P063. Identification of novel non-transcriptionally acting glucocorticoid receptor ligands that suppress T cell activation but lack adipogenic activity
A. Verhaar1, R. Dvorsky2, M. Wildenberg3, M. Löwenberg1, M. Peppelenbosch4, D.W. Hommes5, G. van den Brink1, 1Academic Medical Center, Gastroenterology & Hepatology, Amsterdam, Netherlands, 2Max Planck Institute of Molecular Physiology, Structural Biology, Dortmund, Germany, 3Academic Medical Center, Tytgat Institute for Liver and Intestinal Research, Amsterdam, Netherlands, 4Erasmus MC, Gastroenterology and Hepatology, Rotterdam, Netherlands, 5University of California Los Angeles, Center for Inflammatory Bowel Diseases, Los Angeles, United States
The use of glucocorticoids as immunosuppressives is limited by important side effects such as loss of bone mass, muscle atrophy and adipogenesis. Glucocorticoids bind to the glucocorticoid receptor (GR) that translocates from the cytosol to the nucleus and regulates transcription of target genes. In addition to this transcriptional regulation, there are also more rapid, non-transcriptionally mediated effects of glucocorticoids. We have previously shown that the GR is part of the T cell receptor (TCR) complex and that ligand binding results in dissociation of the GR from this complex and inhibition of canonical TCR signalling through LCK-PLCγ. The dissociation of this complex appears to play an important role in glucocorticoid mediated inhibition of T cell activation as we found that stimuli that bypass LCK-PLCγ signalling render T lymphocytes glucocorticoid resistant. Here we aimed to develop a GR ligand that inactivates LCK-PLCγ signalling without resulting in transcriptional regulation. We examined if this approach could separate the anti-inflammatory effects of glucocorticoids from some of their side effects.
Potential steroidal and nonsteroidal candidates were identified using an in silico docking assay to predict GR affinity. Selected compounds where screened in in vitro for glucocorticoid response element (GRE) mediated transcriptional regulation and their capacity to inhibit phytohemagglutinin (PHA) or Staphylococcus aureus enterotoxin B induced T cell activation. Two lead compounds were examined for GR binding, their capacity to inhibit canonical LCK-PLCγ-mediated TCR signalling and ability to induce adipogenesis and muscle fiber atrophy.
In the first in silico screening round, we screened 9.2 million compounds and selected 20,000 steroidal and non-steroidal compounds based on similarity to the structure of cortisol. In the second round these compounds were virtually docked in the binding pocket of the GR in two conformations and assigned a score that reflected their predicted affinity. The top 100 compounds were further screened for their capacity to inhibit T cell proliferation. This approach led to the discovery of compounds S3.1 and S3.4, which lack a generic cortisol structure but bind the GR in T lymphocytes and inhibit LCK-PLCγ dependent T cell proliferation without causing transcriptional modulation of GR target genes. In contrast to classical glucocorticoids, S3.1 and S3.4 do not induce adipogenesis or cause muscle cell atrophy in vitro.
Our data show that it is possible to develop nonsteroidal GR ligands that dissociate transcriptional from non-transcriptional effects and may have reduced side effects.