Structure-based exploration and pharmacological evaluation of N-substituted piperidin-4-yl-methanamine CXCR4 chemokine receptor antagonists.

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure-activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.

Ligand Residence Time at G-protein-Coupled Receptors-Why We Should Take Our Time To Study It.

Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein-coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells.

A structural chemogenomics analysis of aminergic GPCRs: lessons for histamine receptor ligand design.

BACKGROUND AND PURPOSE: Chemogenomics focuses on the discovery of new connections between chemical and biological space leading to the discovery of new protein targets and biologically active molecules. G-protein coupled receptors (GPCRs) are a particularly interesting protein family for chemogenomics studies because there is an overwhelming amount of ligand binding affinity data available. The increasing number of aminergic GPCR crystal structures now for the first time allows the integration of chemogenomics studies with high-resolution structural analyses of GPCR-ligand complexes. EXPERIMENTAL APPROACH: In this study, we have combined ligand affinity data, receptor mutagenesis studies, and amino acid sequence analyses to high-resolution structural analyses of (hist)aminergic GPCR-ligand interactions. This integrated structural chemogenomics analysis is used to more accurately describe the molecular and structural determinants of ligand affinity and selectivity in different key binding regions of the crystallized aminergic GPCRs, and histamine receptors in particular. KEY RESULTS: Our investigations highlight interesting correlations and differences between ligand similarity and ligand binding site similarity of different aminergic receptors. Apparent discrepancies can be explained by combining detailed analysis of crystallized or predicted protein-ligand binding modes, receptor mutation studies, and ligand structure-selectivity relationships that identify local differences in essential pharmacophore features in the ligand binding sites of different receptors. CONCLUSIONS AND IMPLICATIONS: We have performed structural chemogenomics studies that identify links between (hist)aminergic receptor ligands and their binding sites and binding modes. This knowledge can be used to identify structure-selectivity relationships that increase our understanding of ligand binding to (hist)aminergic receptors and hence can be used in future GPCR ligand discovery and design.

Detailed analysis of biased histamine H4 receptor signalling by JNJ 7777120 analogues.

BACKGROUND AND PURPOSE: The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via ß-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in ß-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or ß-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling. EXPERIMENTAL APPROACH: We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi protein signalling or ß-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure-activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling. KEY RESULTS: One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and ß-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were ß-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as ß-arrestin2-biased indolecarboxamides to induce ß-arrestin2 recruitment could be correlated with different ligand features and hH4R regions. CONCLUSION AND IMPLICATIONS: Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and ß-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-ß-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation.

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H4 receptor.

BACKGROUND AND PURPOSE: The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182(5.46) in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98(3.36) in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98(3.36) by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. EXPERIMENTAL APPROACH: We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and ß-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. KEY RESULTS: VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and ß-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C98(3.36) prevented this covalent interaction. CONCLUSION AND IMPLICATIONS: VUF14480 is thought to bind covalently to the hH4 receptor-C98(3.36) residue and partially induce hH4 receptor-mediated G protein activation and ß-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor.

Inhibition of CXCR3-mediated chemotaxis by the human chemokine receptor-like protein CCX-CKR.

BACKGROUND AND PURPOSE: Induction of cellular migration is the primary effect of chemokine receptor activation. However, several chemokine receptor-like proteins bind chemokines without subsequent induction of intracellular signalling and chemotaxis. It has been suggested that they act as chemokine scavengers, which may control local chemokine levels and contribute to the function of chemokines during inflammation. This has been verified for the chemokine-like receptor proteins D6 and DARC as well as CCX-CKR. Here, we provide evidence for an additional biological function of human (h)CCX-CKR. EXPERIMENTAL APPROACH: We used transfection strategies in HEK293 and human T cells. KEY RESULTS: Co-expression of hCCX-CKR completely inhibits hCXCR3-induced chemotaxis. We found that hCCX-CKR forms complexes with hCXCR3, suggesting a relationship between CCX-CKR heteromerization and inhibition of chemotaxis. Moreover, negative binding cooperativity induced by ligands both for hCXCR3 and hCCX-CKR was observed in cells expressing both receptors. This negative cooperativity may also explain the hCCX-CKR-induced inhibition of chemotaxis. CONCLUSIONS AND IMPLICATIONS: These findings suggest that hCCX-CKR prevents hCXCR3-induced chemotaxis by heteromerization thus representing a novel mechanism of regulation of immune cell migration.

Identification and profiling of CXCR3-CXCR4 chemokine receptor heteromer complexes.

BACKGROUND AND PURPOSE: The C-X-C chemokine receptors 3 (CXCR3) and C-X-C chemokine receptors 4 (CXCR4) are involved in various autoimmune diseases and cancers. Small antagonists have previously been shown to cross-inhibit chemokine binding to CXCR4, CC chemokine receptors 2 (CCR2) and 5 (CCR5) heteromers. We investigated whether CXCR3 and CXCR4 can form heteromeric complexes and the binding characteristics of chemokines and small ligand compounds to these chemokine receptor heteromers. EXPERIMENTAL APPROACH: CXCR3-CXCR4 heteromers were identified in HEK293T cells using co-immunoprecipitation, time-resolved fluorescence resonance energy transfer, saturation BRET and the GPCR-heteromer identification technology (HIT) approach. Equilibrium competition binding and dissociation experiments were performed to detect negative binding cooperativity. KEY RESULTS: We provide evidence that chemokine receptors CXCR3 and CXCR4 form heteromeric complexes in HEK293T cells. Chemokine binding was mutually exclusive on membranes co-expressing CXCR3 and CXCR4 as revealed by equilibrium competition binding and dissociation experiments. The small CXCR3 agonist VUF10661 impaired binding of CXCL12 to CXCR4, whereas small antagonists were unable to cross-inhibit chemokine binding to the other chemokine receptor. In contrast, negative binding cooperativity between CXCR3 and CXCR4 chemokines was not observed in intact cells. However, using the GPCR-HIT approach, we have evidence for specific ß-arrestin2 recruitment to CXCR3-CXCR4 heteromers in response to agonist stimulation. CONCLUSIONS AND IMPLICATIONS: This study indicates that heteromeric CXCR3-CXCR4 complexes may act as functional units in living cells, which potentially open up novel therapeutic opportunities.

High-resolution metabolic profiling towards G protein-coupled receptors: rapid and comprehensive screening of histamine H4 receptor ligands.

In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H(4) receptor (hH(4)R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH(4)R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic 'hotspots' for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

Pharmacological characterization of a small-molecule agonist for the chemokine receptor CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACH: Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with [(125)I]-CXCL10 and [(125)I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [(35)S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and ß-arrestin recruitment. KEY RESULTS: VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated G(i) proteins with the same efficacy as CXCL11 in the [(35)S]-GTPγS binding and cAMP assay, while it recruited more ß-arrestin1 and ß-arrestin2 to CXCR3 receptors than the chemokine. CONCLUSIONS AND IMPLICATIONS: VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or ß-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.

Pharmacological modulation of chemokine receptor function.

G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.

C(X)CR in silico: Computer-aided prediction of chemokine receptor-ligand interactions.

This review will focus on the construction, refinement, and validation of chemokine receptor models for the purpose of structure-based virtual screening and ligand design. The review will present a comparative analysis of ligand binding pockets in chemokine receptors, including a review of the recently released CXCR4 X-ray structures, and their implication on chemokine receptor (homology) modeling. The recommended protein-ligand modeling procedure as well as the use of experimental anchors to steer the modeling procedure is discussed and an overview of several successful structure-based ligand discovery and design studies is provided. This review shows that receptor models, despite structural inaccuracies, can be efficiently used to find novel ligands for chemokine receptors.:

Approaches targeting K(V)10.1 open a novel window for cancer diagnosis and therapy.

K(V)10.1 has recently become generally accepted as a promising cancer target, as it is ectopically expressed in the majority of solid tumors. Due to its cell-surface accessibility, K(V)10.1 has a strong potential for tumor treatment and diagnosis. Given that its mode of action is likely independent of conventional cancer pathways such as tyrosine kinases, K(V)10.1 opens a novel window for treating cancer. In this review we will give an overview of the current status of data linking K(V)10.1 to cancer, and propose techniques that could exploit K(V)10.1's properties for the management of cancer.

Quantitative expression profiling of G-protein-coupled receptors (GPCRs) in metastatic melanoma: the constitutively active orphan GPCR GPR18 as novel drug target.

G-protein-coupled receptors (GPCRs) have been implicated in the tumorigenesis and metastasis of human cancers and are considered amongst the most desirable targets for drug development. Utilizing a robust quantitative PCR array, we quantified expression of 94 human GPCRs, including 75 orphan GPCRs and 19 chemokine receptors, and 36 chemokine ligands, in 40 melanoma metastases from different individuals and benign nevi. Inter-metastatic site comparison revealed that orphan GPR174 and CCL28 are statistically significantly overexpressed in subcutaneous metastases, while P2RY5 is overexpressed in brain metastases. Comparison between metastases (all three metastatic sites) and benign nevi revealed that 16 genes, including six orphan receptors (GPR18, GPR34, GPR119, GPR160, GPR183 and P2RY10) and chemokine receptors CCR5, CXCR4, and CXCR6, were statistically significantly differentially expressed. Subsequent functional experiments in yeast and melanoma cells indicate that GPR18, the most abundantly overexpressed orphan GPCR in all melanoma metastases, is constitutively active and inhibits apoptosis, indicating an important role for GPR18 in tumor cell survival. GPR18 and five other orphan GPCRs with yet unknown biological function may be considered potential novel anticancer targets in metastatic melanoma.

The histamine H4 receptor is functionally expressed on neurons in the mammalian CNS.

BACKGROUND AND PURPOSE: The histamine H4 receptor is the most recently identified of the G protein-coupled histamine receptor family and binds several neuroactive drugs, including amitriptyline and clozapine. So far, H4 receptors have been found only on haematopoietic cells, highlighting its importance in inflammatory conditions. Here we investigated the possibility that H4 receptors may be expressed in both the human and mouse CNS. METHODS: Immunological and pharmacological studies were performed using a novel anti-H4 receptor antibody in both human and mouse brains, and electrophysiological techniques in the mouse brain respectively. Pharmacological tools, selective for the H4 receptor and patch clamp electrophysiology, were utilized to confirm functional properties of the H4 receptor in layer IV of the mouse somatosensory cortex. RESULTS: Histamine H4 receptors were prominently expressed in distinct deep laminae, particularly layer VI, in the human cortex, and mouse thalamus, hippocampal CA4 stratum lucidum and layer IV of the cerebral cortex. In layer IV of the mouse somatosensory cortex, the H4 receptor agonist 4-methyl histamine (20 micromol x L(-1)) directly hyperpolarized neurons, an effect that was blocked by the selective H4 receptor antagonist JNJ 10191584, and promoted outwardly rectifying currents in these cells. Monosynaptic thalamocortical CNQX-sensitive excitatory postsynaptic potentials were not altered by 4-methyl histamine (20 micromol x L(-1)) suggesting that H4 receptors did not act as hetero-receptors on thalamocortical glutamatergic terminals. CONCLUSIONS AND IMPLICATIONS: This is the first demonstration that histamine H4 receptors are functionally expressed on neurons, which has major implications for the therapeutic potential of these receptors in neurology and psychiatry.

[From gene to drug: recent developments in medicinal chemistry]. / Van gen tot medicijn: moderne ontwikkelingen in de farmacochemie.

The development ofa new drug often takes more than ten years. Until recently, researchers were obliged to carry out experimental research on the efficacy of variants of those molecules of which the effect was known. It took a long time before a structure-activity relationship could be established. Currently, the full effect of the stormy developments in the Life Sciences, including the sequencing of the human genome, has not yet been fully felt in clinical practice. However, in laboratories researching medicinal products, the technology and the strategies being applied are changing drastically. Understanding disease processes and pharmaconprotein interactions at molecular level make it possible to develop biologically active pharmaceuticals using efficient developmental techniques involving crystallography and computer models. At the same time it is becoming clear that more research is necessary in order to understand the pharmacokinetic characteristics and toxicological activity of medicines under development. This second phase in the development of medicinal products too, is being strongly stimulated by the recent changes.

Antinociceptive activity of furan-containing congeners of improgan and ranitidine.

Furan-containing congeners of the histamine H(2) receptor antagonist ranitidine were synthesized and tested for improgan-like antinociceptive activity. The most potent ligand of the series, VUF5498, is the most potent improgan-like agent described to date (ED(50)=25 nmol, icv). This compound is approximately equal in potency with morphine. These non-imidazole, improgan-like pain relievers further define the structural requirements for analgesics of this class and are important tools for ongoing mechanism-based studies.

In vitro pharmacology of clinically used central nervous system-active drugs as inverse H(1) receptor agonists.

The human histamine H(1) receptor (H(1)R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H(1)R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H(1)R agonist (Mol Pharmacol 65:538-549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H(1)R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.

Efficient library synthesis of imidazoles using a multicomponent reaction and microwave irradiation.

Optimization of Radziszewski's four-component reaction employing a microwave-assisted protocol, led to a small library of 48 imidazoles with a success rate of 65% (conversion > 45%). All three diversity points of the four-component reaction were varied. Aromatic and aliphatic inputs were successfully implemented and mono-, di-, tri- and tetrasubstituted imidazoles with various substitution patterns were synthesized. Furthermore, unsymmetrical diketones could successfully be used which improved the intrinsic diversity of the method significantly. If the unsymmetrical diketone 1,2-phenylpropanedione (R1 and R2) was used two regioisomers were formed. Depending on the type of amine (R4) and aldehyde (R3) applied, regioselectivity was modest to good. Based on these results, a reaction mechanism is proposed.

Virus-encoded G-protein-coupled receptors: constitutively active (dys)regulators of cell function and their potential as drug target.

G-protein-coupled receptors encoded by herpesviruses such as EBV, HCMV and KSHV are very interesting illustrations of the (patho)physiological importance of constitutive GPCR activity. These viral proteins are expressed on the cell surface of infected cells and often constitutively activate a variety of G-proteins. For some virus-encoded GPCRs, the constitutive activity has been shown to occur in vivo, i.e., in infected cells. In this paper, we will review the occurrence of virus-encoded GPCRs and describe their known signaling properties. Moreover, we will also review the efforts, directed towards the discovery of small molecule antagonist, that so far have been mainly focused on the HCMV-encoded GPCR US28. This virus-encoded receptor might be involved in cardiovascular diseases and cancer and seems an interesting target for drug intervention.