Retinoic acid receptor-related orphan receptor γ (RORγ) is a transcription factor regulating the expression of the pro-inflammatory cytokine IL-17 in human T helper 17 (Th17) cells. Activating RORγ can induce multiple IL-17-mediated autoimmune diseases but may also be useful for anticancer therapy. Its deep immunological functions make RORɣ an attractive drug target. Over 100 crystal structures have been published describing atomic interactions between RORɣ and agonists and inverse agonists. In this review, we focus on the role of dynamic properties and plasticity of the RORɣ orthosteric and allosteric binding sites by examining structural information from crystal structures and simulated models. We discuss the possible influences of allosteric ligands on the orthosteric binding site. We find that high structural plasticity favors the druggability of RORɣ, especially for allosteric ligands.

Autotaxin (ATX) is a secreted lysophospholipase D, catalysing the conversion of lysophosphatidylcholine (LPC) to bioactive lysophosphatidic acid (LPA). LPA acts through two families of G protein-coupled receptors (GPCRs) controlling key cellular responses, and is implicated in many physiological processes and pathologies. ATX has therefore been established as an important drug target in the pharmaceutical industry. Structural and biochemical studies of ATX have shown that it has a bimetallic nucleophilic catalytic site, a substrate-binding (orthosteric) hydrophobic pocket that accommodates the lipid alkyl chain, and an allosteric tunnel that can accommodate various steroids and LPA. Here we first review what is known about ATX-mediated catalysis, crucially in light of allosteric regulation. We then present the known ATX catalysis-independent functions, including binding to cell-surface integrins and proteoglycans. In light of these data we then discuss the four types of ATX inhibitors, as classified depending on their binding to the orthosteric and/or the allosteric site. Finally, we analyse the binding mode of known members of all four types and discuss how mechanistic differences might differentially modulate the activity of the ATX-LPA signalling axis, and clinical applications including cancer.

Background: Although autophagy is a pro-survival process of tumor cells, in particular conditions and when differently regulated by specific signals it can stimulate cell death. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (M2 mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N-8-Iper. Methods: To assess the activation of autophagy, we analyzed the expression of LC3B by Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by Caspases 3 and 9 protein expression. Results: Our data indicate that activation of M2 mAChR by N-8-Iper promotes autophagy in both U251 and GB7 cells lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by the pAMPK expression. Downstream autophagy activation, the increase of apoptosis was also observed in both cell lines after treatment with the two M2 agonists. Conclusions: N-8-Iper treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N-8-Iper.