A virally encoded GPCR drives glioblastoma through feed-forward activation of the SK1-S1P1 signaling axis.

The G protein-coupled receptor (GPCR) US28 encoded by the human cytomegalovirus (HCMV) is associated with accelerated progression of glioblastomas, aggressive brain tumors with a generally poor prognosis. Here, we showed that US28 increased the malignancy of U251 glioblastoma cells by enhancing signaling mediated by sphingosine-1-phosphate (S1P), a bioactive lipid that stimulates oncogenic pathways in glioblastoma. US28 expression increased the abundance of the key components of the S1P signaling axis, including an enzyme that generates S1P [sphingosine kinase 1 (SK1)], an S1P receptor [S1P receptor 1 (S1P1)], and S1P itself. Enhanced S1P signaling promoted glioblastoma cell proliferation and survival by activating the kinases AKT and CHK1 and the transcriptional regulators cMYC and STAT3 and by increasing the abundance of cancerous inhibitor of PP2A (CIP2A), driving several feed-forward signaling loops. Inhibition of S1P signaling abrogated the proliferative and anti-apoptotic effects of US28. US28 also activated the S1P signaling axis in HCMV-infected cells. This study uncovers central roles for S1P and CIP2A in feed-forward signaling that contributes to the US28-mediated exacerbation of glioblastoma.

Exosomal release of the virus-encoded chemokine receptor US28 contributes to chemokine scavenging.

The human cytomegalovirus (HCMV)-encoded chemokine receptor US28 contributes to various aspects of the viral life cycle and promotes immune evasion by scavenging chemokines from the microenvironment of HCMV-infected cells. In contrast to the plasma membrane localization of most human chemokine receptors, US28 has a predominant intracellular localization. In this study, we used immunofluorescence and electron microscopy to determine the localization of US28 upon exogenous expression, as well as in HCMV-infected cells. We observed that US28 localizes to late endosomal compartments called multivesicular bodies (MVBs), where it is sorted in intraluminal vesicles. Live-cell total internal reflection fluorescence (TIRF) microscopy revealed that US28-containing MVBs can fuse with the plasma membrane, resulting in the secretion of US28 on exosomes. Exosomal US28 binds the chemokines CX3CL1 and CCL5, and US28-containing exosomes inhibited the CX3CL1-CX3CR1 signaling axis. These findings suggest that exosomal release of US28 contributes to chemokine scavenging and immune evasion by HCMV.

Discovery of 5-Phenylpyrazolopyrimidinone Analogs as Potent Antitrypanosomal Agents with In Vivo Efficacy.

Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei, is one of the neglected tropical diseases with a continuing need for new medication. We here describe the discovery of 5-phenylpyrazolopyrimidinone analogs as a novel series of phenotypic antitrypanosomal agents. The most potent compound, 30 (NPD-2975), has an in vitro IC50 of 70 nM against T. b. brucei with no apparent toxicity against human MRC-5 lung fibroblasts. Showing good physicochemical properties, low toxicity potential, acceptable metabolic stability, and other pharmacokinetic features, 30 was further evaluated in an acute mouse model of T. b. brucei infection. After oral dosing at 50 mg/kg twice per day for five consecutive days, all infected mice were cured. Given its good drug-like properties and high in vivo antitrypanosomal potential, the 5-phenylpyrazolopyrimidinone analog 30 represents a promising lead for future drug development to treat HAT.

Monitoring Snake Venom-Induced Extracellular Matrix Degradation and Identifying Proteolytically Active Venom Toxins Using Fluorescently Labeled Substrates.

Snakebite envenoming is an important public health issue with devastating consequences and annual mortality rates that range between 81,000 and 138,000. Snake venoms may cause a range of pathophysiological effects affecting the nervous system and the cardiovascular system. Moreover, snake venom may have tissue-damaging activities that result in lifelong morbidities such as amputations, muscle degeneration, and organ malfunctioning. The tissue-damaging components in snake venoms comprise multiple toxin classes with various molecular targets including cellular membranes and the extracellular matrix (ECM). In this study, we present multiple assay formats that enable investigation of snake venom-induced ECM degradation using a variety of (dye-quenched) fluorescently labeled ECM components. Using a combinatorial approach, we were able to characterise different proteolytic profiles for different medically relevant snake venoms, followed by identification of the responsible components within the snake venoms. This workflow could provide valuable insights into the key mechanisms by which proteolytic venom components exert their effects and could therefore prove useful for the development of effective snakebite treatments against this severe pathology.

To Target or Not to Target Schistosoma mansoni Cyclic Nucleotide Phosphodiesterase 4A?

Schistosomiasis is a neglected tropical disease with high morbidity. Recently, the Schistosoma mansoni phosphodiesterase SmPDE4A was suggested as a putative new drug target. To support SmPDE4A targeted drug discovery, we cloned, isolated, and biochemically characterized the full-length and catalytic domains of SmPDE4A. The enzymatically active catalytic domain was crystallized in the apo-form (PDB code: 6FG5) and in the cAMP- and AMP-bound states (PDB code: 6EZU). The SmPDE4A catalytic domain resembles human PDE4 more than parasite PDEs because it lacks the parasite PDE-specific P-pocket. Purified SmPDE4A proteins (full-length and catalytic domain) were used to profile an in-house library of PDE inhibitors (PDE4NPD toolbox). This screening identified tetrahydrophthalazinones and benzamides as potential hits. The PDE inhibitor NPD-0001 was the most active tetrahydrophthalazinone, whereas the approved human PDE4 inhibitors roflumilast and piclamilast were the most potent benzamides. As a follow-up, 83 benzamide analogs were prepared, but the inhibitory potency of the initial hits was not improved. Finally, NPD-0001 and roflumilast were evaluated in an in vitro anti-S. mansoni assay. Unfortunately, both SmPDE4A inhibitors were not effective in worm killing and only weakly affected the egg-laying at high micromolar concentrations. Consequently, the results with these SmPDE4A inhibitors strongly suggest that SmPDE4A is not a suitable target for anti-schistosomiasis therapy.

NanoB2 to monitor interactions of ligands with membrane proteins by combining nanobodies and NanoBRET.

The therapeutic potential of ligands targeting disease-associated membrane proteins is predicted by ligand-receptor binding constants, which can be determined using NanoLuciferase (NanoLuc)-based bioluminescence resonance energy transfer (NanoBRET) methods. However, the broad applicability of these methods is hampered by the restricted availability of fluorescent probes. We describe the use of antibody fragments, like nanobodies, as universal building blocks for fluorescent probes for use in NanoBRET. Our nanobody-NanoBRET (NanoB2) workflow starts with the generation of NanoLuc-tagged receptors and fluorescent nanobodies, enabling homogeneous, real-time monitoring of nanobody-receptor binding. Moreover, NanoB2 facilitates the assessment of receptor binding of unlabeled ligands in competition binding experiments. The broad significance is illustrated by the successful application of NanoB2 to different drug targets (e.g., multiple G protein-coupled receptors [GPCRs] and a receptor tyrosine kinase [RTK]) at distinct therapeutically relevant binding sites (i.e., extracellular and intracellular).

Fusicoccin-A Targets Cancerous Inhibitor of Protein Phosphatase 2A by Stabilizing a C-Terminal Interaction with 14-3-3.

The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein found overexpressed in many types of cancer. CIP2A has been shown to stabilize oncoproteins such as cMYC by shielding them from PP2A-mediated dephosphorylation. Here we report that the penultimate residue Ser904 in the C-terminus of CIP2A can be phosphorylated to create a binding site for the regulatory protein 14-3-3. We demonstrate that 14-3-3 is a new interaction partner of CIP2A. The 14-3-3/CIP2A C-terminal interaction complex can be targeted by the protein-protein interaction (PPI) stabilizer fusicoccin-A (FC-A), resulting in enhanced levels of phosphorylated Ser904. FC-A treatment of TNBC cells leads to the increased association of CIP2A with 14-3-3. We show that the composite interface between 14 and 3-3 and CIP2A's C-terminus can be targeted by the PPI stabilizer FC-A, providing a new interface that could potentially be exploited to modulate CIP2A's activity.

3-nitroimidazo[1,2-b]pyridazine as a novel scaffold for antiparasitics with sub-nanomolar anti-Giardia lamblia activity.

As there is a continuous need for novel anti-infectives, the present study aimed to fuse two modes of action into a novel 3-nitroimidazo[1,2-b]pyridazine scaffold to improve antiparasitic efficacy. For this purpose, we combined known structural elements of phosphodiesterase inhibitors, a target recently proposed for Trypanosoma brucei and Giardia lamblia, with a nitroimidazole scaffold to generate nitrosative stress. The compounds were evaluated in vitro against a panel of protozoal parasites, namely Giardia lamblia, Trypanosoma brucei, T. cruzi, Leishmania infantum and Plasmodium falciparum and for cytotoxicity on MRC-5 cells. Interestingly, selective sub-nanomolar activity was obtained against G. lamblia, and by testing several analogues with and without the nitro group, it was shown that the presence of a nitro group, but not PDE inhibition, is responsible for the low IC50 values of these novel compounds. Adding the favourable drug-like properties (low molecular weight, cLogP (1.2-4.1) and low polar surface area), the key compounds from the 3-nitroimidazo[1,2-b]pyridazine series can be considered as valuable hits for further anti-giardiasis drug exploration and development.

Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor.

The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits ß-arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.

Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.

Differential Involvement of ACKR3 C-Tail in ß-Arrestin Recruitment, Trafficking and Internalization.

Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits ß-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of ß-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of ß-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both ß-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for ß-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that ß-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for ß-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential ß-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when ß-arrestin recruitment is impaired or in the absence of ß-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced ß-arrestin recruitment, ACKR3 trafficking and internalization.

Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.

Herpesviruses are ubiquitous pathogens that establish lifelong, latent infections in their host. Spontaneous reactivation of herpesviruses is often asymptomatic or clinically manageable in healthy individuals, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with multiple proliferative cardiovascular and post-transplant diseases. Herpesviruses encode viral G protein-coupled receptors (vGPCRs) that alter the host cell by hijacking cellular pathways and play important roles in the viral life cycle and these different disease settings. In this review, we discuss the pharmacological and signaling properties of these vGPCRs, their role in the viral life cycle, and their contribution in different diseases. Because of their prominent role, vGPCRs have emerged as promising drug targets, and the potential of vGPCR-targeting therapeutics is being explored. Overall, these vGPCRs can be considered as attractive targets moving forward in the development of antiviral, cancer, and/or cardiovascular disease treatments. SIGNIFICANCE STATEMENT: In the last decade, herpesvirus-encoded G protein-coupled receptors (GPCRs) have emerged as interesting drug targets with the growing understanding of their critical role in the viral life cycle and in different disease settings. This review presents the pharmacological properties of these viral receptors, their role in the viral life cycle and different diseases, and the emergence of therapeutics targeting viral GPCRs.

From recipe to research: introducing undergraduate students to the nature of science using a hybrid practical course centred on drug discovery for neglected diseases.

A hybrid undergraduate practical course involving synthetic medicinal chemistry on neglected diseases bridges the gap between skills, techniques and scientific research, and exposes students to the nature of science.

The CXCL12/CXCR4/ACKR3 Axis in the Tumor Microenvironment: Signaling, Crosstalk, and Therapeutic Targeting.

Elevated expression of the chemokine receptors CXCR4 and ACKR3 and of their cognate ligand CXCL12 is detected in a wide range of tumors and the tumor microenvironment (TME). Yet, the molecular mechanisms by which the CXCL12/CXCR4/ACKR3 axis contributes to the pathogenesis are complex and not fully understood. To dissect the role of this axis in cancer, we discuss its ability to impinge on canonical and less conventional signaling networks in different cancer cell types; its bidirectional crosstalk, notably with receptor tyrosine kinase (RTK) and other factors present in the TME; and the infiltration of immune cells that supporttumor progression. We discuss current and emerging avenues that target the CXCL12/CXCR4/ACKR3 axis. Coordinately targeting both RTKs and CXCR4/ACKR3 and/or CXCL12 is an attractive approach to consider in multitargeted cancer therapies. In addition, inhibiting infiltrating immune cells or reactivating the immune system along with modulating the CXCL12/CXCR4/ACKR3 axis in the TME has therapeutic promise.

Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages.

Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.

Human Cytomegalovirus-Encoded G Protein-Coupled Receptor UL33 Facilitates Virus Dissemination via the Extracellular and Cell-to-Cell Route.

Human cytomegalovirus (HCMV) encodes four G protein-coupled receptor (GPCR) homologs. Three of these receptors, UL78, US27 and US28, are known for their roles in HCMV dissemination and latency. Despite importance of its rodent orthologs for viral replication and pathogenesis, such a function is not reported for the HCMV-encoded GPCR UL33. Using the clinical HCMV strain Merlin, we show that UL33 facilitates both cell-associated and cell-free virus transmission. A UL33-deficient virus derivative revealed retarded virus spread, formation of less and smaller plaques, and reduced extracellular progeny during multi-cycle growth analysis in fibroblast cultures compared to parental virus. The growth of UL33-revertant, US28-deficient, and US28-revertant viruses were similar to parental virus under multistep growth conditions. UL33- and US28-deficient Merlin viruses impaired cell-associated virus spread to a similar degree. Thus, the growth defect displayed by the UL33-deficient virus but not the US28-deficient virus reflects UL33's contribution to extracellular transmission. In conclusion, UL33 facilitates cell-associated and cell-free spread of the clinical HCMV strain Merlin in fibroblast cultures.

Viral G protein-coupled receptors as modulators of cancer hallmarks.

Herpesviruses encode transmembrane G protein-coupled receptors (GPCRs), which share structural homology to human chemokine receptors. These viral GPCRs include KSHV-encoded ORF74, EBV-encoded BILF1, and HCMV-encoded US28, UL33, UL78 and US27. Viral GPCRs hijack various signaling pathways and cellular networks, including pathways involved in the so-called cancer hallmarks as defined by Hanahan and Weinberg. These hallmarks describe cellular characteristics crucial for transformation and tumor progression. The cancer hallmarks involve growth factor-independent proliferation, angiogenesis, avoidance of apoptosis, invasion and metastasis, metabolic reprogramming, genetic instability and immune evasion amongst others. The role of beta herpesviruses modulating these cancer hallmarks is clearly highlighted by the proliferative and pro-angiogenic phenotype associated with KSHV infection which is largely ascribed to the ORF74-mediated modulation of signaling networks in host cells. For HCMV and Epstein-Bar encoded GPCRs, oncomodulatory effects have been described which contribute to the cancer hallmarks, thereby enhancing oncogenic development. In this review, we describe the main signaling pathways controlling the hallmarks of cancer which are affected by the betaherpesvirus encoded GPCRs. Most prominent among these involve the JAK-STAT, PI(3)K-AKT, NFkB and MAPK signaling nodes. These insights are important to effectively target these viral GPCRs and their signaling networks in betaherpesvirus-associated malignancies.

Lead Optimization of Phthalazinone Phosphodiesterase Inhibitors as Novel Antitrypanosomal Compounds.

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.

Discovery of Diaryl Ether Substituted Tetrahydrophthalazinones as TbrPDEB1 Inhibitors Following Structure-Based Virtual Screening.

Several members of the 3',5'-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 µM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.