High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability

Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI

The DNAJB6 and DNAJB8 Protein Chaperones Prevent Intracellular Aggregation of Polyglutamine Peptides

Bio-organic Synthesi

Allosteric modulation, thermodynamics and binding to wild-type and mutant (T277A) adenosine A(1) receptors of LUF5831, a novel nonadenosine-like agonist

1. The interaction of a new nonribose ligand (LUF5831) with the human adenosine A(1) receptor was investigated in the present study. 2. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A(1) receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. 3. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N(6)-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A(1) receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A(1) receptor (K(i)=122±22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively. 4. The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A(1) receptor showed that LUF5831 had a submaximal effect (37±1%) in comparison to CPA (66±5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. 5. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A(1) receptor