Structural and functional characterization of a DARPin which inhibits Ras nucleotide exchange.

Ras mutations are the oncogenic drivers of many human cancers and yet there are still no approved Ras-targeted cancer therapies. Inhibition of Ras nucleotide exchange is a promising new approach but better understanding of this mechanism of action is needed. Here we describe an antibody mimetic, DARPin K27, which inhibits nucleotide exchange of Ras. K27 binds preferentially to the inactive Ras GDP form with a Kd of 4 nM and structural studies support its selectivity for inactive Ras. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK, and slows the growth in soft agar of HCT116 cells. K27 is a potent, non-covalent inhibitor of nucleotide exchange, showing consistent effects across different isoforms of Ras, including wild-type and oncogenic mutant forms.

Phenotypic screening reveals TNFR2 as a promising target for cancer immunotherapy.

Antibodies that target cell-surface molecules on T cells can enhance anti-tumor immune responses, resulting in sustained immune-mediated control of cancer. We set out to find new cancer immunotherapy targets by phenotypic screening on human regulatory T (Treg) cells and report the discovery of novel activators of tumor necrosis factor receptor 2 (TNFR2) and a potential role for this target in immunotherapy. A diverse phage display library was screened to find antibody mimetics with preferential binding to Treg cells, the most Treg-selective of which were all, without exception, found to bind specifically to TNFR2. A subset of these TNFR2 binders were found to agonise the receptor, inducing iκ-B degradation and NF-κB pathway signalling in vitro. TNFR2 was found to be expressed by tumor-infiltrating Treg cells, and to a lesser extent Teff cells, from three lung cancer patients, and a similar pattern was also observed in mice implanted with CT26 syngeneic tumors. In such animals, TNFR2-specific agonists inhibited tumor growth, enhanced tumor infiltration by CD8+ T cells and increased CD8+ T cell IFN-γ synthesis. Together, these data indicate a novel mechanism for TNF-α-independent TNFR2 agonism in cancer immunotherapy, and demonstrate the utility of target-agnostic screening in highlighting important targets during drug discovery.

What matters most for end-of-life care? Perspectives from community-based palliative care providers and administrators.

OBJECTIVES: There has been little research conducted to understand the essential meaning of quality, community-based, end-of-life (EOL) care, despite the expansion of these services. The purpose of this study was to define what matters most for EOL care from the perspective of a diverse range of palliative care providers in the community who have daily encounters with death and dying. METHODS: We used interviews to explore the perceptions of providers and administrators from 14 specialised palliative care teams in Ontario, Canada. Participants were prompted with the question 'What matters most for EOL care?' Responses were analysed using a phenomenological approach to derive themes depicting the universal essence of EOL care. RESULTS: Data from 107 respondents were obtained and analysed, from which 40 formulated concepts emerged; these were further grouped into 9 themes. Of the respondents, 39% were nurses, 19% physicians, 27% were supervisors or executives and 15% other. The most predominate concept was that Patient's Wishes are Fulfilled, cited by almost half the respondents. The most prominent themes were Addressing the Non-physical Needs, Healthcare Teams' Nature of Palliative Care Delivery, Patient Wishes are Honoured, Addressing the Physical Needs, Preparing for and Accepting Death, Communication and Relationship Development, and Involving and Supporting the Family. CONCLUSIONS: 9 critical domains of EOL care evolved from the interviews, indicating that quality EOL care extends beyond managing physical pain, but includes a holistic perspective of care, a healthcare team dedicated to the EOL journey and a patient-centred pathway. Tailoring the provision of care to consider these important elements plays a critical role in supporting a positive EOL experience for patients and families.

Anti-arrhythmic effects of cyclopiazonic acid in Langendorff-perfused murine hearts.

We investigated the effects of reducing sarcoplasmic reticular (SR) Ca(2+) stores using the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) in Langendorff-perfused mouse hearts exposed to different pro-arrhythmic agents all known to produce Ca(2+)-mediated arrhythmogenesis. CPA (100 and 150 nM) produced progressive (beginning over approximately 1 min) and significant (P<0.0001) reductions in peak amplitudes of Ca(2+) transients evoked by regular stimulation in isolated Fluo-3 loaded myocytes from F/F(0)=3.2+/-0.16 (n=12 cells) to 1.62+/-0.012 (n=6 cells) and 1.53+/-0.06 (n=12 cells), respectively, consistent with previous reports describing reductions of store Ca(2+) in other cell systems. The corresponding effects of CPA were then examined in intact hearts exposed to isoproterenol (100 nM), elevated extracellular [Ca(2+)] (5mM) and caffeine (1mM). All three agents produced ventricular tachycardia either when added alone or simultaneously with CPA during programmed electrical stimulation. However, arrhythmogenicity was not observed when such agents were added approximately 10 min after introduction of CPA. CPA thus antagonized this Ca(2+)-mediated arrhythmogenesis but only under circumstances of SR Ca(2+) depletion. These alterations in arrhythmogenic tendency took place despite an absence of alterations in electrogram and monophasic action potential characteristics. This was in sharp contrast to previous observations in murine, DeltaKPQ-Scn5a (LQT3) and KCNE1(-/-) (LQT5), systems where re-entry has been implicated in arrhythmogenesis.