Development of a Plate-Based Optical Biosensor Fragment Screening Methodology to Identify Phosphodiesterase 10A Inhibitors

We describe the development of a novel fragment screening methodology employing a plate-based optical biosensor system that can operate in a 384-well format. The method is based on the “inhibition in solution assay” (ISA) approach using an immobilized target definition compound (TDC) that has been specifically designed for this purpose by making use of available structural information. We demonstrate that this method is robust and is sufficiently sensitive to detect fragment hits as weak as KD 500 μM when confirmed in a conventional surface plasmon resonance approach. The application of the plate-based screen, the identification of fragment inhibitors of PDE10A, and their structural characterization are all discussed in a forthcoming paper

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters

<div><p>It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content.</p><p>Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications.</p></div

Quantification of CD63, CD9 and CD81 content in EVs by ELISA.

<p>Normalization was performed to the EVs protein content (N = 5). * represent statistical significance, and correspondingt p-value description in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187665#pone.0187665.s001" target="_blank">S1 File</a>.</p

EV supplementation to differentiating iPSC clusters in 3D-COL hydrogels.

<p>A) Protocol scheme; B) Relative Luminescence Units (RLU) measured from the Apoptosis assay (N = 3). Quantification of intracellular C) insulin (N = 3) and D) C-peptide (N = 3) by ELISA, normalized to total dsDNA content. “WoEVs” denotes iPSC clusters cultured in 3D-COL without supplementation of EVs. * represent statistical significance. P-value description in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187665#pone.0187665.s001" target="_blank">S1 File</a>.</p

Dosage effect of h-islet-EV supplementation in iPSC clusters differentiated in suspension.

<p>A) Scheme of the timing of EVs supplementation. B) Quantification of C-peptide in iPSC clusters supplemented with increasing concentrations of h-Islet-EVs (N = 4). C) Quantification of C-Peptide in iPSC clusters supplemented with 200 μg/mL of h-Ctr-EVs. ELISA quantification was normalized by total DNA content. * represent statistical significance as compared with 0 μg/mL condition. Corresponding p-value description in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187665#pone.0187665.s001" target="_blank">S1 File</a>.</p

Characterisation of EVs.

<p>A) h-Islet-EVs (N = 11) and h-Ctr-EVs (N = 5) protein concentration per 10 mL of isolation media. For each 10 mL of media containing h-Islets, an average of 1390±511 h-Islets were present. No islets were cultured in the media for the control condition. B) Particle concentration was quantified using the AChE activity assay (N = 11) or nanoparticle tracking analysis (N = 3). C) Size distribution over frequency of events (N = 3). Descriptive statistics listed in table above the graph. D) Representative TEM image. Scale bar = 100 nm. * represent statistical significance, and the corresponding p-value in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187665#pone.0187665.s001" target="_blank">S1 File</a>.</p

h-Islet-EV pancreatic cargo characterization.

<p>A) mRNA levels of pancreatic markers in h-Islet-EVs and h-Ctr-EVs as compared to h-islets. Fold change normalized against h-Islets (N = 3). B) Quantification of C-peptide and glucagon in h-Islet-EVs (N = 4) and h-Ctr-EVs (N = 3), normalized by EV protein content (μg). * represent statistical significance, and corresponding p-value description in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187665#pone.0187665.s001" target="_blank">S1 File</a>.</p

Discovery of AZD2716: A Novel Secreted Phospholipase A₂ (sPLA₂) Inhibitor for the Treatment of Coronary Artery Disease

Expedited structure-based optimization of the initial fragment hit <b>1</b> led to the design of (<i>R</i>)-<b>7</b> (AZD2716) a novel, potent secreted phospholipase A₂ (sPLA₂) inhibitor with excellent preclinical pharmacokinetic properties across species, clear <i>in vivo</i> efficacy, and minimized safety risk. Based on accumulated profiling data, (<i>R</i>)-<b>7</b> was selected as a clinical candidate for the treatment of coronary artery disease