Drug-discovery tools for cystic fibrosis: optical probes to quantify gating and trafficking of ΔF508-CFTR

Cystic fibrosis (CF) is a debilitating disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which codes for the CFTR anion channel. ΔF508, the most common CF-associated mutation, causes both a gating and a trafficking defect in the CFTR protein. This thesis describes the optimisation and use of two fluorescence assays, capable of measuring the two defects caused by the ΔF508 mutation. Yellow fluorescent protein (YFP)-CFTR, in which halide sensitive YFP is tagged to the Nterminal of the CFTR coding sequence, is a functional assay, used to test for ΔF508-CFTR potentiator activity. CFTR-pHTomato, in which the pH sensor is tagged to the fourth extracellular loop of CFTR, is used to measure membrane density and internal CFTR expression. Human embryonic kidney cells (HEK293 cells), expressing YFP-ΔF508-CFTR were used to screen a pilot library of compounds with some structural similarity to known potentiator VX- 770. Ligand-based virtual screening was then used to construct two further libraries, based on VX-770 and the lead hit compound from the pilot screen. A number of novel ΔF508-CFTR potentiators were identified in each of the screens. Recently published studies suggest that chronic treatment with VX-770 decreases ΔF508- CFTR density at the plasma membrane, potentially limiting its clinical effectiveness. ΔF508- CFTR-pHTomato was used to show that a number of hit compounds from our screens did not decrease membrane density of ΔF508-CFTR. The YFP-CFTR assay was also used for studies investigating the mechanism of potentiation by VX-770. We provide evidence that WT-CFTR does not require phosphorylation for potentiation by VX-770, which has not been reported previously. Additionally, studies using gating mutations do not support the current hypothesis that VX-770 stabilises the posthydrolytic open state in the CFTR gating cycle

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients’ age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to “kill two birds with one stone” by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure–activity relationship (SAR) study that led to compound 23a. This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs

Treating frailty-a practical guide

Frailty is a common syndrome that is associated with vulnerability to poor health outcomes. Frail older people have increased risk of morbidity, institutionalization and death, resulting in burden to individuals, their families, health care services and society. Assessment and treatment of the frail individual provide many challenges to clinicians working with older people. Despite frailty being increasingly recognized in the literature, there is a paucity of direct evidence to guide interventions to reduce frailty. In this paper we review methods for identification of frailty in the clinical setting, propose a model for assessment of the frail older person and summarize the current best evidence for treating the frail older person. We provide an evidence-based framework that can be used to guide the diagnosis, assessment and treatment of frail older people

Potentiation of the cystic fibrosis transmembrane conductance regulator by VX‐770 involves stabilization of the pre‐hydrolytic, O₁ state

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a debilitating hereditary disease caused by mutations in the CFTR gene, which encodes an anion channel. WT-CFTR gating is a non-equilibrium process. After ATP binding, CFTR enters a stable open state (O1 ). ATP hydrolysis leads it to a short-lived post-hydrolytic open state (O2 ), from which channels close. Here we use mutations to probe the mechanism of VX-770, the first compound directly targeting the CFTR protein approved for treatment of CF. D1370N and K1250R mutations greatly reduce or abolish catalytic activity, simplifying the gating scheme to an equilibrium (C ↔O1 ); K464A-CFTR has a destabilized O1 state and rarely closes via hydrolysis. EXPERIMENTAL APPROACH: Potentiation by VX-770 was measured using microscopic imaging of HEK293 cells expressing an anion-sensitive YFP-CFTR. A simple mathematical model was used to predict fluorescence quenching following extracellular iodide addition, and estimate CFTR conductance. Membrane density of CFTR channels was measured in a parallel assay, using CFTR-pHTomato. KEY RESULTS: VX-770 strongly potentiates WT-CFTR, D1370N-CFTR and K1250R-CFTR. K464A-CFTR was also strongly potentiated, regardless of whether it retained catalytic activity or not. CONCLUSIONS AND IMPLICATIONS: Similar potentiation of hydrolytic and non-hydrolytic mutants suggests that VX-770 increases CFTR open probability mainly by stabilising the pre-hydrolytic O1 state with respect to the closed state. Potentiation of K464A-CFTR channels suggests action of the drug does not strongly alter conformational dynamics at site 1. Understanding potentiator mechanism could help develop improved treatment for CF patients. The fluorescence assay presented here is a robust tool for such investigations

Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity

The cystic fibrosis transmembrane conductance regulator, CFTR, is a plasma membrane anion channel which plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrhoeas, while CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR – one boosting ion-channel function (potentiators), the other increasing plasma membrane density (correctors) – have provided significant health benefits to CF patients. Here we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein’s proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease

Economic Evaluation of a Multifactorial, Interdisciplinary Intervention Versus Usual Care to Reduce Frailty in Frail Older People

Objective: To compare the costs and cost-effectiveness of a multifactorial interdisciplinary intervention versus usual care for older people who are frail. Design: Cost-effectiveness study embedded within a randomized controlled trial. Setting: Community-based intervention in Sydney, Australia. Participants: A total of 241 community-dwelling people 70 years or older who met the Cardiovascular Health Study criteria for frailty. Intervention: A 12-month multifactorial, interdisciplinary intervention targeting identified frailty characteristics versus usual care. Measurements: Health and social service use, frailty, and health-related quality of life (EQ-5D) were measured over the 12-month intervention period. The difference between the mean cost per person for 12 months in the intervention and control groups (incremental cost) and the ratio between incremental cost and effectiveness were calculated. Results: A total of 216 participants (90%) completed the study. The prevalence of frailty was 14.7% lower in the intervention group compared with the control group at 12 months (95% CI 2.4%-27.0%; P = .02). There was no significant between-group difference in EQ-5D utility scores. The cost for 1 extra person to transition out of frailty was $A15,955 (at 2011 prices). In the very frail subgroup (participants met \u3e3 Cardiovascular Health Study frailty criteria), the intervention was both more effective and less costly than the control. A cost-effectiveness acceptability curve shows that the intervention would be cost-effective with 80$A50,000 per extra person transitioning from frailty. In the very frail subpopulation, this reduced to $25,000. Conclusion: For frail older people residing in the community, a 12-month multifactorial intervention provided better value for money than usual care, particularly for the very frail, in whom it has a high probability of being cost saving, as well as effective