The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT

Abstract

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.

Neural Networks in the Design of Molecules with Affinity to Selected Protein Domains

“…Karaś et al demonstrated that molecular docking can be a powerful tool for identifying active compounds against macromolecular targets [ 35 ]. It was discovered that AZ 5104 is active against ROR receptors [ 35 ].…”

Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity

“…This involved a labor-intensive, costly, and time-consuming period of searching for a compound possessing the desired characteristics. Thus, we decided to use powerful chemoinformatic tools for the virtual screening of previously experimentally analyzed chemical libraries (L1600 Kinase Inhibitor Library, TargetMol) [26]. For the entire library, topological 2048-bit fingerprints were calculated (the 2048-element bit vector reflecting the topological properties of the molecule).…”

“…The reactions were performed under the following conditions: 95 • C for 10 min, 40 cycles of 95 • C for 20 s, 58 • C for 20 s, and 72 • C for 20 s. Primers complementary to IL17A and IL17F were described previously: [29] 5 -GCAGCTCTGCTCAGCTTCTA-3 (forward, IL17A) and 5 -GGGCTTTTCTCCTTCTGTGG-3 (reverse, IL17A); 5 -CTCTGATTTGTGGGCAATGG-3 (forward, IL17F) and 5 -CCGGAGTTACTGACGAATGC-3 (reverse, IL17F). The abundance of a specific promoter sequence was calculated using the dCt method with the Ct obtained for input DNA as a reference value as follows: 1000*2-dCt, where dCt = Ct sample Ct input DNA as described previously [26].…”

“…Therefore, increasingly evolving computer methods aid the process of either screening or rational drug design [25]. In this work, we reanalyzed a previously experimentally screened chemical library [26] using the calculated fingerprints of molecular similarity, and we found compounds that are very similar to the RORγT inverse agonist ursolic acid [27]. Further investigations using a cellular reporter system and analysis of the expression of Th17-specific genes/cytokines in Th17 lymphocytes allowed us to identify compounds exerting stronger biological effects than those that were less effective.…”

Untitled

“…Further, TGF-β is among the commonly reported pro-inflammatory cytokines among COVID-19 infected patients that aggravate lungs injury and ARDs. Sorafenib, a potent kinase inhibitor, as well as Osimertinib, an EGFR inhibitor, have shown significant anti-inflammatory and anti-fibrotic effect hence became a future therapeutics against COVID-19 infection [59]. Apart from EGFR inhibitor, CDK inhibitor i.e, Abemaciclib (an approved drug for advanced breast cancers) showed a significant anti-COVID-19 property and enlisted among the 24 FDA-approved drugs that possess potent inhibitory activity against the COVID-19 virus [48,60].…”