Interaction of small molecule inhibitors of HIV-1 entry with CCR5.

The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands.

[1]  A. Naya,et al.  Design, synthesis, and discovery of a novel CCR1 antagonist. , 2001, Journal of medicinal chemistry.

[2]  Marc Parmentier,et al.  The Core Domain of Chemokines Binds CCR5 Extracellular Domains while Their Amino Terminus Interacts with the Transmembrane Helix Bundle* , 2003, The Journal of Biological Chemistry.

[3]  Timothy N. C. Wells,et al.  New therapeutics that modulate chemokine networks , 2002, Nature Reviews Drug Discovery.

[4]  O. Nishimura,et al.  Discovery of novel, potent, and selective small-molecule CCR5 antagonists as anti-HIV-1 agents: synthesis and biological evaluation of anilide derivatives with a quaternary ammonium moiety. , 2000, Journal of medicinal chemistry.

[5]  T. Dragic.  An overview of the determinants of CCR5 and CXCR4 co-receptor function. , 2001, The Journal of general virology.

[6]  Terry Kenakin,et al.  Recent progress in discovery of small-molecule CCR5 chemokine receptor ligands as HIV-1 inhibitors. , 2003, Bioorganic & medicinal chemistry.

[7]  R. Horuk,et al.  Development and evaluation of pharmacological agents targeting chemokine receptors. , 2003, Methods.

[8]  T. Schwartz,et al.  Molecular Interactions of Cyclam and Bicyclam Non-peptide Antagonists with the CXCR4 Chemokine Receptor* , 2001, The Journal of Biological Chemistry.

[9]  S. Hammer,et al.  HIV fusion and its inhibition. , 2001, Antiviral research.

[10]  R. Doms,et al.  Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1 , 1997, Journal of virology.

[11]  John P. Moore,et al.  Alanine Substitutions of Polar and Nonpolar Residues in the Amino-Terminal Domain of CCR5 Differently Impair Entry of Macrophage- and Dualtropic Isolates of Human Immunodeficiency Virus Type 1 , 1998, Journal of Virology.

[12]  Joseph Sodroski,et al.  Tyrosine Sulfation of the Amino Terminus of CCR5 Facilitates HIV-1 Entry , 1999, Cell.

[13]  William C. Olson,et al.  Amino-Terminal Substitutions in the CCR5 Coreceptor Impair gp120 Binding and Human Immunodeficiency Virus Type 1 Entry , 1998, Journal of Virology.

[14]  Nancy Sullivan,et al.  CCR5 Levels and Expression Pattern Correlate with Infectability by Macrophage-tropic HIV-1, In Vitro , 1997, The Journal of experimental medicine.

[15]  W. Greenlee,et al.  Synthesis, SAR, and biological evaluation of oximino-piperidino-piperidine amides. 1. Orally bioavailable CCR5 receptor antagonists with potent anti-HIV activity. , 2002, Journal of medicinal chemistry.

[16]  Mette M. Rosenkilde,et al.  Molecular Mechanism of AMD3100 Antagonism in the CXCR4 Receptor , 2004, Journal of Biological Chemistry.

[17]  Marc Parmentier,et al.  Regions in β-Chemokine Receptors CCR5 and CCR2b That Determine HIV-1 Cofactor Specificity , 1996, Cell.

[18]  Thomas P. Sakmar,et al.  Analysis of the Mechanism by Which the Small-Molecule CCR5 Antagonists SCH-351125 and SCH-350581 Inhibit Human Immunodeficiency Virus Type 1 Entry , 2003, Journal of Virology.

[19]  T. Kenakin,et al.  The CCR5 Receptor-Based Mechanism of Action of 873140, a Potent Allosteric Noncompetitive HIV Entry Inhibitor , 2005, Molecular Pharmacology.

[20]  B. Neustadt,et al.  Piperazine-based CCR5 antagonists as HIV-1 inhibitors. II. Discovery of 1-[(2,4-dimethyl-3-pyridinyl)carbonyl]-4- methyl-4-[3(S)-methyl-4-[1(S)-[4-(trifluoromethyl)phenyl]ethyl]-1-piperazinyl]- piperidine N1-oxide (Sch-350634), an orally bioavailable, potent CCR5 antagonist. , 2001, Journal of medicinal chemistry.

[21]  M. Paterlini,et al.  Structure modeling of the chemokine receptor CCR5: implications for ligand binding and selectivity. , 2002, Biophysical journal.

[22]  William J Greenlee,et al.  Biological evaluation and interconversion studies of rotamers of SCH 351125, an orally bioavailable CCR5 antagonist. , 2003, Bioorganic & medicinal chemistry letters.

[23]  Stephen C Peiper,et al.  Lipid Bilayer Simulations of CXCR4 with Inverse Agonists and Weak Partial Agonists* , 2003, Journal of Biological Chemistry.

[24]  J. Sodroski,et al.  A Tyrosine-Rich Region in the N Terminus of CCR5 Is Important for Human Immunodeficiency Virus Type 1 Entry and Mediates an Association between gp120 and CCR5 , 1998, Journal of Virology.

[25]  Bryan R. Cullen,et al.  Multiple Residues Contribute to the Inability of Murine CCR-5 To Function as a Coreceptor for Macrophage-Tropic Human Immunodeficiency Virus Type 1 Isolates , 1998, Journal of Virology.

[26]  S. Mccombie,et al.  Piperazine-based CCR5 antagonists as HIV-1 inhibitors. I: 2(S)-methyl piperazine as a key pharmacophore element. , 2001, Bioorganic & medicinal chemistry letters.

[27]  Markus Eilers,et al.  Comparison of Helix Interactions in Membrane and Soluble α-Bundle Proteins , 2002 .

[28]  Marc Parmentier,et al.  Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene , 1996, Nature.

[29]  John P. Moore,et al.  Will Multiple Coreceptors Need To Be Targeted by Inhibitors of Human Immunodeficiency Virus Type 1 Entry? , 1999, Journal of Virology.

[30]  E. Clercq.  New developments in anti-HIV chemotherapy , 2001 .

[31]  S. O. Smith,et al.  Helix packing in polytopic membrane proteins: role of glycine in transmembrane helix association. , 1999, Biophysical journal.

[32]  K. Palczewski,et al.  Crystal Structure of Rhodopsin: A G‐Protein‐Coupled Receptor , 2000, Science.

[33]  Dominique Schols,et al.  Inhibition of Human Immunodeficiency Virus Replication by a Dual CCR5/CXCR4 Antagonist , 2004, Journal of Virology.

[34]  I. Forbes,et al.  CCR2B receptor antagonists: conversion of a weak HTS hit to a potent lead compound. , 2000, Bioorganic & Medicinal Chemistry Letters.

[35]  J J Goedert,et al.  Genetic Restriction of HIV-1 Infection and Progression to AIDS by a Deletion Allele of the CKR5 Structural Gene , 1996, Science.

[36]  S. O. Smith,et al.  A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Maccoss,et al.  Binding of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines to human CCR5: a molecular modeling-guided mutagenesis study of the binding pocket. , 2003, Biochemistry.

[38]  D. D. Perrin,et al.  pKa prediction for organic acids and bases , 1981 .

[39]  P. Carter.  Chemokine receptor antagonism as an approach to anti-inflammatory therapy: 'just right' or plain wrong? , 2002, Current opinion in chemical biology.

[40]  J. Venter,et al.  Site-directed mutagenesis of m1 muscarinic acetylcholine receptors: conserved aspartic acids play important roles in receptor function. , 1989, Molecular pharmacology.

[41]  H. Mitsuya,et al.  Spirodiketopiperazine-Based CCR5 Inhibitor Which Preserves CC-Chemokine/CCR5 Interactions and Exerts Potent Activity against R5 Human Immunodeficiency Virus Type 1 In Vitro , 2004, Journal of Virology.

[42]  Frank Diehl,et al.  Identification of the Binding Site for a Novel Class of CCR2b Chemokine Receptor Antagonists , 2000, The Journal of Biological Chemistry.

[43]  A. Maynard,et al.  Naturally Occurring CCR5 Extracellular and Transmembrane Domain Variants Affect HIV-1 Co-receptor and Ligand Binding Function* , 1999, The Journal of Biological Chemistry.

[44]  J. Farber,et al.  Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. , 1999, Annual review of immunology.

[45]  H. Fujiwara,et al.  A non‐peptide CCR5 antagonist inhibits collagen‐induced arthritis by modulating T cell migration without affecting anti‐collagen T cell responses , 2002, European journal of immunology.

[46]  Serena Xu,et al.  SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Sodroski,et al.  HIV-1 Entry and Macrophage Inflammatory Protein-1β-mediated Signaling Are Independent Functions of the Chemokine Receptor CCR5* , 1997, The Journal of Biological Chemistry.

[48]  Wei Liu,et al.  Comparison of helix interactions in membrane and soluble alpha-bundle proteins. , 2002, Biophysical journal.

[49]  G. Petsko,et al.  Weakly polar interactions in proteins. , 1988, Advances in protein chemistry.

[50]  W. Greenlee,et al.  Discovery of 4-[(Z)-(4-bromophenyl)- (ethoxyimino)methyl]-1'-[(2,4-dimethyl-3- pyridinyl)carbonyl]-4'-methyl-1,4'- bipiperidine N-oxide (SCH 351125): an orally bioavailable human CCR5 antagonist for the treatment of HIV infection. , 2001, Journal of medicinal chemistry.

[51]  J. Moore,et al.  HIV-1 entry inhibitors: Evading the issue , 1999, Nature Medicine.

[52]  Richard A Koup,et al.  Homozygous Defect in HIV-1 Coreceptor Accounts for Resistance of Some Multiply-Exposed Individuals to HIV-1 Infection , 1996, Cell.

[53]  G Vassart,et al.  Multiple Charged and Aromatic Residues in CCR5 Amino-terminal Domain Are Involved in High Affinity Binding of Both Chemokines and HIV-1 Env Protein* , 1999, The Journal of Biological Chemistry.

[54]  C. Strader,et al.  Structure and function of G protein-coupled receptors. , 1994, Annual review of biochemistry.

[55]  John P. Moore,et al.  Use of Inhibitors To Evaluate Coreceptor Usage by Simian and Simian/Human Immunodeficiency Viruses and Human Immunodeficiency Virus Type 2 in Primary Cells , 2000, Journal of Virology.

[56]  John P. Moore,et al.  New targets for inhibitors of HIV-1 replication , 2000, Nature Reviews Molecular Cell Biology.

[57]  W. Olson,et al.  HIV entry inhibitors in clinical development. , 2002, Current opinion in pharmacology.

[58]  S. O’Brien,et al.  The effect of genetic variation in chemokines and their receptorson HIV transmission and progression to AIDS , 2000, Immunological reviews.

[59]  Emmanuel G. Cormier,et al.  The Crown and Stem of the V3 Loop Play Distinct Roles in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Interactions with the CCR5 Coreceptor , 2002, Journal of Virology.

[60]  M. Cadene,et al.  Tyrosine sulfation of CCR5 N-terminal peptide by tyrosylprotein sulfotransferases 1 and 2 follows a discrete pattern and temporal sequence , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[61]  Y. Wan,et al.  The synthesis of substituted bipiperidine amide compounds as CCR3 antagonists. , 2005, Bioorganic & medicinal chemistry letters.

[62]  T. Kenakin.  New Concepts in Drug Discovery: Collateral Efficacy and Permissive Antagonism , 2005, Nature Reviews Drug Discovery.

[63]  F. Miedema,et al.  T cell dynamics in HIV-1 infection. , 1999, Advances in immunology.

[64]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[65]  D. Taub,et al.  Identification and Characterization of Small Molecule Functional Antagonists of the CCR1 Chemokine Receptor* , 1998, The Journal of Biological Chemistry.

[66]  C. Strader,et al.  The family of G‐protein‐coupled receptors , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[67]  E. De Clercq,et al.  Mutation of Asp(171) and Asp(262) of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100. , 2001, Molecular pharmacology.

[68]  D. Taub,et al.  Identification and Characterization of a Potent, Selective, and Orally Active Antagonist of the CC Chemokine Receptor-1* , 2000, The Journal of Biological Chemistry.

[69]  O. Nishimura,et al.  A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[70]  T. Sakmar,et al.  Small-molecule antagonists of CCR5 and CXCR4: a promising new class of anti-HIV-1 drugs. , 2004, Current pharmaceutical design.

[71]  J. Lachowicz,et al.  Piperazine-based CCR5 antagonists as HIV-1 inhibitors. IV. Discovery of 1-[(4,6-dimethyl-5-pyrimidinyl)carbonyl]- 4-[4-[2-methoxy-1(R)-4-(trifluoromethyl)phenyl]ethyl-3(S)-methyl-1-piperazinyl]- 4-methylpiperidine (Sch-417690/Sch-D), a potent, highly selective, and orally bioavailable CCR5 antagonis , 2004, Journal of medicinal chemistry.

[72]  A. Trkola,et al.  The Differential Sensitivity of Human and Rhesus Macaque CCR5 to Small-Molecule Inhibitors of Human Immunodeficiency Virus Type 1 Entry Is Explained by a Single Amino Acid Difference and Suggests a Mechanism of Action for These Inhibitors , 2004, Journal of Virology.

[73]  E. De Clercq,et al.  Determinants for Sensitivity of Human Immunodeficiency Virus Coreceptor CXCR4 to the Bicyclam AMD3100 , 1998, Journal of Virology.

[74]  T. Kenakin.  G-protein coupled receptors as allosteric machines. , 2004, Receptors & channels.

[75]  Hiroaki Mitsuya,et al.  The current status of, and challenges in, the development of CCR5 inhibitors as therapeutics for HIV-1 infection. , 2004, Current opinion in pharmacology.

[76]  S. O. Smith,et al.  The steric trigger in rhodopsin activation. , 1997, Journal of molecular biology.

[77]  Oliver Hartley,et al.  V3: HIV's switch-hitter. , 2005, AIDS research and human retroviruses.