Henry F. Vischer

Crystal structure-based virtual screening for novel fragment-like ligands of the human histamine H1 receptor

The recent crystal structure determinations of druggable class A G protein-coupled receptors (GPCRs) have opened up excellent opportunities in structure-based ligand discovery for this pharmaceutically important protein family. We have developed and validated a customized structure-based virtual fragment screening protocol against the recently determined human histamine H(1) receptor (H(1)R) crystal structure. The method combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. The optimized in silico screening approach was successfully applied to identify a chemically diverse set of novel fragment-like (≤22 heavy atoms) H(1)R ligands with an exceptionally high hit rate of 73%. Of the 26 tested fragments, 19 compounds had affinities ranging from 10 μM to 6 nM. The current study shows the potential of in silico screening against GPCR crystal structures to explore novel, fragment-like GPCR ligand space.

Discrepancy Between Molecular Structure and Ligand Selectivity of a Testicular Follicle-Stimulating Hormone Receptor of the African Catfish (Clarias gariepinus)

Abstract 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.

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.

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.

Cloning and Functional Characterization of a Gonadal Luteinizing Hormone Receptor Complementary DNA from the African Catfish (Clarias gariepinus)

Abstract 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.

The cytomegalovirus-encoded chemokine receptor US28 promotes intestinal neoplasia in transgenic mice.

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3β (GSK-3β) function, promoted accumulation of β-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.

The Epstein-Barr virus-encoded G protein-coupled receptor BILF1 hetero-oligomerizes with human CXCR4, scavenges Gαi proteins and constitutively impairs CXCR4 functioning

Cells express distinct G protein-coupled receptor (GPCR) subtypes on their surface, allowing them to react to a corresponding variety of extracellular stimuli. Cross-regulation between different ligand-GPCR pairs is essential to generate appropriate physiological responses. GPCRs can physically affect each others functioning by forming heteromeric complexes, whereas cross-regulation between activated GPCRs also occurs through integration of shared intracellular signaling networks. Human herpesviruses utilize virally encoded GPCRs to hijack cellular signaling networks for their own benefit. Previously, we demonstrated that the Epstein-Barr virus-encoded GPCR BILF1 forms heterodimeric complexes with human chemokine receptors. Using a combination of bimolecular complementation and bioluminescence resonance energy transfer approaches, we now show the formation of hetero-oligomeric complexes between this viral GPCR and human CXCR4. BILF1 impaired CXCL12 binding to CXCR4 and, consequently, also CXCL12-induced signaling. In contrast, the G protein uncoupled mutant BILF1-K3.50A affected CXCL12-induced CXCR4 signaling to a much lesser extent, indicating that BILF1-mediated CXCR4 inhibition is a consequence of its constitutive activity. Co-expression of Gαi1 with BILF1 and CXCR4 restored CXCL12-induced signaling. Likewise, BILF1 formed heteromers with the human histamine H4 receptor (H4R). BILF1 inhibited histamine-induced Gαi-mediated signaling by H4R, however, without affecting histamine binding to this receptor. These data indicate that functional cross-regulation of Gαi-coupled GPCRs by BILF1 is at the level of G proteins, even though these GPCRs are assembled in hetero-oligomeric complexes.

Llama-derived Single Variable Domains (Nanobodies) Directed against Chemokine Receptor CXCR7 Reduce Head and Neck Cancer Cell Growth in Vivo

Background: The atypical chemokine receptor CXCR7 is highly expressed in various types of cancer. Results: CXCR7 Nanobodies were generated and show inhibition of β-arrestin2 signaling and secretion of angiogenic CXCL1 in vitro. Anti-CXCR7 Nanobodies reduce tumor growth by inhibiting angiogenesis. Conclusion: CXCR7 inhibition by Nanobodies inhibit head and neck tumor formation. Significance: Anti-CXCR7 therapies are potential novel treatments against head and neck cancer. The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the “Nanobody platform” to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced β-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.

Cloning and Spatiotemporal Expression of the Follicle-Stimulating Hormone {beta} Subunit Complementary DNA in the African Catfish (Clarias gariepinus)

Abstract The gene and cDNA encoding a putative follicle-stimulating hormone β subunit (cfFSHβ) from African catfish (Clarias gariepinus) were cloned. Similar to other FSHβ genes, the cfFSHβ gene consisted of three exons interrupted by two introns. The cfFSHβ cDNA coded for a mature protein of 115 amino acids. The 12 cysteines that are required for the typical tertiary folding of glycoprotein hormone β subunits were positionally conserved in cfFSHβ. The cfFSHβ mRNA expression was exclusively detected in the pituitary and was detectable before pubertal development was initiated. The cfFSHβ 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 cfFSHβ mRNA-positive cells were localized in the proximal pars distalis. Castration of mature males caused elevated cfFSHβ 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 cfFSHβ transcript levels opens the possibility for physiologically relevant actions of the putative cfFSH, next to the presumptive bifunctionally acting cfLH.

En route to new blockbuster anti-histamines: surveying the offspring of the expanding histamine receptor family

With the recognition of two new histamine receptors at the start of the new millennium, the field of histamine research has seen a clear revival. In the last 10 years, many academic and industrial groups have taken up the challenge to target these new members of the aminergic G-protein-coupled receptor (GPCR) family. Histamine receptor research nicely illustrates how GPCR research has changed in the post-genomic era. There is a growing understanding of GPCR structure, function and modulation at a molecular level. Emerging concepts such as receptor isoforms, GPCR oligomerization and ligand-biased signaling are all being studied, but their clinical relevance remains to be determined. The histamine H(3) and H(4) drug development programs can help to establish the link between these molecular features and clinical efficacy. Several new anti-histamines are now being tested for diverse clinical applications and are poised to become the next blockbuster drugs targeting histamine receptors.