Dominik Thimm

The four cysteine residues in the second extracellular loop of the human adenosine A2B receptor: Role in ligand binding and receptor function

The adenosine A(2B) receptor is of considerable interest as a new drug target for the treatment of asthma, inflammatory diseases, pain, and cancer. In the present study we investigated the role of the cysteine residues in the extracellular loop 2 (ECL2) of the receptor, which is particularly cysteine-rich, by a combination of mutagenesis, molecular modeling, chemical and pharmacological experiments. Pretreatment of CHO cells recombinantly expressing the human A(2B) receptor with dithiothreitol led to a 74-fold increase in the EC(50) value of the agonist NECA in cyclic AMP accumulation. In the C78(3.25)S and the C171(45.50)S mutant high-affinity binding of the A(2B) antagonist radioligand [(3)H]PSB-603 was abolished and agonists were virtually inactive in cAMP assays. This indicates that the C3.25-C45.50 disulfide bond, which is highly conserved in GPCRs, is also important for binding and function of A(2B) receptors. In contrast, the C166(45.45)S and the C167(45.46)S mutant as well as the C166(45.45)S-C167(45.46)S double mutant behaved like the wild-type receptor, while in the C154(45.33)S mutant significant, although more subtle effects on cAMP accumulation were observed - decrease (BAY60-6583) or increase (NECA) - depending on the structure of the investigated agonist. In contrast to the X-ray structure of the closely related A(2A) receptor, which showed four disulfide bonds, the present data indicate that in the A(2B) receptor only the C3.25-C45.50 disulfide bond is essential for ligand binding and receptor activation. Thus, the cysteine residues in the ECL2 of the A(2B) receptor not involved in stabilization of the receptor structure may have other functions.

The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors.

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A2A and A2B by exchanging the complete EL2 of the human A2BAR for the EL2 of the A2AAR. Furthermore, single amino acid residues (Asp148(45.27), Ser149(45.28), Thr151(45.30), Glu164(45.43), Ser165(45.44), and Val169(45.48)) in the EL2 of the A2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A2A-selective agonist CGS21680, while being completely inactive at A2BARs, showed high affinity for the mutant A2B(EL2-A2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A2B(EL2-A2A) as compared to the wt AR. Thus, the EL2 of the A2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.

8-Benzamidochromen-4-one-2-carboxylic acids: potent and selective agonists for the orphan G protein-coupled receptor GPR35.

8-Amido-chromen-4-one-2-carboxylic acid derivatives were identified as novel agonists at the G protein-coupled orphan receptor GPR35. They were characterized by a β-arrestin recruitment assay and optimized to obtain agonists with nanomolar potency for the human GPR35. The compounds were found to exhibit high selectivity versus the related GPR55. The most potent agonists were 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (85, EC50 12.1 nM) and 6-bromo-8-(2-chloro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (90, EC50 11.1 nM), both of which were >1700-fold selective versus GPR55. Most compounds were considerably less potent at rat and mouse than at human GPR35. 6-Bromo-8-(2-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (87) was the only derivative that activated GPR35 of all three species at similar, low micromolar concentration. Compounds 85 and 90 are the most potent agonists at the human GPR35 known to date and might thus serve as powerful pharmacological tools to further elucidate the receptors (patho)physiological role and its potential as a future drug target.

6-Bromo-8-(4-[3H]methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic Acid: A Powerful Tool for Studying Orphan G Protein-Coupled Receptor GPR35

The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (12) was obtained in tritium-labeled form, designated [(3)H]PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((tert-butyldimethylsilyl)oxy)benzamido)-4-oxo-4H-chromene-2-carboxylate (19) with [(3)H]methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [(3)H]12 labeled the receptor with high affinity (KD = 5.27 nM). Binding was saturable (Bmax = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (83; Ki = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a Ki value in the subnanomolar range.

The nucleobase adenine as a signalling molecule in the kidney

In 2002, the first receptor activated by the nucleobase adenine was discovered in rats. In the past years, two adenine receptors (AdeRs) in mice and one in Chinese hamsters, all of which belong to the family of G protein‐coupled receptors (GPCRs), were cloned and pharmacologically characterized. Based on the nomenclature for other purinergic receptor families (P1 for adenosine receptors and P2 for nucleotide, e.g. ATP, receptors), AdeRs were designated P0 receptors. Pharmacological data indicate the existence of G protein‐coupled AdeRs in pigs and humans as well; however, those have not been cloned so far. Current data suggest a role for adenine and AdeRs in renal proximal tubules. Furthermore, AdeRs are suggested to be functional counterplayers of vasopressin in the collecting duct system, thus exerting diuretic effects. We are only at the beginning of understanding the significance of this new class of purinergic receptors, which might become future drug targets.

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe1103.24, Asn1153.29, Asn1734.60, Phe17945.39, Asn1945.40, Phe1955.41, Leu2015.47, His2526.54, and Tyr2687.32) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [3H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn1945.40 and Leu2015.47, were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that—in contrast to most other rhodopsin-like GPCRs—the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

Bicyclic imidazole-4-one derivatives: a new class of antagonists for the orphan G protein-coupled receptors GPR18 and GPR55

GPR18 and GPR55 are orphan G protein-coupled receptors (GPCRs) that interact with certain cannabinoid (CB) receptor ligands. In the present study bicyclic imidazole-4-one derivatives were discovered as new scaffolds for the development of antagonists for GPR18 and GPR55. Interaction with CB1 and CB2 receptors was also studied to assess selectivity. The presented extensive structure–activity relationship study of 49 derivatives investigated at all four GPCRs revealed structural requirements for the development of potent and selective GPR18 and GPR55 antagonists. (Z)-(2,3-Difluorobenzylidene)-6,7-dihydro-2H-imidazo[2,1b][1,3]thiazin-3(5H)-one (18) was identified as a selective GPR55 antagonist (IC50 3.15 μM). The most potent GPR18 antagonist was (Z)-2-(3-(4-chlorobenzyloxy)benzylidene)-6,7-dihydro-2H-imidazo[2,1b][1,3]-thiazin-3(5H)-one (32, IC50 0.279 μM, >36-fold selective vs. CB1 and GPR55, 14-fold selective vs. CB2) representing the first selective GPR18 antagonist. The new compounds may serve as lead structures and as tools to explore the (patho-)physiological roles of these orphan GPCRs and their potential as drug targets.

6-(Ar)Alkylamino-Substituted Uracil Derivatives: Lipid Mimetics with Potent Activity at the Orphan G Protein-Coupled Receptor 84 (GPR84)

GPR84, a Gi protein-coupled receptor that is activated by medium-chain (hydroxy)fatty acids, appears to play an important role in inflammation, immunity, and cancer. Recently, 6-octylaminouracil (4) has been reported to act as an agonist at GPR84. Here, we describe the synthesis of 69 derivatives and analogs of 4, 66 of which represent new compounds. They were evaluated in (a) cyclic adenosine monophosphate accumulation and (b) β-arrestin assays in human GPR84-expressing cells. Potent nonbiased as well as G protein-biased agonists were developed, e.g., 6-hexylamino-2,4(1H,3H)-pyrimidinedione (20, PSB-1584, EC50 5.0 nM (a), 3.2 nM (b), bias factor: 0) and 6-((p-chloro- and p-bromo-phenylethyl)amino)-2,4(1H,3H)-pyrimidinedione (47, PSB-16434, EC50 7.1 nM (a), 520 nM (b), bias factor: 1.9 = 79-fold Gi pathway-selective; 48, PSB-17365, EC50 2.5 nM (a), 100 nM (b), bias factor 1.3 = 20-fold selective), which were selective versus other free fatty acid-activated receptors. Compounds 20 and 48 were found to be metabolically stable upon incubation with human liver microsomes. A pharmacophore model was created on the basis of structurally diverse lipidlike GPR84 agonists.

The 2016 Frontiers in Medicinal Chemistry Conference in Bonn

Pushing the frontiers of medicinal chemistry: Christa Müller, Dominik Thimm, and Karl-Heinz Baringhaus look back at the events of the 2016 Frontiers in Medicinal Chemistry (FiMC) Conference held in Bonn, Germany. The report highlights the themes & talks in the annual conference hosted by the Joint Division of Medicinal Chemistry of the German Pharmaceutical Society (DPhG) and German Chemical Society (GDCh). It is also an invitation to the 2017 conference in Bern, Switzerland this February 12-15.