopamine 3 (D3) subtype receptor: discovery of novel and potent D3 ligands through a hybrid pharmacophore- and structure-based database searching approach. , 2003, Journal of medicinal chemistry.

[30]  R. Jockers,et al.  Alternative drug discovery approaches for orphan GPCRs. , 2008, Drug discovery today.

[31]  J. Ballesteros,et al.  [19] Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors , 1995 .

[32]  Gebhard F. X. Schertler,et al.  The structural basis of agonist-induced activation in constitutively active rhodopsin , 2011, Nature.

[33]  Michael M. Mysinger,et al.  Structure-based ligand discovery for the protein–protein interface of chemokine receptor CXCR4 , 2012, Proceedings of the National Academy of Sciences.

[34]  S. Rasmussen,et al.  Structure of a nanobody-stabilized active state of the β2 adrenoceptor , 2010, Nature.

[35]  A. Kruse,et al.  Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist , 2011, Nature.

[36]  Ruben Abagyan,et al.  Structure-based discovery of novel chemotypes for adenosine A(2A) receptor antagonists. , 2010, Journal of medicinal chemistry.

[37]  H. Schiöth,et al.  The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. , 2003, Molecular pharmacology.

[38]  M. Trincavelli,et al.  The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinyl‐leukotrienes receptor , 2006, The EMBO journal.

[39]  M. Burghammer,et al.  Crystal structure of the human β2 adrenergic G-protein-coupled receptor , 2007, Nature.

[40]  Christopher G. Tate,et al.  The structural basis for agonist and partial agonist action on a β1-adrenergic receptor , 2010, Nature.

[41]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[42]  R. Taguchi,et al.  Synthesis and evaluation of lysophosphatidylserine analogues as inducers of mast cell degranulation. Potent activities of lysophosphatidylthreonine and its 2-deoxy derivative. , 2009, Journal of medicinal chemistry.

[43]  A. IJzerman,et al.  The crystallographic structure of the human adenosine A2A receptor in a high-affinity antagonist-bound state: implications for GPCR drug screening and design. , 2010, Current opinion in structural biology.

[44]  M. Congreve,et al.  Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine. , 2011, Structure.

[45]  Oliver P. Ernst,et al.  Crystal structure of metarhodopsin II , 2011, Nature.

[46]  R. Stevens,et al.  The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist , 2008, Science.

[47]  T. Schöneberg,et al.  Genomic and supragenomic structure of the nucleotide-like G-protein-coupled receptor GPR34. , 2006, Genomics.

[48]  S. Ishii,et al.  Non-Edg family lysophosphatidic acid (LPA) receptors. , 2009, Prostaglandins & other lipid mediators.

[49]  Yumiko Saito,et al.  Orphan GPCRs and their ligands. , 2006, Pharmacology & therapeutics.

[50]  Avner Schlessinger,et al.  Ligand Discovery from a Dopamine D3 Receptor Homology Model and Crystal Structure , 2011, Nature chemical biology.

[51]  L. Pardo,et al.  Crystal structure of the μ-opioid receptor bound to a morphinan antagonist , 2012, Nature.

[52]  A. Rayan.  New vistas in GPCR 3D structure prediction , 2010, Journal of molecular modeling.

[53]  L. Vallières,et al.  Identification of genes preferentially expressed by microglia and upregulated during cuprizone‐induced inflammation , 2007, Glia.

[54]  Bryan L. Roth,et al.  Structure of the human kappa opioid receptor in complex with JDTic , 2012, Nature.

[55]  Didier Rognan,et al.  Protein‐based virtual screening of chemical databases. II. Are homology models of g‐protein coupled receptors suitable targets? , 2002, Proteins.

[56]  R. Stevens,et al.  High-Resolution Crystal Structure of an Engineered Human β2-Adrenergic G Protein–Coupled Receptor , 2007, Science.

[57]  R. Stevens,et al.  GPCR Engineering Yields High-Resolution Structural Insights into β2-Adrenergic Receptor Function , 2007, Science.

[58]  J. Halford,et al.  Orphan G-Protein-Coupled Receptors , 2007, Drugs in R&D.

[59]  L. de Leval,et al.  t(X;14)(p11.4;q32.33) is recurrent in marginal zone lymphoma and up-regulates GPR34 , 2011, Haematologica.

[60]  Brian K. Shoichet,et al.  Structure-Based Discovery of A2A Adenosine Receptor Ligands , 2010, Journal of medicinal chemistry.

[61]  O. Civelli,et al.  GPCR deorphanizations: the novel, the known and the unexpected transmitters. , 2005, Trends in pharmacological sciences.

[62]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2002, Chembiochem : a European journal of chemical biology.

[63]  John P. Overington,et al.  How many drug targets are there? , 2006, Nature Reviews Drug Discovery.

[64]  Albert C. Pan,et al.  Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor , 2012, Nature.

[65]  Cheng Zhang,et al.  Structure and Function of an Irreversible Agonist-β2 Adrenoceptor complex , 2010, Nature.

[66]  Hyun-Ju Park,et al.  Computer-aided identification of ligands for GPCR anti-obesity targets. , 2009, Current topics in medicinal chemistry.

[67]  Christopher A Reynolds,et al.  Toward the active conformations of rhodopsin and the β2‐adrenergic receptor , 2004, Proteins.

[68]  Pascual Ferrara,et al.  Differential Virtual Screening (DVS) with Active and Inactive Molecular Models for Finding and Profiling GPCR Modulators: Case of the CCK1 Receptor , 2011, Molecular informatics.

[69]  T. Schöneberg,et al.  Structural and functional evolution of the P2Y12-like receptor group , 2007, Purinergic Signalling.

[70]  R. Stevens,et al.  Crystal structure-based virtual screening for fragment-like ligands of the human histamine H(1) receptor. , 2011, Journal of medicinal chemistry.

[71]  Jonathan A. Javitch,et al.  Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist , 2010, Science.

[72]  R. Stevens,et al.  Structure of an Agonist-Bound Human A2A Adenosine Receptor , 2011, Science.

[73]  R. Pathak,et al.  Mining human genome for novel purinergic P2Y receptors: a sequence analysis and molecular modeling approach , 2011, Journal of receptor and signal transduction research.

[74]  Patrick Scheerer,et al.  Crystal structure of the ligand-free G-protein-coupled receptor opsin , 2008, Nature.

[75]  Vadim Cherezov,et al.  Diversity and modularity of G protein-coupled receptor structures. , 2012, Trends in pharmacological sciences.

[76]  S. Rasmussen,et al.  Crystal Structure of the β2Adrenergic Receptor-Gs protein complex , 2011, Nature.

[77]  A. Leslie,et al.  Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation , 2011, Nature.