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.

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.

A viral conspiracy: hijacking the chemokine system through virally encoded pirated chemokine receptors.

Several herpesviruses and poxviruses contain genes encoding for G protein-coupled receptor (GPCR) proteins that are expressed on the surface of infected host cells and/or the viral envelope. Most of these membrane-associated proteins display highest homology to the subfamily of chemokine receptors known to play a key role in the immune system. Virally encoded chemokine receptors have been modified through evolutionary selection both in chemokine binding profile and signaling capacity, ultimately resulting in immune evasion and cellular reprogramming in favor of viral survival and replication. Insight in the role of virally encoded GPCRs during the viral lifecycle may reveal their potential as future drug targets.

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.

Both recombinant African catfish LH and FSH are able to activate the African catfish FSH receptor.

LH and FSH are heterodimeric glycoprotein hormones, composed of a common alpha-subunit non-covalently associated with a hormone-specific beta-subunit. Repeated efforts to isolate catfish FSH (cfFSH) have not been successful and only catfish LH (cfLH) has been purified from catfish pituitaries. Recently, however, we succeeded in cloning the cDNA encoding the putative cfFSHbeta; the cDNAs for the alpha- and beta-subunit of cfLH have been cloned before. Here we report the expression of biologically active cfLH and cfFSH in the soil amoeba, Dictyostelium discoideum. The biological activity of the recombinant hormones was analyzed using cell lines transiently expressing either the cfLH receptor or the cfFSH receptor. Moreover, a primary testis tIssue culture system served to study the steroidogenic potency of the recombinant hormones. Our results demonstrated that Dictyostelium produced biologically active, recombinant catfish gonadotropins, with recombinant cfLH being almost indistinguishable from its native counterpart, purified from pituitaries. Although recombinant cfFSH has significant effects in the bioassays used in this study, the specific function of native cfFSH in the control of reproduction and its expression patterns are not yet understood.

Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus).

A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino acids, specific for the TSH-R subfamily, was also present in the carboxy terminus of the amino-terminal extracellular domain of the cfTSH-R. Next to the testis and thyroid follicles, abundant cfTSH-R expression was detected in cerebellum, brain, ovary, seminal vesicles and pituitary, while weaker expression was found in muscle, stomach, intestine, head-kidney, liver, kidney and heart. HEK-T 293 cells, transiently expressing the cfTSH-R, significantly increased intracellular cAMP levels in response to human TSH. Catfish LH, human choriogonadotropin and human FSH were also able to induce this cfTSH-R-mediated response, although with considerably lower efficiency than human TSH. These results indicated that a functional cfTSH-R had been cloned from the testis of African catfish.

Cloning and spatiotemporal expression of the follicle-stimulating hormone beta subunit complementary DNA in the African catfish (Clarias gariepinus).

The gene and cDNA encoding a putative follicle-stimulating hormone beta subunit (cfFSHbeta) from African catfish (Clarias gariepinus) were cloned. Similar to other FSHbeta genes, the cfFSHbeta gene consisted of three exons interrupted by two introns. The cfFSHbeta cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone beta subunits were positionally conserved in cfFSHbeta. The cfFSHbeta mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSHbeta transcript levels increased in particular during early stages of puberty and reached constantly high levels after the first appearance of spermatids in the testis. The cfFSHbeta mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSHbeta mRNA levels that were decreased by steroid replacement. Previous work indicated that the African catfish is an interesting model to study the regulation of gonadal functions because cfLH is able to activate both the catfish luteinizing hormone receptor (cfLH-R) and follicle-stimulating hormone receptor (cfFSH-R). Because cfFSH purification has failed so far, ongoing studies are directed toward the production of recombinant cfFSH. After all, the developmental and hormonal regulation of cfFSHbeta transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.

Cloning and functional characterization of a gonadal luteinizing hormone receptor complementary DNA from the African catfish (Clarias gariepinus).

A cDNA encoding a putative African catfish (Clarias gariepinus) gonadal LH receptor (cfLH-R) has been cloned. Multiple sequence alignment of the deduced amino acid sequence revealed that the cfLH-R had the highest identity with vertebrate LH receptors (>50%). Overall sequence identity between the cfLH-R and the African catfish FSH receptor (cfFSH-R) is 47%. Sequence analysis of part of the cfLH-R gene revealed the presence of an intron typically found in other vertebrate LH-R genes. Abundant cfLH-R mRNA expression was detected in ovary and testis as well as in head-kidney (the adrenal homologue in fish). Other tissues, such as muscle, brain, cerebellum, stomach, heart, and seminal vesicles, also contained detectable cfLH-R mRNA. Transient expression of the cfLH-R in HEK-T 293 cells resulted in significantly increased basal cAMP levels in the absence of gonadotropic hormone. The cAMP levels could be further elevated in response to catfish LH, salmon LH, human LH, human choriogonadotropin, and human FSH. Salmon FSH and human TSH, however, were inactive. We conclude that we have cloned a cDNA encoding the LH-R of the African catfish. This receptor displays constitutive activity but is still responsive to additional ligand-induced activation.

Gonadotropins, their receptors, and the regulation of testicular functions in fish.

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.

Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus).

A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish FSH-R (cfFSH-R) mRNA expression was observed in testis and ovary; abundant mRNA expression was also detected in seminal vesicles. The isolated cDNA encoded a functional receptor since its transient expression in human embryonic kidney (HEK-T) 293 cells resulted in ligand-dependent cAMP production. Remarkably, African catfish LH (cfLH; the catfish FSH-like gonadotropin has not been purified yet) had the highest potency in this system. From the other ligands tested, only human recombinant FSH (hrFSH) was active, showing a fourfold lower potency than cfLH, while hCG and human TSH (hTSH) were inactive. Human CG (as well as cfLH, hrFSH, eCG, but not hTSH) stimulated testicular androgen secretion in vitro but seemed to be unable to bind to the cfFSH-R. However, it was known that hCG is biologically active in African catfish (e.g., induction of ovulation). This indicated that an LH receptor is also expressed in African catfish testis. We conclude that we have cloned a cDNA encoding a functional FSH-R from African catfish testis. The cfFSH-R appears to be less discriminatory for its species-specific LH than its avian and mammalian counterparts.