Kinetic profiling and functional characterization of 8-phenylxanthine derivatives as A2B adenosine receptor antagonists.

A2B adenosine receptor (A2BAR) antagonists have therapeutic potential in inflammation-related diseases such as asthma, chronic obstructive pulmonary disease and cancer. However, no drug is currently clinically approved, creating a demand for research on novel antagonists. Over the last decade, the study of target binding kinetics, along with affinity and potency, has been proven valuable in early drug discovery stages, as it is associated with improved in vivo drug efficacy and safety. In this study, we report the synthesis and biological evaluation of a series of xanthine derivatives as A2BAR antagonists, including an isothiocyanate derivative designed to bind covalently to the receptor. All 28 final compounds were assessed in radioligand binding experiments, to evaluate their affinity and for those qualifying, kinetic binding parameters. Both structure-affinity and structure-kinetic relationships were derived, providing a clear relationship between affinity and dissociation rate constants. Two structurally similar compounds, 17 and 18, were further evaluated in a label-free assay due to their divergent kinetic profiles. An extended cellular response was associated with long A2BAR residence times. This link between a ligand's A2BAR residence time and its functional effect highlights the importance of binding kinetics as a selection parameter in the early stages of drug discovery.

What happens under the flag of direct observation, and how that matters: A qualitative study in general practice residency.

INTRODUCTION: In competency-based medical education, direct observation (DO) of residents' skills is scarce, notwithstanding its undisputed importance for credible feedback and assessment. A growing body of research is investigating this discrepancy. Strikingly, in this research, DO as a concrete educational activity tends to remain vague. In this study, we concretised DO of technical skills in postgraduate longitudinal training relationships. METHODS: Informed by constructivist grounded theory, we performed a focus group study among general practice residents. We asked residents about their experiences with different manifestations of DO of technical skills. A framework describing different DO patterns with their varied impact on learning and the training relationship was constructed and refined until theoretical sufficiency was reached. RESULTS: The dominant DO pattern was ad hoc, one-way DO. Importantly, in this pattern, various unpredictable, and sometimes unwanted, scenarios could occur. Residents hesitated to discuss unwanted scenarios with their supervisors, sometimes instead refraining from future requests for DO or even for help. Planned bi-directional DO sessions, though seldom practiced, contributed much to collaborative learning in a psychologically safe training relationship. DISCUSSION AND CONCLUSION: Patterns matter in DO. Residents and supervisors should be made aware of this and educated in maintaining an open dialogue on how to use DO for the benefit of learning and the training relationship.

The daily practice of (suspected) coeliac disease management by general practitioners: A qualitative approach.

BACKGROUND: General practitioners (GPs) play a crucial role in diagnosing coeliac disease (CD). However, data on GP management of (suspected) CD patients is sparse. OBJECTIVES: To provide insights into the daily practice of diagnosis, treatment, and follow-up of CD by GPs. METHODS: A qualitative study using topic list-based semi-structured in-depth interviews with Dutch GPs with more than five years' experience carried out between January and March 2017. GPs were purposively sampled. The number of GPs interviewed depended on when data saturation was reached. We applied content analysis to the semi-structured interviews. RESULTS: Seven GPs were interviewed, five of whom were female. Analysis of the interviews resulted in three main themes: 'awareness,' 'diagnostics' and 'management.' Vague gastrointestinal symptoms and diarrhoea were often mentioned as a possible presentation of CD. Antibodies were used in CD diagnosis, although some GPs would start a gluten-free diet as a first diagnostic tool. Some GPs diagnosed CD only based on positive antibodies without referring to secondary care or duodenal biopsy analysis. GPs mentioned no role for primary care physicians in the follow-up of CD and noted the important role of dieticians in CD management. CONCLUSION: The different views of GPs on how to diagnose and monitor CD could be a basis for further research to improve CD detection rate and CD care.

Raising enthusiasm for the medical care of elderly patients: a concept mapping study to find elements for an elderly friendly medical curriculum.

BACKGROUND: To deliver high quality of care for the growing population of older patients more geriatricians are needed. However, the interest of medical students for a career in geriatrics is lagging behind due to a lack of exposure, the nature of the work, and the low status and financial rewards. So far, only isolated interventions aimed at enhancing interest and/or attitudes with regard to geriatrics have been studied, pointing to the need for a broader-based strategy. The goal of this research is to find elements for a curriculum framework that can raise medical students' enthusiasm for the medical care of elderly patients. METHODS: We used the concept mapping method developed by Trochim. This computer-assisted procedure consists of five steps: brainstorming, prioritizing and clustering with several experts, followed by processing by the computer and analysis. RESULTS: The views that were generated were grouped into the following clusters: a patient-centered medical curriculum, a curriculum representative of patient population, geriatrics presented as intellectually challenging and emotionally appealing, senior-friendly role models, a clear professional perspective. The results are presented in the form of a graphic chart. CONCLUSIONS: An agenda to discuss the necessary actions for drastic curricular reforms in medical schools is set. This may give some guidance to this urgent, but highly complicated issue how to make medical student enthusiastic for the medical care for elderly patients.

Patterns of direct observation and their impact during residency: general practice supervisors' views.

CONTEXT: Direct observation (DO) of residents' performance, despite the importance that is ascribed to it, does not readily fit in with the practice of postgraduate medical education (PGME); it is infrequent and the quality of observation may be poor in spite of ongoing efforts towards improvement. In recent literature, DO is mostly portrayed as a means to gather information on the performance of residents for purposes of feedback and assessment. The role of DO in PGME is likely to be more complex and poorly understood in the era of outcome-based education. By exploring the possible complexity of DO in workplace learning, our research aims to contribute to a better use of DO in the practice of PGME. METHODS: Constructivist grounded theory informed our data collection and analysis. Data collection involved focus group sessions with supervisors in Dutch general practice who were invited to discuss the manifestations, meanings and effects of DO of technical skills. Theoretical sufficiency was achieved after four focus groups, with a total of 28 participants being included. RESULTS: We found four patterns of DO of technical skills: initial planned DO sessions; resident-initiated ad hoc DO; supervisor-initiated ad hoc DO, and continued planned DO sessions. Different patterns of DO related to varying meanings, such as checking or trusting, and effects, such as learning a new skill or experiencing emotional discomfort, all of them concerning the training relationship, patient safety or residents' learning. CONCLUSIONS: Direct observation, to supervisors, means much more than gathering information for purposes of feedback and assessment. Planned DO sessions are an important routine during the initiation phase of a training relationship. Continued planned bidirectional DO sessions, although infrequently practised, potentially combine most benefits with least side-effects of DO. Ad hoc DO, although much relied upon, is often hampered by internal tensions in supervisors, residents or both.

[A career in elderly care medicine; an option for today's medical student? : Medical students' interest in elderly care medicine]. / Een carrière als specialist ouderengeneeskunde; iets voor de huidige geneeskundestudent? : Belangstelling geneeskundestudenten voor ouderengeneeskunde.

BACKGROUND: Due to the rise of older patients with multi-morbidity, we need more elderly care physicians. However, not all available training slots for the elderly care medicine specialty have been fully utilized in recent years. To assess medical student interest in this specialty as well as potential causes for this interest we explored the interest of medical students in the profession of elderly care physician, as well their perception of this profession, both in the 'old curriculum' and in a 'new curriculum', where the new curriculum had a mandatory elderly care medicine clerkship and more competency-related learning. METHOD: At VUmc 120 final year medical students were asked to complete a questionnaire in 2014 about professional preferences and professional characteristics. The same questionnaire had been presented five years earlier, in 2009, to 150 medical students at the end of their final year. RESULTS: The response rates were 100% and 85% respectively. Of the students in the new curriculum 16,7% considered a career in elderly care medicine. This percentage was 9,4% for students in the old curriculum (p = 0,087). The characteristics of the profession that appealed most to the students, but were not considered applicable to elderly care medicine were: diagnostics skills, acute complaints, visible results. The professional characteristics that students found to be very much applicable to this specialty, but less attractive for their future profession were: psychosocial, chronic and terminal conditions. DISCUSSION: We observe a trend that students in the new curriculum are more interested in the profession of elderly care physician, even though this interest remains limited. We recommend that the basic medical training, both in the bachelor phase and in a mandatory elderly care medicine clerkship, focus more on demonstrating that the characteristics students find appealing in the medical profession are indeed present in this speciality. Also, the basic training should concentrate more on guidance and treatment of patients with chronic and terminal conditions.

A two-state model for the kinetics of competitive radioligand binding.

BACKGROUND AND PURPOSE: Ligand-receptor binding kinetics is receiving increasing attention in the drug research community. The Motulsky and Mahan model, a one-state model, offers a method for measuring the binding kinetics of an unlabelled ligand, with the assumption that the labelled ligand has no preference while binding to distinct states or conformations of a drug target. As such, the one-state model is not applicable if the radioligand displays biphasic binding kinetics to the receptor. EXPERIMENTAL APPROACH: We extended the Motulsky and Mahan model to a two-state model, in which the kinetics of the unlabelled competitor binding to different receptor states (R1 and R2 ) can be measured. With this extended model, we determined the binding kinetics of unlabelled N-5'-ethylcarboxamidoadenosine (NECA), a representative agonist for the adenosine A1 receptor. Subsequently, an application of the model was exemplified by measuring the binding kinetics of other A1 receptor ligands. In addition, limitations of the model were investigated as well. KEY RESULTS: The kinetic rate constants of unlabelled NECA were comparable with the results of kinetic radioligand binding assays in which [3 H]-NECA was used. The model was further validated by good correlation between simulated results and the experimental data. CONCLUSION: The two-state model is sufficient to analyse the binding kinetics of an unlabelled ligand, when a radioligand shows biphasic association characteristics. We expect this two-state model to have general applicability for other targets as well.

Optimization of the metabolic stability of a fluorinated cannabinoid receptor subtype 2 (CB2) ligand designed for PET studies.

The central CB2 receptor represents a promising target for the treatment of neuroinflammatory diseases as CB2 activation mediates anti-inflammatory effects. Recently, the F-18 labeled PET radiotracer [18F]7a was reported, which shows high CB2 affinity and high selectivity over the CB1 subtype but low metabolic stability due to hydrolysis of the amide group. Based on these findings twelve bioisosteres of 7a were synthesized containing a non-hydrolysable functional group instead of the amide group. The secondary amine 23a (Ki = 7.9 nM) and the ketone 26a (Ki = 8.6 nM) displayed high CB2 affinity and CB2:CB1 selectivity in in vitro radioligand binding studies. Incubation of 7a, 23a and 26a with mouse liver microsomes and LC-quadrupole-MS analysis revealed a slightly higher metabolic stability of secondary amine 23a, but a remarkably higher stability of ketone 26a in comparison to amide 7a. Furthermore, a logD7.4 value of 5.56 ±â€¯0.08 was determined for ketone 26a by micro shake-flask method and LC-MS quantification.

Opportunities for Students to Prescribe: An Evaluation of 185 Consultations in the Student-run Cardiovascular Risk Management Programme.

Involving students in cardiovascular risk management (CVRM) could offer patients a structured CVRM programme and students a valuable learning opportunity. We describe and evaluate a student-run CVRM programme that was set up to offer primary prevention to patients with known risk factors in a general practitioner's practice. During a consultation, two undergraduate medical students assessed the patients' actual risk and formulated a CVRM plan, which they discussed with the patient after approval by a GP. After the consultations, patients were asked to complete evaluation/feedback questionnaires. From December 2014 to December 2015, 185 consultations were carried out by 46 students. Feedback questionnaires of 153 consultations were returned, in which patient satisfaction was 8.43 (1-10, min-max). The cardiovascular risk of 95 patients was determined, and in >50% patients, it was 'high'. Participating students and GPs were enthusiastic about the (pharmacotherapy) learning opportunities and improved CVRM care while contributing to real patient care in this CVRM programme.

Optimization of pharmacokinetic properties by modification of a carbazole-based cannabinoid receptor subtype 2 (CB2) ligand.

Recently, the development of the fluorinated PET tracer [18F]1a for imaging of CB2 receptors in the central nervous system was reported. [18F]1a showed high CB2 affinity and selectivity over the CB1 subtype, but rapid biotransformation in mice. In addition to the amide hydrolysis, oxidative N-dealkylation and carbazole oxidation were postulated as main metabolic pathways. Based on these results, novel carbazole derivatives with additional 6-substituents (23a, 24a), modified hydrogenation state (26a) and enlarged fluoroalkyl substituent (13a, 13b) were synthesized and pharmacologically evaluated. The key step in the synthesis of substituted carbazoles 23a, 24a and 26a was a Fischer indole synthesis. Nucleophilic substitution of tosylated lactate 5 by carbazole anion provided the fluoroisopropyl derivatives 13a and 13b. Partial hydrogenation of the aromatic carbazole system (26a) was not tolerated by the CB2 receptor. A methylsulfonyl moiety in 6-position (24a) led to considerably reduced CB2 affinity, whereas a 6-methoxy moiety (23a) was well tolerated. An additional methyl moiety in the fluoroethyl side chain of 1a resulted in fluoroisopropyl derivatives 13 with unchanged high CB2 affinity and CB2: CB1 selectivity. Compared with the fluoroethyl derivative 1a, the carbazole N-atom of the fluoroisopropyl derivative 13a (Ki(CB2) = 2.9 nM) is better shielded against the attack of CYP enzymes as formation of N-oxides was not observed and N-dealkylation took place to a less amount.

Kinetics of human cannabinoid 1 (CB1) receptor antagonists: Structure-kinetics relationships (SKR) and implications for insurmountable antagonism.

While equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In this study, we therefore conducted kinetic assays for nine 1-(4,5-diarylthiophene-2-carbonyl)-4-phenylpiperidine-4-carboxamide derivatives and included the CB1 antagonist rimonabant as a comparison. For this we newly developed a dual-point competition association assay with [3H]CP55940 as the radioligand. This assay yielded Kinetic Rate Index (KRI) values from which structure-kinetics relationships (SKR) of hCB1 receptor antagonists could be established. The fast dissociating antagonist 6 had a similar receptor residence time (RT) as rimonabant, i.e. 19 and 14 min, respectively, while the slowest dissociating antagonist (9) had a very long RT of 2222 min, i.e. pseudo-irreversible dissociation kinetics. In functional assays, 9 displayed insurmountable antagonism, while the effects of the shortest RT antagonist 6 and rimonabant were surmountable. Taken together, this study shows that hCB1 receptor antagonists can have very divergent RTs, which are not correlated to their equilibrium affinities. Furthermore, their RTs appear to define their mode of functional antagonism, i.e. surmountable vs. insurmountable. Finally, based on the recently resolved hCB1 receptor crystal structure, we propose that the differences in RT can be explained by a different binding mode of antagonist 9 from short RT antagonists that is able to displace unfavorable water molecules. Taken together, these findings are of importance for future design and evaluation of potent and safe hCB1 receptor antagonists.

Structure-Affinity Relationships and Structure-Kinetic Relationships of 1,2-Diarylimidazol-4-carboxamide Derivatives as Human Cannabinoid 1 Receptor Antagonists.

We report on the synthesis and biological evaluation of a series of 1,2-diarylimidazol-4-carboxamide derivatives developed as CB1 receptor antagonists. These were evaluated in a radioligand displacement binding assay, a [35S]GTPγS binding assay, and in a competition association assay that enables the relatively fast kinetic screening of multiple compounds. The compounds show high affinities and a diverse range of kinetic profiles at the CB1 receptor and their structure-kinetic relationships (SKRs) were established. Using the recently resolved hCB1 receptor crystal structures, we also performed a modeling study that sheds light on the crucial interactions for both the affinity and dissociation kinetics of this family of ligands. We provide evidence that, next to affinity, additional knowledge of binding kinetics is useful for selecting new hCB1 receptor antagonists in the early phases of drug discovery.

Synthesis and biological evaluation of chemokine receptor ligands with 2-benzazepine scaffold.

Targeting CCR2 and CCR5 receptors is considered as promising concept for the development of novel antiinflammatory drugs. Herein, we present the development of the first probe-dependent positive allosteric modulator (PAM) of CCR5 receptors with a 2-benzazepine scaffold. Compound 14 (2-isobutyl-N-({[N-methyl-N-(tetrahydro-2H-pyran-4-yl)amino]methyl}phenyl)-1-oxo-2,3-dihydro-1H-2-benzazepine-4-carboxamide) activates the CCR5 receptor in a CCL4-dependent manner, but does not compete with [3H]TAK-779 binding at the CCR5. Furthermore, introduction of a p-tolyl moiety at 7-position of the 2-benzazepine scaffold turns the CCR5 PAM 14 into the selective CCR2 receptor antagonist 26b. The structure affinity and activity relationships presented here offer new insights into ligand recognition by CCR2 and CCR5 receptors.

A novel CCR2 antagonist inhibits atherogenesis in apoE deficient mice by achieving high receptor occupancy.

CC Chemokine Receptor 2 (CCR2) and its endogenous ligand CCL2 are involved in a number of diseases, including atherosclerosis. Several CCR2 antagonists have been developed as potential therapeutic agents, however their in vivo clinical efficacy was limited. In this report, we aimed to determine whether 15a, an antagonist with a long residence time on the human CCR2, is effective in inhibiting the development of atherosclerosis in a mouse disease model. First, radioligand binding assays were performed to determine affinity and binding kinetics of 15a on murine CCR2. To assess the in vivo efficacy, western-type diet fed apoE-/- mice were treated daily with 15a or vehicle as control. Treatment with 15a reduced the amount of circulating CCR2+ monocytes and the size of the atherosclerotic plaques in both the carotid artery and the aortic root. We then showed that the long pharmacokinetic half-life of 15a combined with the high drug concentrations ensured prolonged CCR2 occupancy. These data render 15a a promising compound for drug development and confirms high receptor occupancy as a key parameter when targeting chemokine receptors.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity.

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Synthesis and evaluation of N-substituted 2-amino-4,5-diarylpyrimidines as selective adenosine A1 receptor antagonists.

We report the synthesis and biological evaluation of new 2-amino-4,5-diarylpyrimidines as selective antagonists at the adenosine A1 receptor. The scaffold they are based upon is a deaza variation of a previously reported collection of 3-amino-5,6-diaryl-1,2,4-triazines, members of which had a subnanomolar affinity but limited selectivity over the A2A subtype. Initially, similar structure-affinity relationships at the 5-aryl ring were established, and then emphasis was put on increasing selectivity at the hA1AR by introducing substituents on the N2-position, all the while maintaining a nanomolar affinity. Compound 3z, bearing a trans 4-hydroxycyclohexyl substituent, was identified as a potent (Ki(hA1AR) = 7.7 nM) and selective (Ki(hA2AAR) = 1389 nM) antagonist at the human adenosine A1 receptor. Computational docking was effected at the A1 and A2A subtypes, rationalizing the effect of the 4-hydroxycyclohexyl substituent on selectivity, in relation with the nature of the substituent on the 5-position of the pyrimidine.

Bioisosteric replacement of central 1,2,4-oxadiazole ring of high affinity CB2 ligands by regioisomeric 1,3,4-oxadiazole ring.

It has been reported that bioisosteric replacement of an 1,2,4-oxadiazole ring by an 1,3,4-oxadiazole ring leads to higher polarity, reduced metabolic degradation by human liver microsomes and reduced interaction with hERG channels. In a seven to eight step synthesis 1,3,4-oxadiazles 9a-c were synthesized as bioisosteric analogs of high-affinity but rather lipophilic CB2 ligands 1a-c containing an 1,2,4-oxadiazole ring. The 1,3,4-oxadiazole derivatives 9a and 9b show 10- and 50-fold reduced CB2 affinity compared to the 1,2,4-oxadiazole derivatives 1a and 1b, respectively. However, the 1,3,4-oxadiazole 9a has high CB2 affinity (Ki = 25 nM) and high selectivity over the CB1 receptor.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists.

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer. These disease associations have motivated numerous preclinical studies and clinical trials (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2-chemokine axis. To aid drug discovery efforts, here we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein-protein interactions, receptor-chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.

Predicting Binding Affinities for GPCR Ligands Using Free-Energy Perturbation.

The rapid growth of structural information for G-protein-coupled receptors (GPCRs) has led to a greater understanding of their structure, function, selectivity, and ligand binding. Although novel ligands have been identified using methods such as virtual screening, computationally driven lead optimization has been possible only in isolated cases because of challenges associated with predicting binding free energies for related compounds. Here, we provide a systematic characterization of the performance of free-energy perturbation (FEP) calculations to predict relative binding free energies of congeneric ligands binding to GPCR targets using a consistent protocol and no adjustable parameters. Using the FEP+ package, first we validated the protocol, which includes a full lipid bilayer and explicit solvent, by predicting the binding affinity for a total of 45 different ligands across four different GPCRs (adenosine A2AAR, ß1 adrenergic, CXCR4 chemokine, and δ opioid receptors). Comparison with experimental binding affinity measurements revealed a highly predictive ranking correlation (average spearman ρ = 0.55) and low root-mean-square error (0.80 kcal/mol). Next, we applied FEP+ in a prospective project, where we predicted the affinity of novel, potent adenosine A2A receptor (A2AR) antagonists. Four novel compounds were synthesized and tested in a radioligand displacement assay, yielding affinity values in the nanomolar range. The affinity of two out of the four novel ligands (plus three previously reported compounds) was correctly predicted (within 1 kcal/mol), including one compound with approximately a tenfold increase in affinity compared to the starting compound. Detailed analyses of the simulations underlying the predictions provided insights into the structural basis for the two cases where the affinity was overpredicted. Taken together, these results establish a protocol for systematically applying FEP+ to GPCRs and provide guidelines for identifying potent molecules in drug discovery lead optimization projects.