Synthesis and testing of novel classical cannabinoids: exploring the side chain ligand binding pocket of the CB1 and CB2 receptors

A series of C3 cyclic side-chain analogues of classical cannabinoids were synthesized to probe the ligand binding pocket of the CB1 and CB2 receptors. The analogues were evaluated for CB1 and CB2 receptor binding affinities relative to delta(8)-THC. The C3 side-chain geometries of the analogues were studied using high field NMR spectroscopy and quantum mechanical calculations. The results of these studies provide insights into the geometry of the ligand binding pocket of the CB1 and CB2 receptors.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [3H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in Kd and EC50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

Identification of CC chemokine receptor 7 residues important for receptor activation

The binding pocket of family A GPCRs that bind small biogenic amines is well characterized. In this study we identify residues on CC chemokine receptor 7 (CCR-7) that are involved in agonist-mediated receptor activation but not in high affinity ligand binding. The mutations also affect the ability of the ligands to induce chemotaxis. Two of the residues, Lys3.33(137) and Gln5.42(227), are consistent with the binding pocket described for biogenic amines, while Lys3.26(130) and Asn7.32(305), are found at, or close to, the cell surface. Our observations are in agreement with findings from other peptide and chemokine receptors, which indicate that receptors that bind larger ligands contain contact sites closer to the cell surface in addition to the conventional transmembrane binding pocket. These findings also support the theory that chemokine receptors require different sets of interactions for high affinity ligand binding and receptor activation.     

Activation of nuclear receptors: a perspective from structural genomics

Crystal structures of more than two dozen different nuclear receptor ligand binding domains have defined a simple paradigm of receptor activation, in which agonist binding induces the activation function-2 (AF-2) helix to form a charge clamp for coactivator recruitment. Recent structural studies present a surprising contrast. Activation of the mouse LRH-1 receptor is independent of a bound agonist despite its large ligand binding pocket, whereas the activation of the Drosophila DHR38 receptor is dependent on ecdysteroids even though the receptor lacks a ligand binding pocket. These new findings shed light on the diverse structural mechanisms that nuclear receptors have evolved for activation, and have important implications in their respective signaling pathways.     

Doubling the size of the glucocorticoid receptor ligand binding pocket by deacylcortivazol

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 ų, effectively doubling the size of the GR dexamethasone-binding pocket of 540 ų and yet leaving the structure of the coactivator binding site intact. DAC occupies only ~50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.     

Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A–F. Here we examine the 5-HT₃ receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [³H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC₅₀ when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in K_d and EC₅₀ values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT₃ receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT₃ receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.     

On the benzodiazepine binding pocket in GABAA receptors

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.     

Synthesis,binding and bioactivity of γ-methylene γ-lactam ecdysone receptor ligands: Advantages of QSAR models for flexible receptors

Nuclear hormone receptors, such as the ecdysone receptor, often display a large amount of induced fit to ligands. The size and shape of the binding pocket in the EcR subunit changes markedly on ligand binding, making modelling methods such as docking extremely challenging. It is, however, possible to generate excellent 3D QSAR models for a given type of ligand, suggesting that the receptor adopts a relatively restricted number of binding site configurations or ‘attractors’. We describe the synthesis, in vitro binding and selected in vivo toxicity data for γ-methylene γ-lactams, a new class of high-affinity ligands for ecdysone receptors from Bovicola ovis (Phthiraptera) and Lucilia cuprina (Diptera). The results of a 3D QSAR study of the binding of methylene lactams to recombinant ecdysone receptor protein suggest that this class of ligands is indeed recognised by a single conformation of the EcR binding pocket.     

Ligand Binding to A1 Adenosine Receptors is Influenced by Protonation

Abstract

Studies on the pH dependence of ligand binding to A₁ adenosine receptors revealed that protonation of a histidine residue in the binding pocket is accompanied by high affinity agonist binding.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.     

Inhibition and Reversal of Microbial Attachment by an Antibody with Parasteric Activity against the FimH Adhesin of Uropathogenic E. coli

Attachment proteins from the surface of eukaryotic cells, bacteria and viruses are critical receptors in cell adhesion or signaling and are primary targets for the development of vaccines and therapeutic antibodies. It is proposed that the ligand-binding pocket in receptor proteins can shift between inactive and active conformations with weak and strong ligand-binding capability, respectively. Here, using monoclonal antibodies against a vaccine target protein - fimbrial adhesin FimH of uropathogenic Escherichia coli, we demonstrate that unusually strong receptor inhibition can be achieved by antibody that binds within the binding pocket and displaces the ligand in a non-competitive way. The non-competitive antibody binds to a loop that interacts with the ligand in the active conformation of the pocket but is shifted away from ligand in the inactive conformation. We refer to this as a parasteric inhibition, where the inhibitor binds adjacent to the ligand in the binding pocket. We showed that the receptor-blocking mechanism of parasteric antibody differs from that of orthosteric inhibition, where the inhibitor replaces the ligand or allosteric inhibition where the inhibitor binds at a site distant from the ligand, and is very potent in blocking bacterial adhesion, dissolving surface-adherent biofilms and protecting mice from urinary bladder infection.     

A structural and mutagenic blueprint for molecular recognition of strychnine and d-tubocurarine by different cys-loop receptors

Cys-loop receptors (CLR) are pentameric ligand-gated ion channels that mediate fast excitatory or inhibitory transmission in the nervous system. Strychnine and d-tubocurarine (d-TC) are neurotoxins that have been highly instrumental in decades of research on glycine receptors (GlyR) and nicotinic acetylcholine receptors (nAChR), respectively. In this study we addressed the question how the molecular recognition of strychnine and d-TC occurs with high affinity and yet low specificity towards diverse CLR family members. X-ray crystal structures of the complexes with AChBP, a well-described structural homolog of the extracellular domain of the nAChRs, revealed that strychnine and d-TC adopt multiple occupancies and different ligand orientations, stabilizing the homopentameric protein in an asymmetric state. This introduces a new level of structural diversity in CLRs. Unlike protein and peptide neurotoxins, strychnine and d-TC form a limited number of contacts in the binding pocket of AChBP, offering an explanation for their low selectivity. Based on the ligand interactions observed in strychnine- and d-TC-AChBP complexes we performed alanine-scanning mutagenesis in the binding pocket of the human α1 GlyR and α7 nAChR and showed the functional relevance of these residues in conferring high potency of strychnine and d-TC, respectively. Our results demonstrate that a limited number of ligand interactions in the binding pocket together with an energetic stabilization of the extracellular domain are key to the poor selective recognition of strychnine and d-TC by CLRs as diverse as the GlyR, nAChR, and 5-HT(3)R.     

1-Phenylpyrazolo[3,4-d]pyrimidines; structure-ac:tivity relationships for C6 substituents at A1 and A2A adenosine receptors

Substitution of I-phenylpyrazolo[3,4-d]pyrimidines at C6 with N-alkyl-2-thiopropionamide groups has resulted in a series of 18 compounds which have been evaluated for binding at A1 and A2A adenosine receptors. Introduction of an N-ethyl group gave increased affinity at both A1 and A2A receptors for the amino compound 7b compared to the primary amide 7a. An additional hydrophobic pocket exists for substituents on the amide. This pocket allows an N-ethyl group for increased affinity at both A1 and A2A receptors, allows larger alkyl groups at A2A receptors but not at A1 receptors and there is an H-bond interaction requiring one H-bond donor. Molecular modeling studies have also enabled a proposal of the amino acid residues involved in ligand binding at both the A1 and A2A receptors.     

Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors

The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.     

Recent progress in understanding the interaction between avermectins and ligand-gated ion channels: putting the pests to sleep

Avermectins and milbemycins are an important family of anthelmintics, insecticides and acaricides. Their mode of action is as positive allosteric modulators of ligand-gated chloride channels, and at higher concentrations, they gate some channels directly. Though it has long been known that the avermectins do not compete for the ligand binding site, the actual site at which they interact with their receptors has been unclear. Recent data demonstrate the importance to drug binding of amino acid residues predicted to line a water-filled pocket in the channel domain. This pocket acts as the binding site for anaesthetics and other modulators of mammalian GABA_A and glycine receptors, suggesting similarities in the mode of action between these drugs and the avermectins/milbemycins.