Nephrology Nurses: Essential Professionals in Sustainable Kidney Care.

Purpose: The increasing frequency of extreme climate events underscores the need for urgent action on climate change. The health care system contributes 4.6% of greenhouse gas emissions (GHGs) in Canada; thus, it is a major contributor to the country's carbon footprint. Kidney care in particular can involve high amounts of waste (eg, plastic and consumable waste associated with dialysis, transportation, emissions, energy, and water consumption). Therefore, sustainability initiatives within the health care system, and especially in the context of kidney care, have great potential to make a positive impact on planetary health. Here, we outline ways in which nephrology nurses can expand our duty of care to the environment and incorporate sustainability into our work. Sources of information: A small advisory group of nephrology nurses in partnership with the Canadian Association of Nurses for the Environment (CANE) assessed ways that sustainable practices can be incorporated into nephrology nursing. Drawing on the Planetary Health Care model used by the Canadian Society of Nephrology: Sustainable Nephrology Action Planning (SNAP) committee, we assessed how the model could be adapted in the context of kidney care using 3 main actionable themes in their work: reducing the demand for health services, matching the supply of health services with demand, and reducing emissions from the supply of health services. We also reviewed and selected real-world examples of initiatives pursued by colleagues. Key findings: Through this established framework, we provide recommendations and case examples for nephrology nurses to expand our duty of care to the environment. We describe nursing-led strategies used in Canada to improve environmental sustainability in kidney programs and consider their applicability to other renal programs. In 1 case example, we show how a simple nurse-led initiative at a single dialysis clinic can lower plastic waste and associated costs by $2042.59 per year. More broadly, we provide recommendations and actions for nephrology nurses to improve environmental sustainability in kidney care. Limitations: Nurses in Canada have many responsibilities within limited timeframes, making it essential to choose sustainable practices that do not exacerbate burnout and high workloads. For sustainable practices to be successful, nurses must integrate them into their existing workflows.

Contexte: L'augmentation de la fréquence des phénomènes climatiques extrêmes met en lumière la nécessité de prendre des mesures urgentes pour lutter contre les changements climatiques. Le système de santé est responsable de 4,6 % des émissions de gaz à effet de serre (GES) au Canada et contribue grandement à l'empreinte carbone du pays. Les soins rénaux en particulier peuvent générer de grandes quantités de gaspillage (p. ex., déchets plastiques et consommables associés à la dialyse, transport, émissions, énergie et consommation d'eau). Par conséquent, les initiatives de durabilité dans le système de santé, et en particulier dans le contexte des soins rénaux, pourraient avoir une incidence positive sur la santé de la planète. Dans cet article, nous décrivons comment le personnel infirmier en néphrologie pourrait élargir son devoir de soins à la protection de l'environnement par l'intégration des pratiques durables dans son travail. Sources de l'information: Un petit groupe consultatif constitué d'infirmières et infirmiers en néphrologie, en partenariat avec l'Association canadienne des infirmières et infirmiers pour l'environnement (ACIIE) et le Sustainable Nursing Action Planning (SNAP) Committee, s'est réuni pour évaluer comment les pratiques durables pourraient être intégrées aux soins infirmiers en néphrologie. Nous avons utilisé un modèle de soins favorisant la santé de la planète et décrit de manière itérative les façons dont celui-ci pourrait être adapté au contexte des soins rénaux. Nous avons également examiné quelques exemples concrets d'initiatives prises par des collègues. Principales observations: Nous décrivons comment le personnel infirmier en néphrologie pourrait appliquer un modèle de soins favorisant la santé de la planète dans le cadre de son travail. Ce concept est présenté sous trois principaux thèmes exploitables: réduire la demande pour des services de santé, adapter l'offre de services de santé à la demande et réduire les émissions liées à l'offre de services de santé. Dans ce cadre, nous formulons des recommandations au personnel infirmier en néphrologie et nous lui présentons des exemples de cas pour élargir son devoir de soins à la protection de l'environnement. Nous présentons des stratégies utilisées au Canada, et menées en soins infirmiers, pour améliorer la durabilité environnementale des programmes rénaux. Nous examinons également leur applicabilité à d'autres programmes rénaux. Dans un des exemples de cas, nous montrons comment une simple initiative dirigée par une infirmière, au sein d'une seule clinique de dialyse, a permis de réduire la quantité de déchets plastiques et les coûts connexes de 2 042,59 $ par année. De manière plus générale, nous formulons des recommandations et proposons des actions qui pourraient être posées par le personnel infirmier en néphrologie pour améliorer la durabilité des soins rénaux.

Proteolysis Targeting Chimeras (PROTACs): A Perspective on Integral Membrane Protein Degradation.

Targeted protein degradation (TPD) is a promising therapeutic modality to modulate protein levels and its application promises to reduce the "undruggable" proteome. Among TPD strategies, Proteolysis TArgeting Chimera (PROTAC) technology has shown a tremendous potential with attractive advantages when compared to the inhibition of the same target. While PROTAC technology has had a significant impact in scientific research, its application to degrade integral membrane proteins (IMPs) is still in its beginnings. Among the 15 compounds having entered clinical trials by the end of 2021, only two targets are membrane-associated proteins. In this review we are discussing the potential reasons which may underlie this, and we are presenting new tools that have been recently developed to solve these limitations and to empower the use of PROTACs to target IMPs.

Structural analysis of the LDL receptor-interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site.

Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.

Fragment-based Differential Targeting of PPI Stabilizer Interfaces.

Stabilization of protein-protein interactions (PPIs) holds great potential for therapeutic agents, as illustrated by the successful drugs rapamycin and lenalidomide. However, how such interface-binding molecules can be created in a rational, bottom-up manner is a largely unanswered question. We report here how a fragment-based approach can be used to identify chemical starting points for the development of small-molecule stabilizers that differentiate between two different PPI interfaces of the adapter protein 14-3-3. The fragments discriminately bind to the interface of 14-3-3 with the recognition motif of either the tumor suppressor protein p53 or the oncogenic transcription factor TAZ. This X-ray crystallography driven study shows that the rim of the interface of individual 14-3-3 complexes can be targeted in a differential manner with fragments that represent promising starting points for the development of specific 14-3-3 PPI stabilizers.

Proteolysis-targeting chimeras in drug development: A safety perspective.

Proteolysis-targeting chimeras are a new drug modality that exploits the endogenous ubiquitin proteasome system to degrade a protein of interest for therapeutic benefit. As the first-generation of proteolysis-targeting chimeras have now entered clinical trials for oncology indications, it is timely to consider the theoretical safety risks inherent with this modality which include off-target degradation, intracellular accumulation of natural substrates for the E3 ligases used in the ubiquitin proteasome system, proteasome saturation by ubiquitinated proteins, and liabilities associated with the "hook effect" of proteolysis-targeting chimeras This review describes in vitro and non-clinical in vivo data that provide mechanistic insight of these safety risks and approaches being used to mitigate these risks in the next generation of proteolysis-targeting chimera molecules to extend therapeutic applications beyond life-threatening diseases.

Small-molecule modulation of p53 protein-protein interactions.

Small-molecule modulation of protein-protein interactions (PPIs) is a very promising but also challenging area in drug discovery. The tumor suppressor protein p53 is one of the most frequently altered proteins in human cancers, making it an attractive target in oncology. 14-3-3 proteins have been shown to bind to and positively regulate p53 activity by protecting it from MDM2-dependent degradation or activating its DNA binding affinity. PPIs can be modulated by inhibiting or stabilizing specific interactions by small molecules. Whereas inhibition has been widely explored by the pharmaceutical industry and academia, the opposite strategy of stabilizing PPIs still remains relatively underexploited. This is rather interesting considering the number of natural compounds like rapamycin, forskolin and fusicoccin that exert their activity by stabilizing specific PPIs. In this review, we give an overview of 14-3-3 interactions with p53, explain isoform specific stabilization of the tumor suppressor protein, explore the approach of stabilizing the 14-3-3σ-p53 complex and summarize some promising small molecules inhibiting the p53-MDM2 protein-protein interaction.

Adoption of a Turn Conformation Drives the Binding Affinity of p53 C-Terminal Domain Peptides to 14-3-3σ.

The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding.

IDOL regulates systemic energy balance through control of neuronal VLDLR expression.

Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity.

A Comparative Study of Fluorescence Assays in Screening for BRD4.

Fluorescence assay technologies are commonly used in high-throughput screening because of their sensitivity and ease of use. Different technologies have their characteristics and the rationale for choosing one over the other can differ between projects because of factors such as availability of reagents, assay performance, and cost. Another important factor to consider is the assay susceptibility to artifacts, which is almost as important as the ability of the assay to pick up active compounds. Spending time and money on false positives or missing the opportunity to build chemistry around false negatives is something that every drug project tries to avoid. We used a BET family Bromodomain, BRD4(1), to explore the outcome of a screening campaign using three fluorescent assay technologies as primary assays. A diverse 7,038 compound set was screened in fluorescence lifetime, fluorescence polarization, and homogeneous time-resolved fluorescence to look at primary hit rates, compound overlap, and hit confirmation rates. The results show a difference between the fluorescence assay technologies with three separate hit lists and some overlap. The confirmed hits from each assay were further evaluated for translation into cells (NanoBRET™). Most of the actives confirmed in cells originated from compounds that overlapped between the assays. In addition, a well-annotated set of compounds with undesirable mechanism of inhibition was screened against BRD4(1) to compare the ability to discriminate true hits from artifact compounds. The results indicate a difference between the assays in their ability to generate false positives and negatives.

Prospectively Validated Proteochemometric Models for the Prediction of Small-Molecule Binding to Bromodomain Proteins.

The bromodomain-containing proteins are a ligandable family of epigenetic readers, which play important roles in oncological, cardiovascular, and inflammatory diseases. Achieving selective inhibition of specific bromodomains is challenging, due to the limited understanding of compound and target selectivity features. In this study we build and benchmark proteochemometric (PCM) classification models on bioactivity data for 15,350 data points across 31 bromodomains, using both compound fingerprints and binding site protein descriptors as input variables, achieving a maximum performance as measured by the Matthew's Correlation Coefficient (MCC) of 0.83 on the external test set. We also find that histone peptide binding data can be used as a target descriptor to build a high performing PCM model (MCC 0.80), showing the transferability of peptide interaction information to modeling small-molecule bioactivity. 1,139 compounds were selected for prospective experimental testing by performing a virtual screen using model predictions and implementing conformal prediction, which resulted in 319 correctly predicted compound-target pair actives and the correct prediction for certain selectivity profile combinations of the four bromodomains tested against. We identify that conformal prediction can be used to fine-tune the balance between hit retrieval and hit structural diversity in a virtual screening setting. PCM can be applied to future virtual screening and compound design, including off-target prediction for bromodomains.

Inhibition of low-density lipoprotein receptor degradation with a cyclic peptide that disrupts the homodimerization of IDOL E3 ubiquitin ligase.

Cellular uptake of circulating cholesterol occurs via the low density lipoprotein receptor (LDLR). The E3 ubiquitin ligase IDOL is a mediator of LDLR degradation, with IDOL homodimerization thought to be required for its activity. To probe the possibility of modulating LDLR levels with an inhibitor of IDOL homodimerization, we screened a SICLOPPS library of 3.2 million cyclic peptides for compounds that disrupt this protein-protein interaction. We identified cyclo-CFFLYT as the lead inhibitor, and improved its activity through the incorporation of non-natural amino acids. The activity of the optimized cyclic peptide was assessed in hepatic cells, with a dose-dependent increase in LDLR levels observed in the presence of our IDOL homodimerization inhibitor.

Design of a Chemical Probe for the Bromodomain and Plant Homeodomain Finger-Containing (BRPF) Family of Proteins.

The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.

Small-molecule stabilization of the p53 - 14-3-3 protein-protein interaction.

14-3-3 proteins are positive regulators of the tumor suppressor p53, the mutation of which is implicated in many human cancers. Current strategies for targeting of p53 involve restoration of wild-type function or inhibition of the interaction with MDM2, its key negative regulator. Despite the efficacy of these strategies, the alternate approach of stabilizing the interaction of p53 with positive regulators and, thus, enhancing tumor suppressor activity, has not been explored. Here, we report the first example of small-molecule stabilization of the 14-3-3 - p53 protein-protein interaction (PPI) and demonstrate the potential of this approach as a therapeutic modality. We also observed a disconnect between biophysical and crystallographic data in the presence of a stabilizing molecule, which is unusual in 14-3-3 PPIs.

Impact of Etanercept Therapy on Disease Activity and Health-Related Quality of Life in Moderate Rheumatoid Arthritis Patients Population from a National British Observational Cohort.

OBJECTIVES: To describe a population with moderate rheumatoid arthritis (RA) before biologic initiation and assess change in disease status, health-related quality of life (HRQOL), and adverse events in etanercept (ETN)-treated patients. METHODS: Data on adult patients with moderate RA (3.2 < Disease Activity Score in 28 Joints [DAS28] ≤ 5.1) were retrospectively analyzed from the British Society for Rheumatology Biologics Register comparing a nonbiologic-treated group (nBG) using at least one traditional disease-modifying antirheumatic drug to a biologic group (BG) treated with ETN. The HRQOL was assessed by using the Health Assessment Questionnaire disability index score. To mitigate confounding, we controlled for drivers of progression. Appropriate univariate, multivariate, and regression analyses were used. RESULTS: A total of 1754 patients with RA were assessed (211 BG and 1543 nBG). Compared with the nBG, the BG tended toward higher disease activity, such as significantly higher tender joints and DAS28. The BG compared with the nBG had 1) a greater reduction in DAS28 and Health Assessment Questionnaire scores; 2) disease remission occurring more often (odds ratio = 2.7; P = 0.006); and 3) progression occurring in fewer patients (odds ratio = 0.3; P = 0.002). BG patients had a higher incidence of "other serious infection" and "other central nervous system-related events," with no significant differences in associated hospitalization rates or deaths. CONCLUSIONS: Among patients with moderate RA from a clinical practice registry, ETN-treated patients had significantly higher disease activity at the time of biologic initiation but significantly reduced disease activity and better HRQOL after 6 months compared with nBG patients, although the possibility of unmeasured confounding remains. The ETN group reported significantly higher incidences of "other serious infections" and "other central nervous system-related events" without higher hospitalization rates.

Assessing HTS performance using BioAssay Ontology: screening and analysis of a bacterial phospho-N-acetylmuramoyl-pentapeptide translocase campaign.

With the public availability of biochemical assays and screening data constantly increasing, new applications for data mining and method analysis are evolving in parallel. One example is BioAssay Ontology (BAO) for systematic classification of assays based on screening setup and metadata annotations. In this article we report a high-throughput screening (HTS) against phospho-N-acetylmuramoyl-pentapeptide translocase (MraY), an attractive antibacterial drug target involved in peptidoglycan synthesis. The screen resulted in novel chemistry identification using a fluorescence resonance energy transfer assay. To address a subset of the false positive hits, a frequent hitter analysis was performed using an approach in which MraY hits were compared with hits from similar assays, previously used for HTS. The MraY assay was annotated according to BAO and three internal reference assays, using a similar assay design and detection technology, were identified. Analyzing the assays retrospectively, it was clear that both MraY and the three reference assays all showed a high false positive rate in the primary HTS assays. In the case of MraY, false positives were efficiently identified by applying a method to correct for compound interference at the hit-confirmation stage. Frequent hitter analysis based on the three reference assays with similar assay method identified additional false actives in the primary MraY assay as frequent hitters. This article demonstrates how assays annotated using BAO terms can be used to identify closely related reference assays, and that analysis based on these assays clearly can provide useful data to influence assay design, technology, and screening strategy.

Association between disease activity and risk of serious infections in subjects with rheumatoid arthritis treated with etanercept or disease-modifying anti-rheumatic drugs.

OBJECTIVES: To determine the risk of serious infection in patients with rheumatoid arthritis (RA) receiving etanercept (ETN) or disease-modifying anti-rheumatic drugs (DMARDs) and to identify factors that predict a higher risk. METHODS: Five-year data from the British Society of Rheumatology Biologics Register (BSRBR), a prospective observational study of patients with active RA treated with ETN, were used. These data were compared with a cohort of patients receiving DMARDs with active RA. RESULTS: Total follow-up was 19,964 patient-years (py; ETN, 14,381 py; DMARDs, 5583 py). Over the study period, 651 first-recorded serious infections were reported (ETN, 469 [39.9 per 1000 py]; DMARDs, 182 [35.0 per 1000 py]). Overall the risk of serious infection was similar for the 2 treatments; however, in the first 6 months of treatment the hazard ratio (HR) was higher in the ETN than the DMARD group (1.979; p=0.015). A linear association was observed between the serious infection rate and disease-activity score in 28 joints (DAS28) in patients from each treatment group and overall (DAS28 <4, 27.1 per 1000 py; DAS28 ≥8, 64.4 per 1000 py; 7.5% increase in serious infection for each unit increase of DAS28 score at baseline). In a time-dependent analysis, a DAS28 change of 1 unit during follow-up predicted a 27% increase in serious infection rates. CONCLUSIONS: No significant increase in the risk of serious infection was observed with ETN versus DMARDs over the 5-year study; a linear relationship existed between the serious infection rate and disease activity, as measured by DAS28.

Treatment of rheumatoid arthritis with etanercept with reference to disease-modifying anti-rheumatic drugs: long-term safety and survival using prospective, observational data.

OBJECTIVE: The objective of this study was to examine the long-term safety of etanercept (ETN) in comparison with conventional DMARDs in a large observational cohort of RA patients in the UK. METHODS: Data were made available from the British Society of Rheumatology Biologics Register for a cohort of patients with RA treated with ETN and a reference cohort of RA patients treated with conventional DMARDs (maximum follow-up 10 years). The adjusted risk of events was compared using Cox proportional hazards models. RESULTS: There were 3529 eligible ETN-treated patients (16,919 person-years) and 2864 conventional DMARD-treated patients (11,095 person-years), with notable differences between groups at baseline. Crude mortality rates were 12.0 vs 20.1 events per 1000 person-years for ETN and conventional DMARD patients, respectively, with an adjusted hazard ratio (aHR) of 0.72 (95% CI 0.54, 0.96). There was no difference in the long-term risk of serious infections (aHR = 1.02, 95% CI 0.83, 1.25). However, the risk was increased for ETN in the first 2 years (aHR = 1.56, 95% CI 1.16, 2.09; aHR = 1.32, 95% CI 1.06, 1.65). The aHRs (95% CIs) of various outcomes were cancer, 0.84 (0.68, 1.03); lymphoproliferative malignancy specifically, 0.51 (0.28, 0.95); all other serious adverse events, 0.70 (0.56, 0.87) and cardiac events specifically, 0.52 (0.37, 0.72). CONCLUSION: There was no evidence of adverse outcome from long-term exposure to ETN. There was evidence of improved survival, reduced cardiovascular events and reduced lymphoproliferative malignancies.

Long-Term Drug Survival of TNF Inhibitor Therapy in RA Patients: A Systematic Review of European National Drug Registers.

Objective. The present systematic review of RA registry data was undertaken to analyse the time on treatment of licensed TNF inhibitors in patients with RA in Europe. Methods. English language European registry studies comparing TNF inhibitors were searched using MEDLINE, Embase, Cochrane, and WHO: ICTRP up to 16 April 2012 and proceedings of three selected conferences held between 2010 and 2012. Pooled analysis was performed to determine drug survival rates for each TNF inhibitor. Results. Sixteen studies met the inclusion criteria, of which 11 studies assessed biologic-naive patients and five studies included a mixed population of biologic-naive and biologic pretreated patients. The overall effectiveness of TNF inhibitors diminished with time, leading to decreased drug survival rates. Pooled drug survival rates after 60 months follow-up were 37% (infliximab), 48% (adalimumab), and 52% (etanercept). Further, in an observational study, when TNF inhibitors were used in combination with methotrexate, a longer drug survival was observed compared to TNF inhibitors alone. Conclusion. The findings of this systematic review indicated numerically lower drug discontinuation rates with etanercept than adalimumab, whereas infliximab had the highest rate. Further research is needed to understand the underlying mechanisms of treatment discontinuation with TNF inhibitors.

Fluorescently labeled analogues of dofetilide as high-affinity fluorescence polarization ligands for the human ether-a-go-go-related gene (hERG) channel.

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide. When bound to cell membranes expressing the hERG channel, 10e shows a robust increase in fluorescence polarization (FP) signal. In a FP binding assay using 10e as tracer ligand, Ki values for several known hERG channel blockers were measured and excellent agreement with the literature Ki values was observed over an affinity range of 2 nM to 3 muM. 10e blocks hERG channel current in electrophysiological patch clamp experiments, and computational docking experiments predict that the dofetilide core of 10e binds hERG channel in a conformation similar to that previously predicted for 1. These analogues enable high-throughput hERG channel binding assays that are rapid, economical, and predictive of test compounds' potential for prolonged QT liabilities.

Early evaluation of compound QT prolongation effects: a predictive 384-well fluorescence polarization binding assay for measuring hERG blockade.

INTRODUCTION: A large number of drugs from a variety of pharmacological classes have been demonstrated to cause adverse effects on cardiac rhythm, including the life-threatening arrhythmia Torsades de Pointes. These side effects are often associated with prolongation of the QT interval and are mediated via blockade of the human ether-a-go-go related gene (hERG) encoded potassium channel. In order to manage this risk in the pharmaceutical industry it is desirable to evaluate QT prolongation as early as possible in the drug discovery process. METHODS: Here we describe the development of a 384-well fluorescence polarization (FP) binding assay compatible with high-throughput assessment of compound blockade of the hERG channel during the lead optimisation process. To characterise the fluorescent ligand that was developed, competition binding studies, kinetic studies and electrophysiology studies were performed. Furthermore, to validate the assay as a key screening method a series of competition binding studies were performed and correlated with functional data obtained via patch-clamp. RESULTS: Evaluation of the assay indicates that high quality data is obtained (Z'>0.6), that the K(i) values determined are equivalent to more traditional radiometric methods and that it is predictive for functional hERG blockade as assessed by patch clamp. DISCUSSION: Whilst FP assays, utilizing a variety of fluors, have become well established for the evaluation of G-protein-coupled receptor (GPCRs) and kinase ligand interactions, this technique has not been applied widely to the study of ion channels. Therefore, this represents a novel assay format that is amenable to the evaluation of thousands of compounds per day. Whilst other assay formats have proven predictive or high throughput, this assay represents one of few that combines both attributes, moreover it represents the most cost effective assay, making it truly amenable to early assessment of hERG blockade.