Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells

Recent studies have demonstrated that the actin binding protein, ezrin, and the cAMP-sensor, EPAC1, cooperate to induce cell spreading in response to elevations in intracellular cAMP. To investigate the mechanisms underlying these effects we generated a model of EPAC1-dependent cell spreading based on the stable transfection of EPAC1 into HEK293T (HEK293T–EPAC1) cells. We found that direct activation of EPAC1 with the EPAC-selective analogue, 8-pCPT-2′-O-Me-cAMP (007), promoted cell spreading in these cells. In addition, co-activation of EPAC1 and PKA, with a combination of the adenylate cyclase activator, forskolin, and the cAMP phosphodiesterase inhibitor, rolipram, was found to synergistically enhance cell spreading, in association with cortical actin bundling and mobilisation of ezrin to the plasma membrane. PKA activation was also associated with phosphorylation of ezrin on Thr567, as detected by an electrophoretic band mobility shift during SDS-PAGE. Inhibition of PKA activity blocked ezrin phosphorylation and reduced the cell spreading response to cAMP elevation to levels induced by EPAC1-activation alone. Transfection of HEK293T–EPAC1 cells with inhibitory ezrin mutants lacking the key PKA phosphorylation site, ezrin-Thr567Ala, or the ability to associate with actin, ezrin-Arg579Ala, promoted cell arborisation and blocked the ability of EPAC1 and PKA to further promote cell spreading. The PKA phospho-mimetic mutants of ezrin, ezrin-Thr567Asp had no effect on EPAC1-driven cell spreading. Our results indicate that association of ezrin with the actin cytoskeleton and phosphorylation on Thr567 are required, but not sufficient, for PKA and EPAC1 to synergistically promote cell spreading following elevations in intracellular cAMP

The SULSA Assay Development Fund:accelerating translation of new biology from academia to pharma

With industry increasingly sourcing preclinical drug discovery projects from academia it is important that new academic discoveries are enabled through translation with HTS-ready assays. However, many scientifically interesting, novel molecular targets lack associated high-quality, robust assays suitable for hit finding and development. To bridge this gap, the Scottish Universities Life Sciences Alliance (SULSA) established a fund to develop assays to meet quality criteria such as those of the European Lead Factory. A diverse project portfolio was quickly assembled, and a review of the learnings and successful outcomes showed this fund as a new highly cost-effective model for leveraging significant follow-on resources, training early-career scientists and establishing a culture of translational drug discovery in the academic community

In Vitro Assay Development and HTS of Small-Molecule Human ABAD/17β-HSD10 Inhibitors as Therapeutics in Alzheimer's Disease

This research was funded by the Scottish Universities Life Science Alliance (SULSA) assay development fund. This research was also kindly supported by The Rosetrees Trust and The Alzheimer’s Society, specifically The Barcopel Foundation, and partly funded by the MSD Scottish Life Sciences fund. As part of an ongoing contribution to Scottish life sciences, MSD Limited, a global health care leader, has given substantial monetary funding to the Scottish Funding Council for distribution via SULSA to develop and deliver a high-quality drug discovery research and training program.A major hallmark of Alzheimer’s disease (AD) is the formation of neurotoxic aggregates composed of the amyloid-β peptide (Aβ). Aβ has been recognized to interact with numerous proteins, resulting in pathological changes to the metabolism of patients with AD. One such mitochondrial metabolic enzyme is amyloid-binding alcohol dehydrogenase (ABAD), where altered enzyme function caused by the Aβ-ABAD interaction is known to cause mitochondrial distress and cytotoxic effects, providing a feasible therapeutic target for AD drug development. Here we have established a high-throughput screening platform for the identification of modulators to the ABAD enzyme. A pilot screen with a total of 6759 compounds from the NIH Clinical Collections (NCC) and SelleckChem libraries and a selection of compounds from the BioAscent diversity collection have allowed validation and robustness to be optimized. The pilot screen revealed 16 potential inhibitors in the low µM range against ABAD with favorable physicochemical properties for blood-brain barrier penetration.PostprintPeer reviewe

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC

Lead optimization of a pyrazole sulfonamide series of trypanosoma brucei N -myristoyltransferase inhibitors:Identification and evaluation of CNS penetrant compounds as potential treatments for stage 2 human african trypanosomiasis

[Image: see text] Trypanosoma bruceiN-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood–brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe