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Academic skills are essential in most future professions for students, yet frequently receive sporadic or ineffective attention throughout higher education. Therefore, we set out to implement the ALACT reflection model in a second-year academic skills course within the bachelor’s program in Pharmaceutical Sciences. The model was applied to both presentation and teamwork skills which are practiced in a relevant academic context. As such, the aim was to improve the timeliness and level of insight students have into their strengths, growth, and points for improvement. The ALACT model is a spiral approach to reflection where students begin with (1) a concrete experience, (2) observing and reflecting, and then (3) forming abstract concepts and generalizations about the experience. These are used (4) to create an action plan which is then (5) enacted. The last step forms the new experience from which the cycle repeats. Previous research suggests that the conscious and consistent implementation of the ALACT model can provide a basis for meaningful self-evaluation and increased competency and confidence. Therefore, we redesigned the month-long course to ensure the five steps were well-integrated and that students would complete the spiral at least twice for each skill. Results from lecturer interviews and a student questionnaire indicate that the intervention leads to increased awareness of existing competencies and steps for improvement, and a very positive view of reflection and its role in skills development. A review of the practical considerations for implementing the ALACT model in academic skills courses within science education is also provided.

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.

In recent years, the technical possibilities of educational technologies regarding online peer feedback have developed rapidly. However, the impact of online peer feedback activities compared to traditional offline variants has not specifically been meta-analyzed. Therefore, the aim of the current meta-analysis is to do an in-depth comparison between online versus offline peer feedback approaches. An earlier and broader meta-analysis focusing on technology-facilitated peer feedback in general, was used as a starting point. We synthesized 12 comparisons between online and offline peer feedback in higher education, from 10 different studies. Moreover, we reviewed student perceptions of online peer feedback when these were included in the studies. The results show that online peer feedback is more effective than offline peer feedback, with an effect size of 0.33. Moreover, online peer feedback is more effective when the outcome measure is competence rather than self-efficacy for skills. In addition, students are mostly positive towards online peer feedback but also list several downsides. Finally, implications for online peer feedback in teaching practice are discussed and leads are identified for further research on this topic.

Chemokine receptors belong to the class of G protein-coupled receptors and are important in the host defense against infections and inflammation. However, aberrant chemokine signaling is linked to different disorders such as cancer, central nervous system and immune disorders, and viral infections [Scholten DJ et al. (2012) Br J Pharmacol 165(6):1617–1643]. Modulating the chemokine receptor function provides new ways of targeting specific diseases. Therefore, discovery and development of drugs targeting chemokine receptors have received considerable attention from the pharmaceutical industry in the past decade. Along with that, the determination of bioactivities of individual metabolites derived from lead compounds towards chemokine receptors is crucial for drug selectivity, pharmacodynamics, and potential toxicity issues. Therefore, advanced analytical methodologies are in high demand. This study is aimed at the optimization of a new analytical method for metabolic profiling with parallel bioaffinity assessment of CXCR3 ligands of the azaquinazolinone and piperazinyl-piperidine class and their metabolites. The method is based on mass spectrometric (MS) identification after liquid chromatographic (LC) separation of metabolic mixtures. The bioaffinity assessment is performed “at-line” via high-resolution nanofractionation onto 96-well plates allowing direct integration of radioligand binding assays. This new method enables identification of metabolites from lead compounds with associated estimation of their individual bioaffinity. Moreover, the identification of the metabolite structures via accurate mass measurements and MS2 allows the identification of liable metabolic “hotspots” for further lead optimization. The efficient combination of chemokine receptor ligand binding assays with analytical techniques, involving nanofractionation as linking technology, allows implementation of comprehensive metabolic profiling in an early phase of the drug discovery process. Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8867-z) contains supplementary material, which is available to authorized users.

This study describes an analytical method for bioaffinity and selectivity assessment of CXCR2 antagonists and their metabolites. The method is based on liquid chromatographic separation (LC) of metabolic mixtures followed by parallel mass spectrometry (MS) identification and bioaffinity determination. The bioaffinity is assessed using radioligand binding assays in 96-well plates after at-line nanofractionation. The described method was optimized for chemokines and low-molecular weight CXCR2 ligands. The limits of detection (LODs; injected amounts) for MK-7123, a high affinity binder to both CXCR1 and CXCR2 receptors belonging to the diaminocyclobutendione chemical class, were 40pmol in CXCR1 binding and 8pmol in CXCR2 binding. For CXCL8, the LOD was 5pmol in both binding assays. A control compound was always taken along with each bioassay plate as triplicate dose-response curve. For MK-7123, the calculated IC50 values were 314±59nM (CXCR1 binding) and 38±11nM (CXCR2 binding). For CXCL8, the IC50 values were 6.9±1.4nM (CXCR1 binding) and 2.7±1.3nM (CXCR2 binding). After optimization, the method was applied to the analysis of metabolic mixtures of eight LMW CXCR2 antagonists generated by incubation with pig liver microsomes. Moreover, metabolic profiling of the MK-7123 compound was described using the developed method. Three bioactive metabolites were found, two of which were (partially) identified. This method is suitable for bioaffinity and selectivity assessment of mixtures targeting the CXCR2. In contrary to conventional LC-MS based metabolic profiling studies done at the early lead discovery stage, additional qualitative bioactivity information of drug metabolites is obtained with the method described.

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. Crown Copyright©2012 Published by Elsevier Ltd. All rights reserved.

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.

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure-activity relationship and structure-function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of ortho-substituents play an important role in determining agonist efficacies. Compounds 38 (VUF11222) and 39 (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling. © 2012 American Chemical Society.

Targeting of chemokine receptors by small molecules has been widely pursued. This review highlights recent illustrative disclosures of clinical relevance that could further shape our appreciation, and add to our understanding, of the therapeutic value of chemokine receptor targeting. Disclosures include new structures, announcements of new trials, or results of conducted trials (including setbacks). This review shows how most of the discussed disclosures seem to be concentrated on selected receptors, for example, CCR1, CCR2, CCR5, CCR9, CXCR2 and CXCR4, with a wide variety of associated ligand chemotypes and diseases. With two approved antagonist drugs and several in Phase III trials, as well as new antagonist chemotypes entering the pipeline, the chemokine receptor field proves dynamic and upcoming results will further fuel the field.© 2012 Elsevier Ltd. All rights reserved.

Chemokine receptor CXCR3 has attracted much attention, as it is thought to be associated with a wide range of immune-related diseases. As such, several small molecules with different chemical structures targeting CXCR3 have been discovered. Despite limited clinical success so far, these compounds serve as interesting tools for investigating receptor activation and antagonism. Accumulating evidence suggests that many of these compounds are allosteric modulators for CXCR3. One feature of allosteric ligands is that the magnitude of the mediated allosteric effect is dependent on the orthosteric probe that is used. Consequently, there is a risk for incorrect assessment of affinity for allosteric modulators with orthosteric radioligands, which has so far been the most applied approach for chemokine receptors. Therefore, we aimed to use a small-molecule allosteric ligand from the piperazinyl-piperidine class, also known as VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide]. VUF11211 acts as an inverse agonist at a constitutively active mutant of CXCR3. Radiolabeling of VUF11211 gave [(3)H]VUF11211, which in radioligand binding studies shows high affinity for CXCR3 (Kd = 0.65 nM) and reasonably fast association (kon= 0.03 minute(-1)nM(-1)) and dissociation kinetics (koff = 0.02 minute(-1)). The application of the [(3)H]VUF11211 to assess CXCR3 pharmacology was validated with diverse classes of CXCR3 compounds, including both antagonists and agonists, as well as VUF11211 analogs. Interestingly, VUF11211 seems to bind to a different population of CXCR3 conformations compared with the CXCR3 agonists CXC chemokine ligand 11 (CXCL11), VUF11418 [1-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-N-((2'-iodobiphenyl-4-yl)methyl)-N,N-dimethylmethanaminium Iodide], and VUF10661 [N-(6-amino-1-(2,2-diphenylethylamino)-1-oxohexan-2-yl)-2-(4-oxo-4-phenylbutanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide]. These findings, taken together, indicate that this allosteric inverse agonist radioligand for CXCR3 may facilitate the discovery, characterization, and optimization of allosteric modulators for the chemokine receptor CXCR3.

CXCR3 is a CXC chemokine receptor that, together with its three major ligands, CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC), is involved in inflammatory responses, mediated mainly by T cells. In several immune-related diseases, including chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), rheumatoid arthritis, multiple sclerosis, and atherosclerosis, CXCR3 and/or its ligands are found to be overexpressed, potentially indicating a role for this receptor in these diseases. Animal models have confirmed the therapeutic potential of targeting CXCR3 in the treatment of such diseases. Several peptidic, peptidomimetic, and small non-peptidomimetic CXCR3 ligands have been disclosed in the past 10 years. These ligands have served as chemical tools for the investigation of CXCR3 activation, blocking, and signaling, and some of these ligand series have been developed as potential therapeutic agents against inflammation. Computational modeling studies, facilitated by the recent developments in GPCR structural biology, together with mutagenesis and pharmacological studies, have aided in understanding how these ligands interact with CXCR3.

CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have beenmade to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3- ethylpiperazin-1-yl)-N-ethylnicotinamide] (piperazinyl-piperidine) with a rigid elongated structure containing two basic groups andNBI-74330 [(R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[ 2,3-d]pyrimidin-2-yl) ethyl)-2-(4-fluoro-3-(trifluoromethyl)phenyl)- N-(pyridin-3-ylmethyl)acetamide] (8-azaquinazolinone) without any basic group. Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico-guided studies fits well with the already published structure-activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.

Recent reports of G protein-coupled receptor (GPCR) X-ray structures, including the structure of CXCR4 chemokine receptor, have advanced the aim of elucidating with chemical precision GPCR ligand binding, activation and interactions. Structure-based drug design is also becoming increasingly important as a tool to find new GPCR-targeted drugs. As new structural information becomes available, complementary experimental strategies are required to address questions related to receptor dynamics and organization in membranes. To this end, the introduction of unnatural amino acids (UAAs) into GPCR ligands and expressed receptors has opened up new opportunities to study GPCRs in native systems. Because UAAs can contain a wide range of chemically diverse functional groups, genetic incorporation of UAAs into proteins provides an important new tool of chemical biology to the study of GPCRs, including chemokine receptors. For example, UAAs enable the incorporation of spectroscopic probes and functional groups for selective chemical modification that facilitates studies of (real-time) protein dynamics and interactions. In this review, we highlight recent methods developed to use UAAs to study chemokine receptors and we describe examples of their use to probe chemokine receptor function.

The chemokine receptor CXCR7 is an atypical G protein-coupled receptor as it preferentially signals through the β-arrestin pathway rather than through G proteins. CXCR7 is thought to be of importance in cancer and the development of CXCR7-targeting ligands is of huge importance to further elucidate the pharmacology and the therapeutic potential of CXCR7. In the present study, we synthesized 24 derivatives based on a compound scaffold patented by Chemocentryx and obtained CXCR7 ligands with pK(i) values ranging from 5.3 to 8.1. SAR studies were supported by computational 3D Fingerprint studies, revealing several important affinity descriptors. Two key compounds (29 and 30, VUF11207 and VUF11403) were found to be high-potency ligands that induce recruitment of β-arrestin2 and subsequent internalization of CXCR7, making them important tool compounds in future CXCR7 research.

The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gα(i) class of heterotrimeric G-proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G-protein and non-G-protein (β-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gα(i) inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G-protein signaling, but not β-arrestin2 recruitment.

The chemokine receptor CXCR3 is involved in various inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, and allograft rejection in transplantation patients. The CXCR3 ligands CXCL9, CXCL10, and CXCL11 are expressed at sites of inflammation, and they attract CXCR3-bearing lymphocytes, thus contributing to the inflammatory process. In this study, we characterize five nonpeptidergic compounds of different chemical classes that block the action of CXCL10 and CXCL11 at the human CXCR3, i.e., the 3H-pyrido[2,3-d]pyrimidin-4-one derivatives N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4- dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-fluoro-3- trifluoromethylphenyl)-acetamide (VUF10472/NBI-74330) and N-1R-[3-(4-ethoxy- phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3- ylmethyl-2-(4-trifluoromethoxy-phenyl)-acetamide (VUF10085/AMG-487), the 3H-quinazolin-4-one decanoic acid {1-[3-(4-cyano-phenyl)-4-oxo-3,4-dihydro- quinazolin-2-yl]-ethyl}-(2-dimethylamino-ethyl)-amide (VUF5834), the imidazolium compound 1,3-bis-[2-(3,4-dichloro-phenyl)-2-oxo-ethyl]-3H-imidazol-1-ium bromide (VUF10132), and the quaternary ammonium anilide N,N-dimethyl-N-[4-[[[2- (4-methylphenyl)-6,7-dihydro-5H-benzocyclohepten-8-yl]-carbonyl]amino]benzyl] tetrahydro-2H-pyran-4-aminium chloride (TAK-779). To understand the action of these CXCR3 antagonists in various animal models of disease, the compounds were also tested at rat and mouse CXCR3, as well as at CXCR3 from rhesus macaque, which was cloned and characterized for the first time in this study. Except for TAK-779, all compounds show slightly lower affinity for rodent CXCR3 than for primate CXCR3. In addition, we have characterized the molecular mechanism of action of the various antagonists at the human CXCR3 receptor. All tested compounds act as noncompetitive antagonists at CXCR3. Moreover, this noncompetitive behavior is accompanied by inverse agonistic properties of all five compounds as determined on an identified constitutively active mutant of CXCR3, CXCR3 N3.35A. It is interesting to note that all compounds except TAK-779 act as full inverse agonists at CXCR3 N3.35A. TAK-779 shows weak partial inverse agonism at CXCR3 N3.35A, and it probably has a different mode of interaction with CXCR3 than the other two classes of small-molecule inverse agonists. Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics.

The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gα(i)-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.