Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)-Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and ß2-microglobulin (ß2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to ß2m, both of which participate in the FcRnECD-IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

A prospective multi-centre study assessing the safety and effectiveness following the implementation of an accelerated chest pain pathway using point-of-care troponin for use in New Zealand rural hospital and primary care settings.

AIMS: Most rural hospitals and general practices in New Zealand (NZ) are reliant on point-of-care troponin. A rural accelerated chest pain pathway (RACPP), combining an electrocardiogram (ECG), a structured risk score (Emergency Department Assessment of Chest Pain Score), and serial point-of-care troponin, was designed for use in rural hospital and primary care settings across NZ. The aim of this study was to evaluate the safety and effectiveness of the RACPP. METHODS AND RESULTS: A prospective multi-centre evaluation following implementation of the RACPP was undertaken from 1 July 2018 to 31 December 2020 in rural hospitals, rural and urban general practices, and urgent care clinics. The primary outcome measure was the presence of 30-day major adverse cardiac events (MACEs) in low-risk patients. The secondary outcome was the percentage of patients classified as low-risk that avoided transfer or were eligible for early discharge. There were 1205 patients enrolled in the study. 132 patients were excluded. Of the 1073 patients included in the primary analysis, 474 (44.0%) patients were identified as low-risk. There were no [95% confidence interval (CI): 0-0.3%] MACE within 30 days of the presentation among low-risk patients. Most of these patients (91.8%) were discharged without admission to hospital. Almost all patients who presented to general practice (99%) and urgent care clinics (97.6%) were discharged to home directly. CONCLUSION: The RACPP is safe and effective at excluding MACEs in NZ rural hospital and primary care settings, where it can identify a group of low-risk patients who can be safely discharged home without transfer to hospital.

Implementation and evaluation of a rural general practice assessment pathway for possible cardiac chest pain using point-of-care troponin testing: a pilot study.

OBJECTIVES: To assess the feasibility and acceptability, and additionally to preliminarily evaluate, the effectiveness and safety of an accelerated diagnostic chest pain pathway in rural general practice using point-of-care troponin to identify patients at low risk of acute myocardial infarction, avoiding unnecessary patient transfer to hospital and enabling early discharge home. DESIGN: A prospective observational pilot evaluation. SETTING: Twelve rural general (family) practices in the Midlands region of New Zealand. PARTICIPANTS: Patients aged ≥18 years who presented acutely to rural general practice with suspected ischaemic chest pain for whom the doctor intended transfer to hospital for serial troponin measurement. OUTCOME MEASURES: The proportion of patients managed using the low-risk pathway without transfer to hospital and without 30-day major adverse cardiac event (MACE); pathway adherence; rate of 30-day MACE; patient satisfaction with care; and agreement between point-of-care and laboratory measured troponin concentrations. RESULTS: A total of 180 patients were assessed by the pathway. The pathway classified 111 patients (61.7%) as low-risk and all were managed in rural general practice with no 30-day MACE (0%, 95% CI 0.0% to 3.3%). Adherence to the low-risk pathway was 95.5% (106 out of 111). Of the 56 patients classified as non-low-risk and referred to hospital, 9 (16.1%) had a 30-day MACE. A further 13 non-low-risk patients were not transferred to hospital, with no events. The sensitivity of the pathway for 30-day MACE was 100.0% (95% CI 70.1% to 100%). Of low-risk patients, 94% reported good to excellent satisfaction with care. Good concordance was observed between point-of-care and duplicate laboratory measured troponin concentrations. CONCLUSIONS: The use of an accelerated diagnostic chest pain pathway incorporating point-of-care troponin in a rural general practice setting was feasible and acceptable, with preliminary results suggesting that it may safely and effectively reduce the urgent transfer of low-risk patients to hospital.

Study protocol for an observational study to evaluate an accelerated chest pain pathway using point-of-care troponin in New Zealand rural and primary care populations.

INTRODUCTION Accelerated diagnostic chest pain pathways are used widely in urban New Zealand hospitals. These pathways use laboratory-based troponin assays with good analytical precision. Widespread implementation has not occurred in many of New Zealand's rural hospitals and general practices as they are reliant on point-of-care troponin assays, which are less sensitive and precise. An accelerated chest pain pathway using point-of-care troponin has been adapted for use in rural settings. A pilot study in a low-risk rural population showed no major adverse cardiac events at 30 days. A larger study is required to be confident that the pathway is safe. AIMS To assess the safety and effectiveness of an accelerated chest pain pathway adapted for rural settings and general practice using point-of-care troponin to identify low-risk patients and allow early discharge. METHODS This is a prospective observational study of an accelerated chest pain pathway using point-of-care troponin in rural hospitals and general practices in New Zealand. A total of 1000 patients, of whom we estimate 400 will be low risk, will be enrolled in the study. OUTCOME MEASURES The primary outcome is the proportion of patients identified by the pathway as low risk for a 30-day major adverse cardiac event. Secondary outcomes include the proportion of low-risk patients who were discharged directly from general practice or rural hospitals, the proportion of patients reclassified as having acute myocardial infarction by the pathway and the proportion of patients with low and intermediate risk safely managed in the rural hospital.

Small molecules that inhibit TNF signalling by stabilising an asymmetric form of the trimer.

Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.