Development of isoform-selective Affimers as novel high affinity agents to study EPAC1 function

Exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular cyclic AMP (cAMP) sensor involved in a wide variety of cellular and physiological processes. Despite an increased understanding of the different roles of EPAC1 in health and disease, reagents are lacking to study its association with cAMP nanodomains intracellularly. In this work, the non-antibody Affimer protein scaffold was used to develop isoform-selective Affimers of EPAC1. Phage-display screens against purified, biotinylated human recombinant EPAC1ΔDEP protein (149-811) identified five potential EPAC1-selective Affimers. Through dot blot and indirect ELISA assays, Affimer 780A was confirmed to be the top EPAC1-binder. Thermal shift assays revealed that 780A had a stabilising effect on the EPAC1 protein conformation in vitro and was found to not discriminate between the active and inactive conformations of EPAC1 in the presence of the EPAC1-specific agonist 007. HSQC NMR confirmed that the binding of 780A to the cyclic-nucleotide binding domain (CNBD) of EPAC1 caused a subtle conformational change. Peptide array technology and mutagenesis studies further revealed a potential interaction site for 780A in the CNBD. In EPAC1-expressing U2OS cells, 780A was shown to co-precipitate EPAC1 protein in immunoprecipitation studies. Through fluorescent and immunofluorescent confocal microscopy, 780A was able to interact with EPAC1 in COS1 cells and co-localised with both EPAC1 WT and a non-targeting EPAC1-SUMOmutant predominantly in the perinuclear and cytosolic regions, respectively. As a novel EPAC1- selective binder, 780A has the potential to be used in super resolution microscopy studies to further our understanding of the cAMP-EPAC1 signalling pathway

The Potential of a Novel Class of EPAC-Selective Agonists to Combat Cardiovascular Inflammation

The cyclic 3′,5′-adenosine monophosphate (cAMP) sensor enzyme, EPAC1, is a candidate drug target in vascular endothelial cells (VECs) due to its ability to attenuate proinflammatory cytokine signalling normally associated with cardiovascular diseases (CVDs), including atherosclerosis. This is through the EPAC1-dependent induction of the suppressor of cytokine signalling gene, SOCS3, which targets inflammatory signalling proteins for ubiquitinylation and destruction by the proteosome. Given this important role for the EPAC1/SOCS3 signalling axis, we have used high throughput screening (HTS) to identify small molecule EPAC1 regulators and have recently isolated the first known non-cyclic nucleotide (NCN) EPAC1 agonist, I942. I942 therefore represents the first in class, isoform selective EPAC1 activator, with the potential to suppress pro-inflammatory cytokine signalling with a reduced risk of side effects associated with general cAMP-elevating agents that activate multiple response pathways. The development of augmented I942 analogues may therefore provide improved research tools to validate EPAC1 as a potential therapeutic target for the treatment of chronic inflammation associated with deadly CVDs

Apoptosis signal-regulating kinase 1 inhibition in in vivo and in vitro models of pulmonary hypertension

Pulmonary arterial hypertension, group 1 of the pulmonary hypertension disease family, involves pulmonary vascular remodelling, right ventricular dysfunction and cardiac failure. Oxidative stress, through activation of mitogen-activated protein kinases is implicated in these changes. Inhibition of apoptosis signal-regulating kinase 1, an apical mitogen-activated protein kinase, prevented pulmonary arterial hypertension developing in rodent models. Here, we investigate apoptosis signal-regulating kinase 1 in pulmonary arterial hypertension by examining the impact that its inhibition has on the molecular and cellular signalling in established disease. Apoptosis signal-regulating kinase 1 inhibition was investigated in in vivo pulmonary arterial hypertension and in vitro pulmonary hypertension models. In the in vivo model, male Sprague Dawley rats received a single subcutaneous injection of Sugen SU5416 (20 mg/kg) prior to two weeks of hypobaric hypoxia (380 mmHg) followed by three weeks normoxia (Sugen/hypoxic), then animals were either maintained for three weeks on control chow or one containing apoptosis signal-regulating kinase 1 inhibitor (100 mg/kg/day). Cardiovascular measurements were carried out. In the in vitro model, primary cultures of rat pulmonary artery fibroblasts and rat pulmonary artery smooth muscle cells were maintained in hypoxia (5% O2) and investigated for proliferation, migration and molecular signalling in the presence or absence of apoptosis signal-regulating kinase 1 inhibitor. Sugen/hypoxic animals displayed significant pulmonary arterial hypertension compared to normoxic controls at eight weeks. Apoptosis signal-regulating kinase 1 inhibitor decreased right ventricular systolic pressure to control levels and reduced muscularised vessels in lung tissue. Apoptosis signal-regulating kinase 1 inhibition was found to prevent hypoxia-induced proliferation, migration and cytokine release in rat pulmonary artery fibroblasts and also prevented rat pulmonary artery fibroblast-induced rat pulmonary artery smooth muscle cell migration and proliferation. Apoptosis signal-regulating kinase 1 inhibition reversed pulmonary arterial hypertension in the Sugen/hypoxic rat model. These effects may be a result of intrinsic changes in the signalling of adventitial fibroblast

Identification and characterization of an affimer affinity reagent for the detection of the cAMP sensor, EPAC1

An exchange protein directly activated by cAMP 1 (EPAC1) is an intracellular sensor for cAMP that is involved in a wide variety of cellular and physiological processes in health and disease. However, reagents are lacking to study its association with intracellular cAMP nanodomains. Here, we use non-antibody Affimer protein scaffolds to develop isoform-selective protein binders of EPAC1. Phage-display screens were carried out against purified, biotinylated human recombinant EPAC1ΔDEP protein (amino acids 149–811), which identified five potential EPAC1-selective Affimer binders. Dot blots and indirect ELISA assays were next used to identify Affimer 780A as the top EPAC1 binder. Mutagenesis studies further revealed a potential interaction site for 780A within the EPAC1 cyclic nucleotide binding domain (CNBD). In addition, 780A was shown to co-precipitate EPAC1 from transfected cells and co-localize with both wild-type EPAC1 and a mis-targeting mutant of EPAC1(K212R), predominantly in perinuclear and cytosolic regions of cells, respectively. As a novel EPAC1-selective binder, 780A therefore has the potential to be used in future studies to further understand compartmentalization of the cAMP-EPAC1 signaling system

Utilisation of beds on the general medical unit by 'non-acute medical' patients: a retrospective study of incidence and cost in two Tasmanian regional medical hospital units

BACKGROUND: Demand for healthcare services threatens to overwhelm the Australian healthcare system. Public hospitals have the largest component of expenditure growth and as such represent the largest opportunity for efficiency gains. Utilisation of inpatient hospital beds and in particular those on general medical units has not been studied in Australia. AIM: To undertake a retrospective patient medical record review of 200 sequential admissions to the medical wards in two regional Tasmanian hospitals to determine the incidence of non-acute medical patient admission to the medical unit, and the subsequent days in hospital that were not required for medical reasons. The cost of these days was estimated. RESULTS: Sixteen patient admissions (8%) could not be justified on medical grounds. Forty-eight (24%) patient admissions had at least one day hospital day that could not be justified on medical grounds. Of the 1438 total bed days, 475 (33%) were for non-medical reasons. The estimated cost of those non-medical bed days for this cohort was $764 800. CONCLUSIONS: The incidence of non-acute medical admissions and non-acute medical bed days to the medical unit and associated cost was significant. Further research is needed to design alternative care provision for such patients particularly in regional Australia. The potential savings to the Australian healthcare system could be significant.M. D. Buist, L. Jaffray, E. Bell, L. Hanna, P. Weinstein, S. Kumar, and K. Grimme