Inhibition of Ral GTPases Using a Stapled Peptide Approach

Aberrant Ras signalling drives numerous cancers and drugs to inhibit this are urgently required. This compelling clinical need, combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly and the focus has moved to the main downstream Ras-signalling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets, which were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development and there have therefore been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This therefore provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.This work was supported by a Cambridge Cancer Centre Pump Priming award to CA, DO and HRM, a BBSRC Studentship to NSC, and a National Institutes for Health grant (CA71443) and the Welch Foundation (grant number I-1414) to MAW.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.M116.72024

Inhibition of Ral GTPases Using a Stapled Peptide Approach.

Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of α-helical-stapled peptides that bind selectively to active, GTP-bound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.This work was supported by a Cambridge Cancer Centre Pump Priming award to CA, DO and HRM, a BBSRC Studentship to NSC, and a National Institutes for Health grant (CA71443) and the Welch Foundation (grant number I-1414) to MAW.This is the final version of the article. It first appeared from the American Society for Biochemistry and Molecular Biology via https://doi.org/10.1074/jbc.M116.72024

On-site bundled rapid HIV/HCV testing in substance use disorder treatment programs: study protocol for a hybrid design randomized controlled trial

Background More than 1.2 million people in the United States are living with human immunodeficiency virus (HIV), and 3.2 million are living with hepatitis C virus (HCV). An estimated 25 % of persons living with HIV also have HCV. It is therefore of great public health importance to ensure the prompt diagnosis of both HIV and HCV in populations that have the highest prevalence of both infections, including individuals with substance use disorders (SUD). Methods/design In this theory-driven, efficacy-effectiveness-implementation hybrid study, we will develop and test an on-site bundled rapid HIV/HCV testing intervention for SUD treatment programs. Its aim is to increase the receipt of HIV and HCV test results among SUD treatment patients. Using a rigorous process involving patients, providers, and program managers, we will incorporate rapid HCV testing into evidence-based HIV testing and linkage to care interventions. We will then test, in a randomized controlled trial, the extent to which this bundled rapid HIV/HCV testing approach increases receipt of HIV and HCV test results. Lastly, we will conduct formative research to understand the barriers to, and facilitators of, the adoption, implementation, and sustainability of the bundled rapid testing strategy in SUD treatment programs. Discussion Novel approaches that effectively integrate on-site rapid HIV and rapid HCV testing are needed to address both the HIV and HCV epidemics. If feasible and efficacious, bundled rapid HIV/HCV testing may offer a scalable, potentially cost-effective approach to testing high-risk populations, such as patients of SUD treatment programs. It may ultimately lead to improved linkage to care and progress through the HIV and HCV care and treatment cascades. Trial registration ClinicalTrials.gov: NCT02355080. (30 January 2015

Optimizing linkage and retention to hypertension care in rural Kenya (LARK hypertension study): study protocol for a randomized controlled trial

BACKGROUND: Hypertension is the leading global risk factor for mortality. Hypertension treatment and control rates are low worldwide, and delays in seeking care are associated with increased mortality. Thus, a critical component of hypertension management is to optimize linkage and retention to care. METHODS/DESIGN: This study investigates whether community health workers, equipped with a tailored behavioral communication strategy and smartphone technology, can increase linkage and retention of hypertensive individuals to a hypertension care program and significantly reduce blood pressure among them. The study will be conducted in the Kosirai and Turbo Divisions of western Kenya. An initial phase of qualitative inquiry will assess facilitators and barriers of linkage and retention to care using a modified Health Belief Model as a conceptual framework. Subsequently, we will conduct a cluster randomized controlled trial with three arms: 1) usual care (community health workers with the standard level of hypertension care training); 2) community health workers with an additional tailored behavioral communication strategy; and 3) community health workers with a tailored behavioral communication strategy who are also equipped with smartphone technology. The co-primary outcome measures are: 1) linkage to hypertension care, and 2) one-year change in systolic blood pressure among hypertensive individuals. Cost-effectiveness analysis will be conducted in terms of costs per unit decrease in blood pressure and costs per disability-adjusted life year gained. DISCUSSION: This study will provide evidence regarding the effectiveness and cost-effectiveness of strategies to optimize linkage and retention to hypertension care that can be applicable to non-communicable disease management in low- and middle-income countries. TRIAL REGISTRATION: This trial is registered with (NCT01844596) on 30 April 2013

Increasing frailty is associated with higher prevalence and reduced recognition of delirium in older hospitalised inpatients: results of a multi-centre study

Purpose: Delirium is a neuropsychiatric disorder delineated by an acute change in cognition, attention, and consciousness. It is common, particularly in older adults, but poorly recognised. Frailty is the accumulation of deficits conferring an increased risk of adverse outcomes. We set out to determine how severity of frailty, as measured using the CFS, affected delirium rates, and recognition in hospitalised older people in the United Kingdom. Methods: Adults over 65 years were included in an observational multi-centre audit across UK hospitals, two prospective rounds, and one retrospective note review. Clinical Frailty Scale (CFS), delirium status, and 30-day outcomes were recorded. Results: The overall prevalence of delirium was 16.3% (483). Patients with delirium were more frail than patients without delirium (median CFS 6 vs 4). The risk of delirium was greater with increasing frailty [OR 2.9 (1.8–4.6) in CFS 4 vs 1–3; OR 12.4 (6.2–24.5) in CFS 8 vs 1–3]. Higher CFS was associated with reduced recognition of delirium (OR of 0.7 (0.3–1.9) in CFS 4 compared to 0.2 (0.1–0.7) in CFS 8). These risks were both independent of age and dementia. Conclusion: We have demonstrated an incremental increase in risk of delirium with increasing frailty. This has important clinical implications, suggesting that frailty may provide a more nuanced measure of vulnerability to delirium and poor outcomes. However, the most frail patients are least likely to have their delirium diagnosed and there is a significant lack of research into the underlying pathophysiology of both of these common geriatric syndromes

Multimeric complexes among Ankyrin-Repeat and SOCS-box Protein 9 (ASB9), ElonginBC, and Cullin 5:insights into the structure and assembly of ECS-type Cullin-RING E3 Ubiquitin Ligases

[Image: see text] Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC–Cullin–SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM–MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5(NTD)) but not to Cul2(NTD). The structure of the monomeric ASB9–EloBC–Cul5(NTD) quaternary complex is revealed by molecular modeling and is consistent with IM–MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems

Contract Report: Australian Indigenous Road Safety

Indigenous Australians are over-represented in road fatalities by approximately 3.5 times 2. Until recently, there has been a lack of research in this area, and quantification of the road safety problem has been difficult due to poor reporting of crashes and complexities with the identification of Indigenous people