A group of cationic amphiphilic drugs activates MRGPRX2 and induces scratching behavior in mice.

BACKGROUND: Mas gene-related G protein-coupled receptors (MRGPRs) are a G protein-coupled receptor family responsive to various exogenous and endogenous agonists, playing a fundamental role in pain and itch sensation. The primate-specific family member MRGPRX2 and its murine orthologue MRGPRB2 are expressed by mast cells mediating IgE-independent signaling and pseudoallergic drug reactions. OBJECTIVES: Our aim was to increase knowledge about the function and regulation of MRGPRX2/MRGPRB2, which is of major importance in prevention of drug hypersensitivity reactions and drug-induced pruritus. METHODS: To identify novel MRGPR (ant)agonists, we screened a library of pharmacologically active compounds by utilizing a high-throughput calcium mobilization assay. The identified hit compounds were analyzed for their pseudoallergic and pruritogenic effects in mice and human. RESULTS: We found a class of commonly used drugs activating MRGPRX2 that, to a large extent, consists of antidepressants, antiallergic drugs, and antipsychotics. Three-dimensional pharmacophore modeling revealed structural similarities of the identified agonists, classifying them as cationic amphiphilic drugs. Mast cell activation was investigated by using the 3 representatively selected antidepressants clomipramine, paroxetine, and desipramine. Indeed, we were able to show a concentration-dependent activation and MRGPRX2-dependent degranulation of the human mast cell line LAD2 (Laboratory of Allergic Diseases-2). Furthermore, clomipramine, paroxetine, and desipramine were able to induce degranulation of human skin and murine peritoneal mast cells. These substances elicited dose-dependent scratching behavior following intradermal injection into C57BL/6 mice but less so in MRGPRB2-mutant mice, as well as wheal-and-flare reactions following intradermal injections in humans. CONCLUSION: Our results contribute to the characterization of structure-activity relationships and functionality of MRGPRX2 ligands and facilitate prediction of adverse reactions such as drug-induced pruritus to prevent severe drug hypersensitivity reactions.

A Cellular Assay for the Identification and Characterization of Connexin Gap Junction Modulators.

Connexin gap junctions (Cx GJs) enable the passage of small molecules and ions between cells and are therefore important for cell-to-cell communication. Their dysfunction is associated with diseases, and small molecules acting as modulators of GJs may therefore be useful as therapeutic drugs. To identify GJ modulators, suitable assays are needed that allow compound screening. In the present study, we established a novel assay utilizing HeLa cells recombinantly expressing Cx43. Donor cells additionally expressing the Gs protein-coupled adenosine A2A receptor, and biosensor cells expressing a cAMP-sensitive GloSensor luciferase were established. Adenosine A2A receptor activation in the donor cells using a selective agonist results in intracellular cAMP production. The negatively charged cAMP migrates via the Cx43 gap junctions to the biosensor cells and can there be measured by the cAMP-dependent luminescence signal. Cx43 GJ modulators can be expected to impact the transfer of cAMP from the donor to the biosensor cells, since cAMP transit is only possible via GJs. The new assay was validated by testing the standard GJ inhibitor carbenoxolon, which showed a concentration-dependent inhibition of the signal and an IC50 value that was consistent with previously reported values. The assay was demonstrated to be suitable for high-throughput screening.

Targeting the Main Protease of SARS-CoV-2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors.

The main protease of SARS-CoV-2 (Mpro ), the causative agent of COVID-19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed Mpro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of Mpro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards Mpro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (kinac /Ki =37 500 m-1 s-1 , Ki =24.0 nm) and pyridyl ester 17 (kinac /Ki =29 100 m-1 s-1 , Ki =10.0 nm), promising drug candidates for further development have been discovered.

Ligand-specific binding and activation of the human adenosine A(2B) receptor.

Adenosine A(2B) receptors, which play a role in inflammation and cancer, are of considerable interest as novel drug targets. To gain deeper insights into ligand binding and receptor activation, we exchanged amino acids predicted to be close to the binding pocket. The alanine mutants were stably expressed in CHO cells and characterized by radioligand binding and cAMP assays using three structural classes of ligands: xanthine (antagonist), adenosine, and aminopyridine derivatives (agonists). Asn282(7.45) and His280(7.43) were found to stabilize the binding site by intramolecular hydrogen bond formation as in the related A(2A) receptor subtype. Trp247(6.48), Val250(6.51), and particularly Ser279(7.42) were shown to be important for binding of nucleosidic agonists. Leu81(3.28), Asn186(5.42), and Val250(6.51) were discovered to be crucial for binding of the xanthine-derived antagonist PSB-603. Leu81(3.28), which is not conserved among adenosine receptor subtypes, may be important for the high selectivity of PSB-603. The N186(5.42)A mutant resulted in an increased potency for agonists. The interactions of the non-nucleosidic agonist BAY60-6583 were different from those of the nucleosides: while BAY60-6583 appeared not to interact with Ser279(7.42), its interactions with Trp247(6.48) and Val250(6.51) were significantly weaker compared to those of NECA. Moreover, our results discount the hypothesis of Trp247(6.48) serving as a "toogle switch" because BAY60-6583 was able to activate the corresponding mutant. This study reveals distinct interactions of structurally diverse ligands with the human A(2B) receptor and differences between closely related receptor subtypes (A(2B) and A(2A)). It will contribute to the understanding of G protein-coupled receptor function and advance A(2B) receptor ligand design.

Characterization of new G protein-coupled adenine receptors in mouse and hamster.

The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [(3)H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins.

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 (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]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, Asn194(5.40) and Leu201(5.47), 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.

Methods of Conservative Intra-Articular Treatment for Osteoarthritis of the Hip and Knee.

BACKGROUND: Osteoarthritis is a degenerative joint disease that is becoming increasingly common as the population ages. Conservative treatment for hip or knee osteoarthritis has been limited to pain control. Intra-articular injections for targeted local treatment have been widely used in clinical practice for many years. METHODS: This review is based on publications retrieved by a selective literature search, including recent meta-analyses, systematic reviews, randomized controlled trials (RCTs), and current guidelines. RESULTS: In Germany, the 12-month prevalence of osteoarthritis in adults is 17.9%. Conservative treatments are intended to alleviate symptoms and do not affect the progression of the disease. Glucocorticoids can be used to relieve otherwise intractable pain in the short term, but their prolonged use increases the risk of cartilage loss and progression of osteoarthritis. According to multiple guidelines, there is only weak evidence for the use of hyaluronic acid. Evidence does exist that high-molecular-weight hyaluronic acid may lead to better outcomes than the low-molecular-weight form. RCTs have revealed no more than short-term clinical efficacy for a variety of specific therapeutic approaches, including the use of cytokine inhibitors. Other treatments, e.g., with platelet-enriched plasma, aspirates from bone marrow or adipose tissue, or expanded mesenchymal stromal cells (MSC), have not been found to have clinically relevant long-term effects. CONCLUSION: In view of the scant available evidence, further standardized RCTs will be needed to give a more comprehensive picture of the efficacy of intra-articular treatments for hip and knee osteoarthritis.

MAS-related G protein-coupled receptors X (MRGPRX): Orphan GPCRs with potential as targets for future drugs.

MAS-related G protein-coupled receptors (GPCRs) of subfamily X, designated MRGPRX, are primate-specific orphan receptors that belong to the δ-branch of rhodopsin-like, class A GPCRs. Four distinct subtypes exist, MRGPRX1, -2, -3, and -4, MRGPRX2 having the lowest degree of similarity with the others. Due to their expression on sensory neurons and immune cells, and their roles in pain perception and transmission, itch, inflammation, immune defense, pseudo-allergic reactions, wound healing, and possibly cancer, they have recently attracted much attention as novel drug targets. In particular MRGPRX2 was identified as an important mast cell receptor, responsible for anaphylactoid drug reactions and involved in skin and mucosal diseases, e.g. urticaria, atopic dermatitis, rosacea, and allergic rhinitis. A major hurdle has been the lack of animal models for studying these primate-specific receptors. However, recently humanized mice have been created. Moreover, a mouse ortholog of MRGPRX2, MRGPRB2, was identified, both receptors having a certain degree of similarity. MRGPRX1 and -2 can be activated by various peptides and small (partly peptidomimetic) molecules. MRGPRX2 is additionally activated by a very broad range of basic molecules, positively charged at physiologic pH value of 7.4, including many drugs. MRGPRX4 is activated by small acidic molecules including bile acids. For MRGPRX3, no ligands have been reported yet. Antagonists with reasonable potency and selectivity have been described for MRGPRX1, and few antagonists also for MRGPRX2, but not for the other subtypes. The recent elucidation of cryogenic electron microscopy structures of MRGPRX2 and -4 is expected to facilitate and advance drug development for these receptors. Currently, research on MRGPRX is still in its infancy, and exciting discoveries can be awaited. These receptors have great potential as future drug targets.

Increased antigen cross-presentation but impaired cross-priming after activation of peroxisome proliferator-activated receptor gamma is mediated by up-regulation of B7H1.

Dendritic cells are able to take up exogenous Ags and present Ag-derived peptides on MHC class I molecules, a process termed cross-presentation. The mannose receptor (MR), an endocytic receptor expressed on a variety of APCs, has been demonstrated to target soluble Ags exclusively toward cross-presentation. In this study, we investigated the role of the murine nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor with immunomodulatory properties, in MR-mediated endocytosis and cross-presentation of the model Ag OVA. We could demonstrate both in vitro and in vivo that activation of PPARgamma resulted in increased MR expression, which in consequence led to enhanced MR-mediated endocytosis and elevated cross-presentation of soluble OVA. Concomitantly, activation of PPARgamma in dendritic cells induced up-regulation of the coinhibitory molecule B7H1, which, despite enhanced cross-presentation, caused an impaired activation of naive OVA-specific CD8(+) T cells and the induction of T cell tolerance. These data provide a mechanistic basis for the immunomodulatory action of PPARgamma which might open new possibilities in the development of therapeutic approaches aimed at the control of excessive immune responses, e.g., in T cell-mediated autoimmunity.

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) ß-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.

Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

The Gi protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and ß-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC50 41.3 nM). In ß-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

6-Bromo-8-(4-[(3)H]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 ß-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.

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 ß-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 receptor's (patho)physiological role and its potential as a future drug target.

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.

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.