The chemokine CXCR4 receptor and epidermal growth factor receptor (EGFR) are cell surface receptors that are overexpressed in numerous types of cancer. In this study, we have investigated the formation of CXCR4-EGFR heteromers using bioluminescence resonance energy transfer (BRET) and nanobody-based proximity ligation assays (PLAs). The present study demonstrated that EGFR and CXCR4 can form oligomeric complexes that were capable of coupling to PLCγ, Gi proteins and β-arrestin-2. The presence of these oligomeric complexes was detected by PLA in native HeLa cells at endogenous levels of expression using receptor-selective nanobody-oligonucleotide conjugates. Furthermore, the individual receptor components of the oligomer underwent conformation changes in response to EGF and CXCL12, which in the case of Gi-signalling led to altered responses to combinations of CXCL12 and EGF. This may have clinical implications in those cancers where both EGFR and CXCR4 are overexpressed.

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remains elusive. Using a nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and prosurvival signaling via changes in the phosphoproteome, indicating the existence of constitutive CXCR4 oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B cells to venetoclax is of clinical relevance. Taken together, we established a noncanonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that their selective targeting has clinical potential.

CXCR4 is an important therapeutic target for hematopoietic stem cell mobilization, which enhances the success of autologous stem cell transplantation for treating blood cancers such as lymphomas and myeloma. As CXCR4 has been shown to be involved in various inflammatory diseases, cancer progression, and cell entry by the human immunodeficiency virus, understanding the molecular mechanism of CXCR4 inhibitors has potential implications in a wide area of diseases. Here, we present an exploratory study which involves the molecular pharmacological characterization of TG-0 054 (burixafor, GPC-10 0), a clinical candidate for hematopoietic stem cell mobilization. TG-0 054 inhibited CXCL12 binding at CXCR4, and antagonized both Gai and b-arrestin2 recruitment as well as the downstream Gai-attenuation of cAMP signaling pathway, with pIC50 of 7.7, 8.0, and 7.9, respectively. Compared with the clinically used antagonist AMD310 0 and the prototypical inverse agonist Isothiourea-1t (IT1t), TG-0 054 displayed a unique pharmacological profile. Like IT1t, TG-0 054 inhibited the constitutive Gai signaling of CXCR4. However, in contrast to IT1t and other reported inverse agonists, TG-0 054 was not able to induce monomerization of CXCR4 oligomeric complexes. Considering the unique properties of TG-0054 on CXCR4, TG-0 054 is an interesting tool compound for studying the relevance of inverse agonism as well as CXCR4 monomerization in various pathologies. Statement: CXCR4-targeted therapeutics hold important potential for the treatment of blood cancers. TG-0 054 has inverse agonistic properties and is a non-CXCR4-monomerizing small molecule antagonist, unlike other well studied CXCR4 small molecule antagonists. (c) 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

CXCR4 is an important therapeutic target for hematopoietic stem cell mobilization, which enhances the success of autologous stem cell transplantation for treating blood cancers such as lymphomas and myeloma. As CXCR4 has been shown to be involved in various inflammatory diseases, cancer progression, and cell entry by the human immunodeficiency virus, understanding the molecular mechanism of CXCR4 inhibitors has potential implications in a wide area of diseases. Here, we present an exploratory study which involves the molecular pharmacological characterization of TG-0054 (burixafor, GPC-100), a clinical candidate for hematopoietic stem cell mobilization. TG-0054 inhibited CXCL12 binding at CXCR4, and antagonized both Gαi and β-arrestin2 recruitment as well as the downstream Gαi-attenuation of cAMP signaling pathway, with pIC50 of 7.7, 8.0, and 7.9, respectively. Compared with the clinically used antagonist AMD3100 and the prototypical inverse agonist Isothiourea-1t (IT1t), TG-0054 displayed a unique pharmacological profile. Like IT1t, TG-0054 inhibited the constitutive Gαi signaling of CXCR4. However, in contrast to IT1t and other reported inverse agonists, TG-0054 was not able to induce monomerization of CXCR4 oligomeric complexes. Considering the unique properties of TG-0054 on CXCR4, TG-0054 is an interesting tool compound for studying the relevance of inverse agonism as well as CXCR4 monomerization in various pathologies. SIGNIFICANCE STATEMENT: CXCR4-targeted therapeutics hold important potential for the treatment of blood cancers. TG-0054 has inverse agonistic properties and is a non-CXCR4-monomerizing small molecule antagonist, unlike other well studied CXCR4 small molecule antagonists.

The chemokine receptor CXCR4 is involved in the development and migration of stem and immune cells but is also implicated in tumor progression and metastasis for a variety of cancers. Antagonizing ligand (CXCL12)-induced CXCR4 signaling is, therefore, of therapeutic interest. Currently, there are two small-molecule CXCR4 antagonists on the market for the mobilization of hematopoietic stem cells. Other molecules with improved potencies and safety profiles are being developed for different indications, including cancer. Moreover, multiple antagonistic nanobodies targeting CXCR4 displayed similar or better potencies as compared to the CXCR4-targeting molecule AMD3100 (Plerixafor), which was further enhanced through avid binding of bivalent derivatives. In this study, we aimed to compare the affinities of various multivalent nanobody formats which might be differently impacted by avidity. By fusion to a flexible GS-linker, Fc-region of human IgG1, different C4bp/CLR multimerization domains, or via site-directed conjugation to a trivalent linker scaffold, we generated different types of multivalent nanobodies with varying valencies ranging from bivalent to decavalent. Of these, C-terminal fusion, especially to human Fc, was most advantageous with a 2-log-fold and 3-log-fold increased potency in inhibiting CXCL12-mediated Gαi- or β-arrestin recruitment, respectively. Overall, we describe strategies for generating multivalent and high-potency CXCR4 antagonistic nanobodies able to induce receptor clustering and conclude that fusion to an Fc-tail results in the highest avidity effect irrespective of the hinge linker.

Stimulation of atypical chemokine receptor 3 (ACKR3) by chemokines does not activate G proteins but recruits arrestin. It is a chemokine scavenger that indirectly influences responses by restricting the availability of CXCL12, an agonist shared with the canonical receptor CXCR4. ACKR3 is upregulated in numerous disorders. Due to limited insights in chemokine-activated ACKR3 signaling, it is unclear how ACKR3 contributes to pathological phenotypes. One explanation may be that constitutive activity of ACKR3 drives non-canonical signaling through a basal receptor state. Here we characterize the constitutive responses of ACKR3 using inverse agonistic nanobodies to suppress its basal activity. These tools promote an inactive receptor conformation which decreased arrestin engagement and inhibited constitutive internalization. Basal non-chemotactic, cancer cell motility was also suppressed, suggesting a role for ACKR3 in this process. The basal receptor activity in pathophysiology may provide an alternate therapeutic approach for targeting ACKR3.

Dysregulated intercellular communication is a key feature driving cancer progression. Recently, extracellular vesicles (EVs) have added a new channel to this dense communication network. Despite solid evidence that EVs are central mediators of dysregulated signaling in onco-pathological settings, this has yet to be translated into clinically actionable strategies. The heterogeneity of EV cargo molecules, plasticity of biogenesis routes, and large overlap with their role in physiological communication, complicate a potential targeting strategy. However, recent work has linked EV biology to perhaps the "most druggable" proteins - G protein-coupled receptors (GPCRs). GPCR targeting accounts for ~60% of drugs in development and more than a third of all currently approved drugs, spanning almost all areas of medicine. Although several GPCRs have been linked to cancer initiation and progression, relatively few agents have made it into oncological regimes, suggesting that their potential is underexploited. Herein, we examine the molecular mechanisms linking GPCRs to EV communication in cancer settings. We propose that GPCRs hold potential in the search for EV-targeting in oncology.

The chemokine receptor CXCR4 is overexpressed in many cancers and contributes to pathogenesis, disease progression, and resistance to therapies. CXCR4 is known to form oligomers, but the potential functional relevance in malignancies remain elusive. Using a newly established nanobody-based BRET method, we demonstrate that oligomerization of endogenous CXCR4 on lymphoid cancer cell lines correlates with enhanced expression levels. Specific disruption of CXCR4 oligomers reduced basal cell migration and pro-survival signaling via changes in the phosphoproteome, indicating the existence of basal CXCR4-oligomer-mediated signaling. Oligomer disruption also inhibited growth of primary CLL 3D spheroids and sensitized primary malignant cells to clinically used Bcl-2 inhibitor venetoclax. Given its limited efficacy in some patients and the ability to develop resistance, sensitizing malignant B-cells to venetoclax is of clinical relevance. Taken together, we established a new, non-canonical and critical role for CXCR4 oligomers in lymphoid neoplasms and demonstrated that selective targeting thereof has clinical potential. ### Competing Interest Statement SMA and RH are employed by QVQ, a CRO offering Nb services and reagents. A provisional patent application has been filed on the research described in this manuscript (P135924EP00).

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 (S1P 1 )], 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.

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 biolumines-cence 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 experi-ments. 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 thera-peutically relevant binding sites (i.e., extracellular and intracellular).

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.

Human African Trypanosomiasis (HAT), caused by Trypanosomabrucei, is one of the neglected tropical diseaseswith a continuing need for new medication. We here describe the discoveryof 5-phenylpyrazolopyrimidinone analogs as a novel series of phenotypicantitrypanosomal 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 humanMRC-5 lung fibroblasts. Showing good physicochemical properties, lowtoxicity potential, acceptable metabolic stability, and other pharmacokineticfeatures, 30 was further evaluated in an acute mousemodel of T. b. brucei infection. Afteroral dosing at 50 mg/kg twice per day for five consecutive days, allinfected mice were cured. Given its good drug-like properties andhigh in vivo antitrypanosomal potential, the 5-phenylpyrazolopyrimidinoneanalog 30 represents a promising lead for future drugdevelopment to treat HAT.

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.

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.

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.

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.

•A hybrid undergraduate laboratory course combines laboratory skills and exposure to research.•Students experience the nature of science first-hand by synthesising hit derivatives.•A neglected disease drug discovery project is well suited as research topic.•A daily interactive session fosters scientific discussion amongst students.

Incorporation of increased flexibility and adaptivity in courses can support freshmen with taking responsibility for their learning process. This is especially beneficial in the case of chemistry courses given their typically abstract nature and associated need for engaging in practice and higher order thinking. Blended learning can be used to achieve course flexibility and adaptivity, and increased student engagement. Adopting blended learning requires teachers to reconsider and redesign their course setup. The willingness for this will largely be dictated by perceived practicality, however. To this end, our aim is to develop practical redesign guidelines for flexible blended learning. Such guidelines need to be instrumental, congruent, and of 'low cost' for a teacher. The resulting four guidelines were fine-tuned in two phases over 5 years using two introductory chemistry courses and incorporated various blended learning elements, such as slide-cast lectures, live lectures, formative in-cast multiple choice questions, tutorials, formative electronic tutorial questions, and student voting on the course schedule. The outcome of our multiyear endeavor was positive and sustainable by all metrics used, including online engagement by students, learning outcomes, and student evaluations. The teachers in phase II largely embraced the guidelines emerging from phase I, and in their course experienced them predominantly as practical while limitations were also identified. We hope that our guidelines and experiences can provide fellow teachers with a blueprint for practical incorporation of flexibility and adaptivity using blended learning in their chemistry courses.

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.